WO2025034978A1

WO2025034978A1 - Enhanced dna dendrimers and methods of use thereof

Info

Publication number: WO2025034978A1
Application number: PCT/US2024/041488
Authority: WO
Inventors: Robert C. Getts; Jennifer M. LUKE; Samantha Z. Brown; Casey J. Massena; Lori A. Getts
Original assignee: Code Biotherapeutics Inc
Current assignee: Code Biotherapeutics Inc
Priority date: 2023-08-09
Filing date: 2024-08-08
Publication date: 2025-02-13
Anticipated expiration: 2026-02-09

Abstract

The embodiments provide compositions comprising DNA dendrimers, targeting molecules, therapeutic agents, adaptor molecules, support molecules, or combinations thereof. Pharmaceutical compositions, kits, and methods using and producing the same are also provided.

Description

ENHANCED DNA DENDRIMERS AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/518,457, filed on August 9, 2023, which is hereby incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on August 2, 2024, is named “COB-015WO_SL” and is 51,965 bytes in size.

FIELD

The present embodiments relate to DNA dendrimers and other molecules linked to, or encoding, targeting molecules, cargo molecules, adaptor molecules, support molecules, or combinations thereof, and methods of using and producing the same.

BACKGROUND

In many research, diagnostic, and therapeutic applications, the targets of action of a molecule, such as a reporter probe, a small molecule drug, a peptide or a nucleic acid, are intracellular. For nucleic acids that require transcription and/or translation inside a target cell, the nucleic acid must be transported safety into the cell nucleus. However, targeting of a molecule to selected cell types, achieving intracellular delivery to the cytosol and subsequent trafficking to the desired intracellular compartments such as the nucleus represent a challenging goal. Further, therapeutic nucleic acid cargo must be kept safe from degradation, and must express in the nucleus in quantities that are neither too limited or too much. Thus, there is a need for compositions that can reliably deliver molecule such as therapeutic nucleic acids to the nucleus of a target cell without degradation, and with the ability to optimize expression. The present disclosure addresses this need and others.

SUMMARY

In some embodiments, a composition is provided, the composition comprising a DNA dendrimer, a targeting moiety, and an adaptor molecule-cargo polynucleotide complex. In some embodiments, the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest. In some embodiments, cargo polynucleotide is a nicked circular polynucleotide, a linear polynucleotide with a closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the polynucleotide.

In some embodiments, the cargo polynucleotide further comprises at least one DNA targeting sequence (DTS).

In some embodiments, the cargo polynucleotide further comprises at least one nuclear localization signal (NLS).

In some embodiments, the cargo polynucleotide comprises at least one DTS and at least one NLS.

In some embodiments, the adaptor molecule comprises a DNA dendrimer binding sequence (DBS) and a cargo binding region (CBR). In some embodiments, the DBS comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3, 4, 10, 11, 22, or 23.

In some embodiments, the CBR is linked or conjugated to the cargo polynucleotide by DNA ligation. In some embodiments, the CBR is linked or conjugated to the cargo polynucleotide by chemical coupling.

In some embodiments, the adaptor molecule further comprises at least one NLS. In some embodiments, the at least one NLS comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 1, 2, 15, or 43.

In some embodiments, the at least one NLS further comprises at least one spacer located before or after the at least one NLS. In some embodiments, the at least one spacer comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 5 or 29.

In some embodiments, the adaptor molecule further comprises at least one DTS. In some embodiments, the adaptor molecule further comprises a cell penetrating peptide sequence (CPP). In some embodiments, the CPP comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6.

In some embodiments, the adaptor molecule further comprises at least one cleavage site. In some embodiments, the cleavage site is a val-cit linker.

In some embodiments, the adaptor molecule further comprises at least one flexible linker. In some embodiments, the at least one flexible linker is polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), poly lactic-co-glycolic acid (PGLA), (GG)n, (GGGGS)n, or (GGGGA)n, wherein each n is, independently, 1-5.

In some embodiments, the composition further comprises a support molecule. In some embodiments, the support molecule comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48. In some embodiments, the support molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C-terminus of the support molecule. In some embodiments, the PEG molecule is PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000.

In some embodiments, the support molecule comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19. In some embodiments, the support molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C- terminus of the support molecule. In some embodiments, the PEG molecule is PEG2000.

In some embodiments, the support molecule comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19 and PEG2000 conjugated at the N-terminus or C-terminus of SEQ ID NO: 19. In some embodiments, PEG2000 is conjugated at the N- terminus of SEQ ID NO: 19. In some embodiments, PEG2000 is conjugated at the C- terminus of SEQ ID NO: 19. In some embodiments, the support molecule comprises the sequence of SEQ ID NO:

30.

In some embodiments, the targeting moiety is an antibody, a naturally-occurring ligand for the receptor or a functional derivative thereof, a vitamin, a hormone, a small molecule mimetic of a naturally-occurring ligand, a peptide, a polypeptide, a peptidomimetic, a carbohydrate, a lipid, an aptamer, a nucleic acid, a toxin, a component of a microorganism, any other molecule provided it binds specifically to the cell surface molecule and induces endocytosis of the bound moiety, or any combination thereof.

In some embodiments, a composition is provided, the composition comprising a cargo polynucleotide, a support molecule, and a DNA dendrimer linked to a targeting moiety. In some embodiments, the support molecule is as provided for herein. In some embodiments, the targeting moiety is as provided for herein.

In some embodiments, the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest. In some embodiments, the cargo polynucleotide optionally comprises a DBS. In some embodiments, the cargo polynucleotide is a full circular polynucleotide (e.g., plasmid), nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the cargo polynucleotide.

In some embodiments, the cargo polynucleotide further comprises at least one DTS.

In some embodiments, the cargo polynucleotide further comprises at least one NLS.

In some embodiments, the cargo polynucleotide comprises a DBS, wherein the DBS comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 3, 4, 10, 11, 22, or 23.

In some embodiments, the support molecule comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48. In some embodiments, the support molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C-terminus of the support molecule. In some embodiments, the PEG molecule is PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000.

In some embodiments, the support molecule comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19 and PEG2000 conjugated at the N-terminus or C-terminus of SEQ ID NO: 19. In some embodiments, PEG2000 is conjugated at the N- terminus of SEQ ID NO: 19. In some embodiments, PEG2000 is conjugated at the C- terminus of SEQ ID NO: 19.

In some embodiments, the support molecule comprises the sequence of SEQ ID NO: 30.

In some embodiments, any of the compositions provided for herein may comprise a nitrogen/phosphate (N/P) ratio within a set threshold. In some embodiments, the N/P ratio is between 0.5 and 10. In some embodiments, the N/P ratio is between 2 and 5.

In some embodiments, a pharmaceutical composition is provided comprising a composition as provided for herein and a pharmaceutically acceptable carrier.

In some embodiments, a method of delivering a molecule of interest to the nucleus of a target cell is provided, the method comprising contacting the target cell with a composition or pharmaceutical composition as provided for herein, wherein the targeting moiety binds to the target cell.

In some embodiments, a method of treating a disease is provided, the method comprising administering a composition or pharmaceutical composition as provided for herein to a subject to treat the disease, wherein the targeting moiety binds to the target cell.

In some embodiments, a plasmid is provided, the plasmid comprising a plasmid backbone comprising at least two enzyme restriction recognition sites, at least one promoter, at least one coding sequence for at least one molecule of interest, and, optionally, a DBS. In some embodiments, the plasmid is capable of forming a cargo polynucleotide with various structures depending on whether the plasmid has one or more, or none of, a 5' end and a 3' end. In some embodiments, the plasmid is a full circular polynucleotide, a nicked circular nucleotide, a linear nucleotide with a closed 5’ and 3’ end, a linear nucleotide with open 5’ and 3’ ends, or a linear nucleotide with one open and one closed end.

In some embodiments, the plasmid further comprises at least one DTS.

In some embodiments, the plasmid further comprises at least one NLS.

In some embodiments, the plasmid comprises at least one DTS and at least one NLS.

In some embodiments, the DBS is present and comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 3, 4, 10, 11, 22, or 23.

In some embodiments, a method of manufacturing a composition as provided for herein is provided, the method comprising contacting a plasmid as provided for herein with one or more restriction enzymes that cut at the restriction enzyme recognition sites to form a nicked circular polynucleotide, a linear polynucleotide with a closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, linking the cargo polynucleotide to the adaptor molecule to form an adaptor molecule-cargo polynucleotide complex, and linking the adaptor molecule- cargo polynucleotide complex and a targeting moiety to a DNA dendrimer to form the composition.

In some embodiments, a method of manufacturing a composition as provided for herein is provided, the method comprising contacting a plasmid as provided for herein that comprises a DNA dendrimer binding sequence (DBS) with one or more restriction enzymes that cut at the restriction enzyme recognition sites to form a nicked circular polynucleotide, a linear polynucleotide with a closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, and linking the cargo polynucleotide and a targeting moiety to a DNA dendrimer to form the composition.

In some embodiments, a method of manufacturing a composition as provided for herein is provided, the method comprising contacting a circular uncut plasmid as provided for herein with a DNA dendrimer linked to a targeting moiety with a support molecule capable of condensing the size of the cargo polynucleotide and the DNA dendrimer, such that the cargo polynucleotide and the DNA dendrimer are associated together to form the composition.

In some embodiments, a kit is provided, the kit comprising a composition as provided for herein, a pharmaceutical composition as provided for herein, a plasmid as provided for herein, or any combination thereof. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a diagram showing a plasmid map, and individual topologies the plasmid can be changed into by contacting or not contacting certain restriction enzymes with the plasmid.

FIG. 2 depicts GFP fluorescence percentages for various polynucleotide topologies.

FIG. 3 depicts a graph showing the number of GFP positive cells per unit area for various polynucleotide topologies.

FIG. 4 depicts relative mean fluorescence of a polynucleotide containing a DNA targeting sequence (DTS) compared to a polynucleotide without a DTS in CH0-K1 cells.

FIG. 5 depicts relative mean fluorescence of a polynucleotide containing a DNA targeting sequence (DTS) compared to a polynucleotide without a DTS in A427 cells.

FIG. 6 depicts relative mean fluorescence of a polynucleotide containing a DNA targeting sequence (DTS) compared to a polynucleotide without a DTS in C2C12 cells.

FIG. 7 depicts relative integral fluorescence of a polynucleotide containing a DNA targeting sequence (DTS) compared to a polynucleotide without a DTS in C2C12 cells.

FIG. 8 depicts relative peak fluorescence of a polynucleotide containing a DNA targeting sequence (DTS) compared to a polynucleotide without a DTS in C2C12 cells.

FIG. 9A-FIG. 9D shows brightfield and GFP imaging of HepG2 cells 48 hours posttransfection a Genl A Control plasmid (FIG. 9A); a Genl B Control plasmid (FIG. 9B); a Gen3 a(-) plasmid (FIG. 9C); and a Gen3 c(-) plasmid (FIG. 9D).

FIG. 10 illustrates the total fluorescent signal (integrated density) for HepG2 cells transfected with various plasmids after 48 hours.

FIG. 11 shows results of a cell titer gio assay using 0.2 ug of plasmid per well.

FIG. 12 shows results of a cell titer gio assay using 1 ug of plasmid per well.

FIG. 1 a illustrates the ability of various support molecules provided for herein to complex a nucleotide cargo. FIG. 13b illustrates the ability of various support molecules provided for herein to protect a nucleotide cargo from DNAse 1 degradation.

FIG. 14a illustrates the ability of various support molecules provided for herein to complex a nucleotide cargo. FIG. 14b illustrates the ability of various support molecules provided for herein to protect a nucleotide cargo from DNAse 1 degradation.

FIG. 15a illustrates the ability of various support molecules provided for herein to complex a nucleotide cargo. FIG. 15b illustrates the ability of various support molecules provided for herein to protect a nucleotide cargo from DNAse 1 degradation. FIG. 16a illustrates the ability of blends of various support molecules provided for herein to complex a nucleotide cargo. FIG. 14b illustrates the ability of blends of various support molecules provided for herein to protect a nucleotide cargo from DNAse 1 degradation.

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing, suitable methods and materials are described herein. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the embodiments provided for herein will be apparent from the present detailed description and claims.

The techniques and procedures for recombinant manipulations, including nucleic acid and peptide synthesis, are generally performed according to conventional methods in the art and various general references (e.g., Sambrook et al, 2001, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al., eds, 2005, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.; and Gerhardt et al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, D.C.), which are provided throughout this document.

The term “about” or “approximately” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%.

The singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise.

The terms “comprise,” “have,” and “include” and their conjugates, as used herein, mean “including but not limited to.” While various compositions, methods, and devices are described in terms of “comprising” various components or steps (interpreted as meaning “including, but not limited to”), the compositions, methods, and devices can also “consist essentially of’ or “consist of’ the various components and steps.

The term “antibody,” as used herein, refers to an immunoglobulin molecule which can specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies useful in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), Fv, Fab and F(ab)2, as well as single chain antibodies (scFv), camelid antibodies and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

“Complementary” as used herein refers to the broad concept of subunit sequence complementarity between two nucleic acids, e.g., two DNA molecules. When a nucleotide position in both of the molecules is occupied by nucleotides normally capable of base pairing with each other, then the nucleic acids are considered to be complementary to each other at this position. Thus, two nucleic acids are substantially complementary to each other when at least about 50%, preferably at least about 60% and more preferably at least about 80% of corresponding positions in each of the molecules are occupied by nucleotides which normally base pair with each other (e.g., A:T and G:C nucleotide pairs).

As used herein, a “DNA-based carrier” refers to a delivery system that comprises a deoxyribonucleic acid molecule. A non-limiting example of a DNA-based carrier is a DNA dendrimer. Other DNA-based carriers include double- stranded DNA, single-stranded DNA, and single-stranded hairpin DNA, or multimers thereof.

As used herein, a “DNA dendrimer” or “dendrimer” refers to a matrix of polynucleotides, exhibiting branching, formed by the sequential or generational addition of branched layers to or from a core molecule, such as an initiating monomer.

As used herein, an “initiating monomer” is a polynucleotide compound that serves to nucleate the formation of a dendrimer.

As used herein, an “extending monomer” is a polynucleotide compound that can bind to the initiating monomer and/or to each other during assembly of a dendrimer. Extending monomers form the layers of the dendrimer. The first layer of a dendrimer is the layer of extending monomers closest to the initiating monomer. The outer layer is the layer furthest from the initiating monomer and forming the surface of the dendrimer. Extending monomers are also referred to in the art as matrix monomers, matrix extending monomers and matrix polynucleotide monomers.

The terms “substituting,” “substituted,” “mutating,” or “mutated” as used herein refer to altering, deleting, or inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to generate a variant of that sequence. The terms “polynucleotide” or “nucleic acid molecule” means a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. Double and single- stranded DNAs and RNAs are non-limiting examples of polynucleotides.

The term “polypeptide” or “protein” means a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. In some embodiments, the term “peptide” can also be used.

The term “variant” as used herein refers to a polypeptide or polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications, including substitutions, insertions, or deletions.

Conventional notation is used herein to describe polynucleotide sequences: the lefthand end of a single-stranded polynucleotide sequence is the 5 '-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5 '-direction.

As used herein, the term “promoter/regulatory sequence” means a nucleic acid sequence which controls or facilitates the expression of a gene product operably linked to the promoter/regulator sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which control or facilitate the expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in an inducible manner. An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced substantially only when an inducer which corresponds (e.g., binds) to the promoter is present.

As used herein, a “targeting moiety” refers to a molecule that binds to a molecule present on the cell surface of a target cell.

As used herein, a “targeted DNA-based carrier” and “a DNA-based carrier comprising a targeting moiety” refers to a composition comprising a DNA-based carrier and a targeting moiety. The targeting moiety may be linked directly, or by means of a linker, to the DNA- based carrier. Alternatively, the targeting moiety may be linked to another molecule, such as a cargo molecule or a secondary, non-nucleic acid carrier in the composition. The DNA- based carrier is targeted by virtue of being present in the same composition with the targeted cargo or targeted secondary carrier.

The term “vector” as used herein refers to a nucleic acid which can infect, transfect, transiently or permanently transduce a cell. It will be recognized that a vector can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. Vectors include, but are not limited to, replicons (such as RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (i.e., “plasmids”), and include both the expression and non-expression plasmids. Where a recombinant microorganism or cell culture is described as hosting an “expression vector” this includes both extra- chromosomal circular and linear DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure or is incorporated within the host’s genome.

“Promoter” as used herein means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. Such promoterenhancers can modify, for example, but not limited to, tissue specificity or transduction efficiency. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.

The term “topology” as used herein refers to different structural arrangement of any vector, plasmid, or polynucleotide disclosed herein. For example, polynucleotides can have a circular topology, such as a plasmid, where the polynucleotide has no 5’ or 3’ end. Polynucleotides disclosure herein can also have linear topologies, where the polynucleotide has a 5’ and 3’ end. Polynucleotides with a liner topology may have one or both ends of the polynucleotide covalently closed. In some embodiments, certain restriction enzymes can covalently close a DNA end, for example, TelN Protelomerase.

Compositions

A. DNA-Based Carriers In some embodiments, compositions disclosed herein comprise a DNA-based carrier. DNA-based carriers include, but are not limited to, DNA dendrimers, double- stranded linear DNA, single- stranded linear DNA, and single- stranded hairpin DNA, formulated either as monomolecular structures or including several units of the conformation cross-linked together (e.g., multimers). In some embodiments, the composition comprises a DNA dendrimer. DNA dendrimers are spheroid particles (diameter typically about 130 to about 150 nm, although they can be designed to be other sizes) of flexible branches formed by inter-hybridized DNA monomers. Each DNA monomer is composed of two polynucleotide strands that share a central region of complementary sequences where the two strands hybridize to each other forming a double-stranded region, leaving 4 terminal single-stranded polynucleotide portions. These terminal sequences are complementary among themselves; hence, they can hybridize (in layers) to terminal sequences of other DNA monomers. Optionally, DNA dendrimers comprise covalently cross-linked strands of DNA.

The double-stranded region formed by the region of complementary sequence between the first and second oligonucleotides is from about 4 to about 2000 bases in length. In some embodiments, the double- stranded region is from about 5 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 6 to about 2000 bases in length. In some embodiments, the double- stranded region is from about 7 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 8 to about 2000 bases in length. In some embodiments, the double- stranded region is from about 9 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 10 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 15 to about 2000 bases in length. In some embodiments, the double- stranded region is from about 20 to about 2000 bases in length. In some embodiments, the doublestranded region is from about 25 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 30 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 35 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 40 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 45 to about 2000 bases in length. In some embodiments, the double- stranded region is from about 50 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 60 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 70 to about 2000 bases in length. In some embodiments, the double- stranded region is from about 80 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 90 to about 2000 bases in length. In some embodiments, the double- stranded region is from about 100 to about 2000 bases in length. In some embodiments, the doublestranded region is from about 200 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 300 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 400 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 500 to about 2000 bases in length. In some embodiments, the double-stranded region is from about 1000 to about 2000 bases in length. In some embodiments, the double- stranded region is from about 1500 to about 2000 bases in length.

In some embodiments, the double-stranded region is from about 4 to about 1500 bases in length. In some embodiments, the double-stranded region is from about 4 to about 1000 bases in length. In some embodiments, the double- stranded region is from about 4 to about 500 bases in length. In some embodiments, the double-stranded region is from about 4 to about 400 bases in length. In some embodiments, the double- stranded region is from about 4 to about 300 bases in length. In some embodiments, the double- stranded region is from about 4 to about 200 bases in length. In some embodiments, the double-stranded region is from about 4 to about 100 bases in length. In some embodiments, the double- stranded region is from about 4 to about 90 bases in length. In some embodiments, the double- stranded region is from about 4 to about 80 bases in length. In some embodiments, the double-stranded region is from about 4 to about 70 bases in length. In some embodiments, the double- stranded region is from about 4 to about 60 bases in length. In some embodiments, the double-stranded region is from about 4 to about 50 bases in length. In some embodiments, the double-stranded region is from about 4 to about 45 bases in length. In some embodiments, the double-stranded region is from about 4 to about 40 bases in length. In some embodiments, the double- stranded region is from about 4 to about 35 bases in length. In some embodiments, the double- stranded region is from about 4 to about 30 bases in length. In some embodiments, the double- stranded region is from about 4 to about 25 bases in length. In some embodiments, the double- stranded region is from about 4 to about 20 bases in length. In some embodiments, the double- stranded region is from about 4 to about 15 bases in length. In some embodiments, the double- stranded region is from about 4 to about 10 bases in length. In some embodiments, the double- stranded region is from about 4 to about 9 bases in length. In some embodiments, the double- stranded region is from about 4 to about 8 bases in length. In some embodiments, the double- stranded region is from about 4 to about 7 bases in length. In some embodiments, the double- stranded region is from about 4 to about 6 bases in length. In some embodiments, the double- stranded region is from about 4 to about 5 bases in length.

In some embodiments, the double-stranded region is from about 5 to about 200 bases in length. In some embodiments, the double-stranded region is from about 6 to about 200 bases in length. In some embodiments, the double-stranded region is from about 7 to about 200 bases in length. In some embodiments, the double-stranded region is from about 8 to about 200 bases in length. In some embodiments, the double-stranded region is from about 9 to about 200 bases in length. In some embodiments, the double- stranded region is from about 10 to about 200 bases in length. In some embodiments, the double- stranded region is from about 11 to about 200 bases in length. In some embodiments, the double-stranded region is from about 12 to about 200 bases in length. In some embodiments, the double- stranded region is from about 13 to about 200 bases in length. In some embodiments, the doublestranded region is from about 14 to about 200 bases in length. In some embodiments, the double-stranded region is from about 15 to about 200 bases in length. In some embodiments, the double-stranded region is from about 20 to about 200 bases in length. In some embodiments, the double-stranded region is from about 25 to about 200 bases in length. In some embodiments, the double- stranded region is from about 30 to about 200 bases in length. In some embodiments, the double- stranded region is from about 35 to about 200 bases in length. In some embodiments, the double-stranded region is from about 40 to about 200 bases in length. In some embodiments, the double- stranded region is from about 45 to about 200 bases in length. In some embodiments, the double-stranded region is from about 50 to about 200 bases in length. In some embodiments, the double- stranded region is from about 60 to about 200 bases in length. In some embodiments, the double- stranded region is from about 70 to about 200 bases in length. In some embodiments, the double-stranded region is from about 80 to about 200 bases in length. In some embodiments, the double- stranded region is from about 90 to about 200 bases in length. In some embodiments, the doublestranded region is from about 100 to about 200 bases in length. In some embodiments, the double-stranded region is from about 125 to about 200 bases in length. In some embodiments, the double-stranded region is from about 150 to about 200 bases in length. In some embodiments, the double- stranded region is from about 175 to about 200 bases in length.

In some embodiments, the double-stranded region is from about 5 to about 175 bases in length. In some embodiments, the double-stranded region is from about 5 to about 150 bases in length. In some embodiments, the double- stranded region is from about 5 to about 125 bases in length. In some embodiments, the double-stranded region is from about 5 to about 100 bases in length. In some embodiments, the double- stranded region is from about 5 to about 90 bases in length. In some embodiments, the double- stranded region is from about 5 to about 80 bases in length. In some embodiments, the double-stranded region is from about 5 to about 70 bases in length. In some embodiments, the double-stranded region is from about 5 to about 60 bases in length. In some embodiments, the double-stranded region is from about 5 to about 50 bases in length. In some embodiments, the double-stranded region is from about 5 to about 45 bases in length. In some embodiments, the doublestranded region is from about 5 to about 40 bases in length. In some embodiments, the double-stranded region is from about 5 to about 35 bases in length. In some embodiments, the double-stranded region is from about 5 to about 30 bases in length. In some embodiments, the double-stranded region is from about 5 to about 25 bases in length. In some embodiments, the double-stranded region is from about 5 to about 20 bases in length. In some embodiments, the double- stranded region is from about 5 to about 15 bases in length. In some embodiments, the double- stranded region is from about 5 to about 14 bases in length. In some embodiments, the double- stranded region is from about 5 to about 13 bases in length. In some embodiments, the double-stranded region is from about 5 to about 12 bases in length. In some embodiments, the double- stranded region is from about 5 to about 11 bases in length. In some embodiments, the double-stranded region is from about 5 to about 10 bases in length. In some embodiments, the double- stranded region is from about 5 to about 9 bases in length. In some embodiments, the double-stranded region is from about 5 to about 8 bases in length. In some embodiments, the double- stranded region is from about 5 to about 7 bases in length. In some embodiments, the double- stranded region is from about 5 to about 6 bases in length.

In some embodiments, the double-stranded region is from about 25 to about 50 bases in length. In some embodiments, the double-stranded region is from about 26 to about 50 bases in length. In some embodiments, the double-stranded region is from about 27 to about 50 bases in length. In some embodiments, the double-stranded region is from about 28 to about 50 bases in length. In some embodiments, the double- stranded region is from about 29 to about 50 bases in length. In some embodiments, the double-stranded region is from about 30 to about 50 bases in length. In some embodiments, the double-stranded region is from about 31 to about 50 bases in length. In some embodiments, the double- stranded region is from about 32 to about 50 bases in length. In some embodiments, the double- stranded region is from about 33 to about 50 bases in length. In some embodiments, the double-stranded region is from about 34 to about 50 bases in length. In some embodiments, the double- stranded region is from about 35 to about 50 bases in length. In some embodiments, the double-stranded region is from about 36 to about 50 bases in length. In some embodiments, the double-stranded region is from about 37 to about 50 bases in length. In some embodiments, the double-stranded region is from about 38 to about 50 bases in length. In some embodiments, the double- stranded region is from about 39 to about 50 bases in length. In some embodiments, the double- stranded region is from about 40 to about 50 bases in length. In some embodiments, the double-stranded region is from about 45 to about 50 bases in length.

In some embodiments, the double-stranded region is from about 25 to about 45 bases in length. In some embodiments, the double-stranded region is from about 25 to about 40 bases in length. In some embodiments, the double-stranded region is from about 25 to about 39 bases in length. In some embodiments, the double-stranded region is from about 25 to about 38 bases in length. In some embodiments, the double-stranded region is from about 25 to about 37 bases in length. In some embodiments, the double- stranded region is from about 25 to about 36 bases in length. In some embodiments, the double- stranded region is from about 25 to about 35 bases in length. In some embodiments, the double- stranded region is from about 25 to about 34 bases in length. In some embodiments, the double-stranded region is from about 25 to about 33 bases in length. In some embodiments, the double-stranded region is from about 25 to about 32 bases in length. In some embodiments, the doublestranded region is from about 25 to about 31 bases in length. In some embodiments, the double-stranded region is from about 25 to about 30 bases in length. In some embodiments, the double-stranded region is from about 25 to about 29 bases in length. In some embodiments, the double-stranded region is from about 25 to about 28 bases in length. In some embodiments, the double-stranded region is from about 25 to about 27 bases in length. In some embodiments, the double- stranded region is from about 25 to about 26 bases in length.

In some embodiments, the double-stranded region is from about 25 to about 35 bases in length. In some embodiments, the double-stranded region is from about 26 to about 35 bases in length. In some embodiments, the double- stranded region is from about 27 to about 35 bases in length. In some embodiments, the double-stranded region is from about 28 to about 35 bases in length. In some embodiments, the double-stranded region is from about 29 to about 35 bases in length. In some embodiments, the double-stranded region is from about 30 to about 35 bases in length. In some embodiments, the double-stranded region is from about 31 to about 35 bases in length. In some embodiments, the double- stranded region is from about 32 to about 35 bases in length. In some embodiments, the double- stranded region is from about 33 to about 35 bases in length. In some embodiments, the double-stranded region is from about 34 to about 35 bases in length.

In some embodiments, the double-stranded region is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, or more than 200 bases in length or any length in-between any of the recited values. In some embodiments, the double-stranded region is 4 bases in length. In some embodiments, the double-stranded region is 5 bases in length. In some embodiments, the double- stranded region is 6 bases in length. In some embodiments, the double- stranded region is 7 bases in length. In some embodiments, the double-stranded region is 8 bases in length. In some embodiments, the double-stranded region is 9 bases in length. In some embodiments, the double-stranded region is 10 bases in length. In some embodiments, the double- stranded region is 11 bases in length. In some embodiments, the double- stranded region is 12 bases in length. In some embodiments, the double-stranded region is 13 bases in length. In some embodiments, the double-stranded region is 14 bases in length. In some embodiments, the double-stranded region is 15 bases in length. In some embodiments, the double-stranded region is 20 bases in length. In some embodiments, the double- stranded region is 21 bases in length. In some embodiments, the double-stranded region is 22 bases in length. In some embodiments, the double-stranded region is 23 bases in length. In some embodiments, the double-stranded region is 24 bases in length. In some embodiments, the double-stranded region is 25 bases in length. In some embodiments, the double- stranded region is 26 bases in length. In some embodiments, the double-stranded region is 27 bases in length. In some embodiments, the double-stranded region is 28 bases in length. In some embodiments, the double-stranded region is 29 bases in length. In some embodiments, the double- stranded region is 30 bases in length. In some embodiments, the double- stranded region is 31 bases in length. In some embodiments, the double-stranded region is 32 bases in length. In some embodiments, the double-stranded region is 33 bases in length. In some embodiments, the double-stranded region is 34 bases in length. In some embodiments, the double- stranded region is 35 bases in length. In some embodiments, the double- stranded region is 36 bases in length. In some embodiments, the double-stranded region is 37 bases in length. In some embodiments, the double-stranded region is 38 bases in length. In some embodiments, the double-stranded region is 39 bases in length. In some embodiments, the double- stranded region is 40 bases in length. In some embodiments, the double- stranded region is 41 bases in length. In some embodiments, the double-stranded region is 42 bases in length. In some embodiments, the double-stranded region is 43 bases in length. In some embodiments, the double-stranded region is 44 bases in length. In some embodiments, the double- stranded region is 45 bases in length. In some embodiments, the double- stranded region is 46 bases in length. In some embodiments, the double-stranded region is 47 bases in length. In some embodiments, the double-stranded region is 48 bases in length. In some embodiments, the double-stranded region is 49 bases in length. In some embodiments, the double- stranded region is 50 bases in length. In some embodiments, the double- stranded region is 55 bases in length. In some embodiments, the double-stranded region is 60 bases in length. In some embodiments, the double-stranded region is 65 bases in length. In some embodiments, the double-stranded region is 70 bases in length. In some embodiments, the double- stranded region is 75 bases in length. In some embodiments, the double- stranded region is 80 bases in length. In some embodiments, the double-stranded region is 85 bases in length. In some embodiments, the double-stranded region is 90 bases in length. In some embodiments, the double-stranded region is 95 bases in length. In some embodiments, the double- stranded region is 100 bases in length. In some embodiments, the double-stranded region is 125 bases in length. In some embodiments, the double-stranded region is 150 bases in length. In some embodiments, the double-stranded region is 175 bases in length. In some embodiments, the double-stranded region is 200 bases in length. In some embodiments, the double-stranded region is more than 200 bases in length.

Each single-stranded arm is each, independently, from about 4 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 5 to about 200 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 6 to about 200 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 7 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 8 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 9 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 10 to about 200 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 11 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 12 to about 200 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 13 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 14 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 15 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 20 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 25 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 30 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 35 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 40 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 45 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 50 to about 200 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 60 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 70 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 80 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 90 to about 200 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 100 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 125 to about 200 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 150 to about 200 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 175 to about 200 bases in length.

In some embodiments, each single-stranded arm is each, individually, from about 5 to about 175 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 5 to about 150 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 5 to about 125 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 5 to about 100 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 5 to about 90 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 5 to about 80 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 5 to about 70 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 5 to about 60 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 5 to about 50 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 5 to about 45 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 5 to about 40 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 5 to about 35 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 5 to about 30 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 5 to about 25 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 5 to about 20 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 5 to about 15 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 5 to about 14 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 5 to about 13 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 5 to about 12 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 5 to about 11 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 5 to about 10 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 5 to about 9 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 5 to about 8 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 5 to about 7 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 5 to about 6 bases in length.

In some embodiments, each single-stranded arm is each, individually, from about 5 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 6 to about 50 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 7 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 8 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 9 to about 50 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 10 to about 50 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 11 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 12 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 13 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 14 to about 50 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 15 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 16 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 17 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 18 to about 50 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 19 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 20 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 21 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 22 to about 50 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 23 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 24 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 25 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 30 to about 50 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 35 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 40 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 45 to about 50 bases in length.

In some embodiments, each single-stranded arm is each, individually, from about 25 to about 50 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 26 to about 50 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 27 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 28 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 29 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 30 to about 50 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 31 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 32 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 33 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 34 to about 50 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 35 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 36 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 37 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 38 to about 50 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 39 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 40 to about 50 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 45 to about 50 bases in length.

In some embodiments, each single-stranded arm is each, individually, from about 25 to about 45 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 25 to about 40 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 25 to about 39 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 25 to about 38 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 25 to about 37 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 25 to about 36 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 25 to about 35 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 25 to about 34 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 25 to about 33 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 25 to about 32 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 25 to about 31 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 25 to about 30 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 25 to about 29 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 25 to about 28 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 25 to about 27 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 25 to about 26 bases in length.

In some embodiments, each single-stranded arm is each, individually, from about 25 to about 35 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 26 to about 35 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 27 to about 35 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 28 to about 35 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 29 to about 35 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 30 to about 35 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 31 to about 35 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 32 to about 35 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 33 to about 35 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 34 to about 35 bases in length.

In some embodiments, each single-stranded arm is each, individually, from about 12 to about 18 bases in length. In some embodiments, each single- stranded arm is each, individually, from about 13 to about 18 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 14 to about 18 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 15 to about 18 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 16 to about 18 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 17 to about 18 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 12 to about 17 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 12 to about 16 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 12 to about 15 bases in length. In some embodiments, each single-stranded arm is each, individually, from about 12 to about 14 bases in length. In some embodiments, each singlestranded arm is each, individually, from about 12 to about 13 bases in length.

In some embodiments, each single-stranded arm is each, individually, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 ,19 ,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, or more than 200 bases in length or any length in-between any of the recited values. In some embodiments, each single-stranded arm is each, individually, 4 bases in length. In some embodiments, each single-stranded arm is each, individually, 5 bases in length. In some embodiments, each single-stranded arm is each, individually, 6 bases in length. In some embodiments, each single-stranded arm is each, individually, 7 bases in length. In some embodiments, each single-stranded arm is each, individually, 8 bases in length. In some embodiments, each single-stranded arm is each, individually, 9 bases in length. In some embodiments, each single-stranded arm is each, individually, 10 bases in length. In some embodiments, each single-stranded arm is each, individually, 11 bases in length. In some embodiments, each single-stranded arm is each, individually, 12 bases in length. In some embodiments, each single- stranded arm is each, individually, 13 bases in length. In some embodiments, each single-stranded arm is each, individually, 14 bases in length. In some embodiments, each single-stranded arm is each, individually, 15 bases in length. In some embodiments, each single- stranded arm is each, individually, 16 bases in length. In some embodiments, each single-stranded arm is each, individually, 17 bases in length. In some embodiments, each single-stranded arm is each, individually, 18 bases in length. In some embodiments, each single-stranded arm is each, individually, 19 bases in length. In some embodiments, each single-stranded arm is each, individually, 20 bases in length. In some embodiments, each single-stranded arm is each, individually, 21 bases in length. In some embodiments, each single-stranded arm is each, individually, 22 bases in length. In some embodiments, each single-stranded arm is each, individually, 23 bases in length. In some embodiments, each single-stranded arm is each, individually, 24 bases in length. In some embodiments, each single- stranded arm is each, individually, 25 bases in length. In some embodiments, each single-stranded arm is each, individually, 26 bases in length. In some embodiments, each single-stranded arm is each, individually, 27 bases in length. In some embodiments, each single-stranded arm is each, individually, 28 bases in length. In some embodiments, each single-stranded arm is each, individually, 29 bases in length. In some embodiments, each single-stranded arm is each, individually, 30 bases in length. In some embodiments, each single-stranded arm is each, individually, 31 bases in length. In some embodiments, each single-stranded arm is each, individually, 32 bases in length. In some embodiments, each single-stranded arm is each, individually, 33 bases in length. In some embodiments, each single- stranded arm is each, individually, 34 bases in length. In some embodiments, each single-stranded arm is each, individually, 35 bases in length. In some embodiments, each single-stranded arm is each, individually, 36 bases in length. In some embodiments, each single-stranded arm is each, individually, 37 bases in length. In some embodiments, each single- stranded arm is each, individually, 38 bases in length. In some embodiments, each single-stranded arm is each, individually, 39 bases in length. In some embodiments, each single-stranded arm is each, individually, 40 bases in length. In some embodiments, each single-stranded arm is each, individually, 41 bases in length. In some embodiments, each single-stranded arm is each, individually, 42 bases in length. In some embodiments, each single-stranded arm is each, individually, 43 bases in length. In some embodiments, each single-stranded arm is each, individually, 44 bases in length. In some embodiments, each single-stranded arm is each, individually, 45 bases in length. In some embodiments, each single-stranded arm is each, individually, 46 bases in length. In some embodiments, each single- stranded arm is each, individually, 47 bases in length. In some embodiments, each single-stranded arm is each, individually, 48 bases in length. In some embodiments, each single-stranded arm is each, individually, 49 bases in length. In some embodiments, each single- stranded arm is each, individually, 50 bases in length. In some embodiments, each single-stranded arm is each, individually, 55 bases in length. In some embodiments, each single-stranded arm is each, individually, 60 bases in length. In some embodiments, each single-stranded arm is each, individually, 65 bases in length. In some embodiments, each single-stranded arm is each, individually, 70 bases in length. In some embodiments, each single-stranded arm is each, individually, 75 bases in length. In some embodiments, each single-stranded arm is each, individually, 80 bases in length. In some embodiments, each single-stranded arm is each, individually, 85 bases in length. In some embodiments, each single-stranded arm is each, individually, 90 bases in length. In some embodiments, each single- stranded arm is each, individually, 95 bases in length. In some embodiments, each single-stranded arm is each, individually, 100 bases in length. In some embodiments, each single-stranded arm is each, individually, 125 bases in length. In some embodiments, each single-stranded arm is each, individually, 150 bases in length. In some embodiments, each single-stranded arm is each, individually, 175 bases in length. In some embodiments, each single-stranded arm is each, individually, 200 bases in length. In some embodiments, each single-stranded arm is each, individually, more than 200 bases in length.

In some embodiments, the oligonucleotides may comprise one or two hinge segments joining any of the single-stranded arms to the double-stranded region. Each hinge segment may be from 1 to 4 nucleotides in length, from 2 to 4 nucleotides in length, 1 nucleotide in length, 2 nucleotides in length, 3 nucleotides in length, or 4 nucleotides in length. In some embodiments, the nucleotide sequence of the hinge segment 5' to the double-stranded region is CA, and the nucleotide sequence of the hinge segment 3' to the double-stranded region is AC. It is to be understood that the hinge segments are separate from the double- stranded portion and the single- stranded arm. For example, a first oligonucleotide may comprise a first single stranded arm of 50 bases in length, a complementary portion of 35 bases in length, and a second single stranded arm of 50 bases in length, thus comprising a first oligonucleotide of 135 bases in length. Alternatively, the first oligonucleotide may comprise a first single stranded arm of 50 bases in length, a first hinge segment of 4 bases in length, a complementary portion of 35 bases in length, a second hinge segment of 4 bases in length, and a second single stranded arm of 50 bases in length, thus comprising a first oligonucleotide of 143 bases in length. These examples are exemplary only to demonstrate how the length of the oligonucleotide may be determined and are not meant to be construed as limiting in any way. Each of the single- stranded arms has a nucleic acid sequence that can be the same as or different from any other single-stranded arm. In some embodiments, the nucleotide sequence of each of the four single- stranded arms is identical. In some embodiments, the nucleotide sequences of three of the four single-stranded arms are identical to each other and different than the nucleotide sequence of the fourth single- stranded arm. In some embodiments, the nucleotide sequences of two of the four single-stranded arms are identical to each other and different than the nucleotide sequences of the other two single-stranded arms. In some embodiments, the nucleotide sequences of two of the four single-stranded arms are identical to each other and the nucleotide sequences of the other two of the four singlestranded arms are identical to each other, wherein the nucleotide sequence of the first two single- stranded arms is different than the nucleotide sequences of the second two singlestranded arms. In some embodiments, the nucleotide sequence of each of the four singlestranded arms is different. Thus, the nucleotide sequence of any particular single- stranded arm provides flexibility in design choice and can be used to specifically bind to or be conjugated to a particular linking oligonucleotide (which may or may not be further conjugated to a targeting agent or a cargo molecule as provided for herein) or to another single- stranded arm of another monomeric nucleic acid carrier. In some embodiments, one or more of the single stranded arms is complementary to a linking oligonucleotide that is conjugated to a targeting agent. In some embodiments, one or more of the single stranded arms is complementary to a DNA dendrimer binding sequence (DBS) as provided for herein. In some embodiments, one or more of the single stranded arms is complementary to a linking oligonucleotide that is conjugated to a targeting agent and one or more of the single stranded arms is complementary to a DNA dendrimer binding sequence (DBS) as provided for herein. Any two nucleic sequences having a degree of complementarity may be used to attach a single stranded arm of the DNA dendrimer to a linking oligonucleotide or a DBS, wherein the two nucleic acid strands comprise the DBS and the corresponding single stranded arm. The skilled artisan would readily recognize which nucleic acid base pairs are complementary and which are not and thus be able to envisage two nucleic acid sequences having a desired degree of complementarity. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 30% to 100% complementary, from 35% to 100% complementary, from 40% to 100% complementary, from 45% to 100% complementary, from 50% to 100% complementary, from 55% to 100% complementary, from 60% to 100% complementary, from 65% to 100% complementary, from 70% to 100% complementary, from 75% to 100% complementary, from 80% to 100% complementary, from 85% to 100% complementary, from 90% to 100% complementary, from 95% to 100% complementary, or any value or range in-between. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 30% to 95% complementary, from 30% to 90% complementary, from 30% to 85% complementary, from 30% to 80% complementary, from 30% to 75% complementary, from 30% to 70% complementary, from 30% to 65% complementary, from 30% to 60% complementary, from 30% to 55% complementary, from 30% to 50% complementary, from 30% to 45% complementary, from 30% to 40% complementary, from 30% to 35% complementary, or any value or range in-between.

In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are less than 30% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 30%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary, or any value or range inbetween. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 30% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 35% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 40% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 45% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 50% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 55% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 60% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 61% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 62% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 63% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 64% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 65% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 66% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 67% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 68% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 69% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 70% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 71% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 72% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 73% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 74% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 75% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 76% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 77% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 78% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 79% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 80% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 81% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 82% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 83% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 84% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 85% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 86% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 87% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 88% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 89% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 90% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 91% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 92% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 93% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 94% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 95% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 96% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 97% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 98% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 99% complementary. In some embodiments, the two nucleic acid sequences (e.g. the single stranded arm and the linking oligonucleotide or the single stranded arm and the DBS) comprise sequences which are 100% complementary.

DNA dendrimers are commercially available. In addition, the structural design and assembly of DNA dendrimers is generally known in the art. See, for instance, U.S. Pat. Nos. 5,175,270 and 6,274,723, each of which are incorporated herein by reference in their entireties. An initiating monomer constitutes the approximate center of a dendrimer, depending on the type of branching in the dendrimer. The three-dimensional assembly of extending monomers around the initiating monomer forms the interior volume of the dendrimer. The last, outer layer of extending monomers forms the surface of the dendrimer. Thus, the assembly of a dendrimer results in a three-dimensional shape, typically, but not exclusively, a roughly spherical shape comprising layers of extending monomers. The outer layer comprises numerous binding sites.

More specifically, DNA dendrimers may be prepared by protocols having the following features, (i) The starting material is a double-stranded duplex of DNA (i.e. the double-stranded region) with 5' and 3' single-stranded overhangs, or “binding arms,” attached to the duplex trunk (e.g., four binding arms total), called the “initiating monomer,” which is descriptive of its role in the assembly of a dendrimer. Each initiating monomer's 5' and 3' binding arms are annealed to complementary binding arms on “extending monomers” that have similar composition and morphology, (ii) A subset of the four binding arms on each extending monomer is complementary to the binding arms on the initiating monomer. The non-complementary binding arms of the extending monomers are inactive for annealing to the initiating monomer. Typically, four extending monomers can anneal to the initiating monomer to yield a single-layer, or one-layer, dendrimer in solution, (iii) To add another layer of extending monomers to dendrimers, one typically adds similar but distinguishable extending monomers, in which each monomer has a subset of its four binding arms that is complementary to binding arms on the dendrimer. Thus, a one-layer dendrimer can be converted to a two-layer dendrimer, and so on, stepwise, until a desired size of dendrimer is reached. The table below provides details of the structural elements of varying dendrimer preparations. These are exemplary only and are not meant to be limiting in any way.

After assembly, a DNA dendrimer can be crosslinked to maintain and stabilize the structure of the dendrimer. Crosslinking hybridized regions between monomers (i.e., intermonomer crosslinking) or between monomers and the nucleic acids that carry detectable labels, as well as between trunk portions (intra-monomer crosslinking), can stabilize the structure of the polynucleotide dendrimer. Similarly, any hybridized region of any DNA- based carrier may be crosslinked to stabilize the carrier. Individual units of carrier may also be crosslinked into a polymolecular carrier. Such crosslinking chemistries are well known in the art. See, e.g., Cimino et al., Annu. Rev. Biochem. 54: 1151-1193 (1985); Shi et al., Biochemistry 25:5895-5902 (1986); and Cimino et al., Biochemistry 25:3013-3020 (1986). See also U.S. Pat. No. 4,196,281. Non-limiting examples of suitable crosslinking agents include: psoralens (including but not limited to 8-methoxypsoralen and angelicin), mitomycin C, daunomycin, ethidium diazide, cisplatin, transplatin, carboplatin, 8-methoxypsoralen, mechlorethamine, oxaliplatin, and carbodiimide compounds, among others.

The polynucleotide strands used in the monomers of dendrimers or in the other DNA- based carriers, can be made using standard techniques for synthesis of nucleic acids. These techniques can be biological or chemical. The techniques and procedures are generally performed according to conventional methods in the art and in various general references (e.g., Sambrook et al., 2001, supra; Ausubel et al., eds., 2005, supra, and Gerhardt et al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, D.C.).

In some embodiments, polynucleotides are chemically synthesized using methods known in the art. See, e.g., Gait, 1985, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, England. In another embodiment, polynucleotides are synthesized enzymatically using the polymerase chain reaction (PCR). One PCR method suitable for generating single- stranded polynucleotides is multi-cycle PCR using a single primer, which thereby amplifies a single strand. Nucleic acids may be purified by any suitable means, as are well known in the art, prior to their use. For example, the nucleic acids can be purified by reverse phase or ion exchange HPLC, size exclusion chromatography or gel electrophoresis. The skilled artisan will recognize that the method of purification will depend in part on the size of the nucleic acid to be purified.

The outer layer of a DNA dendrimer can have at least two types of binding arms. These binding arms can be used for attaching one or more types of moieties. In some embodiments, the DNA dendrimer of the compositions can bind or attach to targeting moieties, cargoes, support molecules, and the like.

In some embodiments, the DNA-based carrier is associated with a secondary carrier, which may serve as a scaffold for the DNA-based carriers. Examples of such secondary carriers include, but are not limited to, liposomes, non-DNA dendrimers, polymer carriers, microbubbles, paramagnetic and ferromagnetic particles, self-assembled polymers, polymersomes, filomicelles, albumin particles, lipoproteins, and the like. A self- assembled polymer is one that is formed by self-assembly of monomolecular building blocks. These building blocks can be amphiphilic copolymers, which comprise a hydrophilic component (such as, but not limited to, polyethylenimine or polyethyleneglycol) and a hydrophobic component (such a, but not limited to, aliphatic polyesters) into a core-shell-type structure. These structures that are not maintained by direct conjugation or crosslink of the building blocks are known to as micelles and can vary in their morphology, for instance from spherical micelles (“polymersomes”) to elongated or filamentous rods (“filomicelles”). The sizes of these structures may also vary from the nanometer to the micrometer size range. Once formed by self-assembly, these structures may alternatively be further crosslinked chemically to increase their stability. Hydrophobic cargoes can be embedded into the hydrophobic regions of the carrier, while hydrophilic cargoes can be incorporated into an internal aqueous core or to the external hydrophilic corona.

In some embodiments, the DNA-based carrier is linked to the surface of the secondary carrier. In some embodiments, the DNA-based carrier is not linked but is associated with the secondary carrier by virtue of being in the same composition. In some embodiments, the cargos and targeting moieties can be linked to the DNA-based carrier, or to the scaffold carrier, using methods described herein and known in the art.

B. Targeting Moieties

In some embodiments, the DNA-based carrier is directed to a specific cell by linking a targeting moiety to the carrier, to a secondary carrier and/or a cargo. In some embodiments, the targeting moiety is linked to the DNA-based carrier. In some embodiments, the targeting moiety is linked to a secondary carrier. A targeting moiety may be an antibody, a naturally- occurring ligand for the receptor or a functional derivative thereof, a vitamin, a hormone, a small molecule mimetic of a naturally-occurring ligand, a peptide, a polypeptide, a peptidomimetic, a carbohydrate, a lipid, a FN3 domain, an aptamer, a nucleic acid, a toxin, a component of a microorganism, or any other molecule provided it binds specifically to the cell surface molecule and induces endocytosis of the bound moiety.

Without being bound to any particular theory, the targeting moiety can bind specifically to a molecule on the cell surface of a target cell. The targeting moiety can be bind to a cell surface molecule, which can, in some embodiments, induce endocytosis of the DNA- based carrier. Non-limiting examples of cell surface molecule that may be targeted include cell surface proteins, carbohydrates, and lipids. Cell surface molecules that may be targeted include molecules associated with classical endocytosis and those associated with non- classical endocytosis. In some embodiments, the target cell surface molecule is a cell adhesion molecule (CAM). Cell adhesion molecules useful in the invention include, but are not limited to, neural specific adhesion molecules (e.g., NCAM) and systemic intercellular adhesion molecules. Systemic CAMs include intercellular adhesion molecules (e.g., ICAM-1, ICAM-2, ICAM-3), platelet-endothelial cell adhesion molecule (PECAM), activated leukocyte cell adhesion molecule (ALCAM), B-lymphocyte cell adhesion molecule (BL-CAM), vascular cell adhesion molecule (VCAM), mucosal vascular addressin cell adhesion molecule (MAdCAM), CD44, LFA-2 (CD2), LFA-3 (CD58), basigin (CD147) and the like. In some embodiments, the cell surface molecule is CD71.

In some embodiments, the targeting moiety is an antibody that specifically binds to a target cell surface molecule. In some embodiments, the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a synthetic antibody, a heavy chain antibody, and a biologically active fragment of an antibody, wherein the biologically active fragment is a Fab fragment, a F(ab')2 fragment, an sc-Fv fragment.

When the antibody used as a targeting moiety in the compositions and methods of the invention is a polyclonal antibody (IgG), the antibody is generated by inoculating a suitable animal with the targeted cell surface molecule. Antibodies produced in the inoculated animal which specifically bind to the cell surface molecule are then isolated from fluid obtained from the animal. Antibodies may be generated in this manner in several non-human mammals such as, but not limited to goat, sheep, horse, camel, rabbit, and donkey. Methods for generating polyclonal antibodies are well known in the art and are described, for example in Harlow, et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.).

Monoclonal antibodies directed against a full-length targeted cell surface molecule or fragments thereof may be prepared using any well-known monoclonal antibody preparation procedures, such as those described, for example, in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, N.Y.) and in Tuszynski et al. (1988, Blood, 72:109- 115). Human monoclonal antibodies may be prepared by the method described in U.S. patent publication 2003/0224490. Monoclonal antibodies directed against an antigen are generated from mice immunized with the antigen using standard procedures as referenced herein. Nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. in Immunol. 12(3,4): 125-168) and the references cited therein. To prepare a targeted DNA-based carrier, such as a DNA dendrimer, the targeting moiety can be linked to the DNA-based carrier, or a secondary carrier or a cargo that is linked to, or associated with, a composition comprising the DNA-based carrier. A single targeting moiety may be linked to the DNA-based carrier or secondary carrier. Alternatively, a plurality of (e.g., two or more) targeting moieties are linked to the DNA-based carrier, secondary carrier or cargo. When a plurality of targeting moieties are linked to a carrier or cargo, the moieties may target the same cell surface molecule or may target different cell surface molecules. If targeting different cell surface molecules, these molecules may be associated with the same endocytic pathway or different endocytic pathways. The targeting moiety may also not be associated with an endocytic pathway. If targeting different cell surface molecules, the cell surface molecules may be present on the same cell type or may be present on different cell types.

Linking may be non-covalent or covalent. A targeting moiety may be linked directly to one or more of the polynucleotide strands comprising the DNA-based carrier. Alternatively, a targeting moiety is linked to a linker molecule which is in turn linked to the DNA-based carrier. In some embodiments, wherein the DNA-based carrier is a DNA dendrimer, the linker molecule is an oligonucleotide comprising a sequence substantially complementary to a sequence present in one of the binding arms on the surface of the DNA dendrimer. Thus, the targeting moiety is linked indirectly and non-covalently to the DNA dendrimer by hybridization of the oligonucleotide to a binding arm. This approach is also applicable to other DNA-based carriers. Optionally, the hybridized oligonucleotide is also cross-linked to the DNA dendrimer. Cross-linking chemistries are disclosed elsewhere herein. When not cross-linked, hybridization between the DNA dendrimer and an oligonucleotide linked to a targeting moiety should be sufficiently long-lived under the conditions of use. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp. 123-133; Freier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chern. Soc. 109:3783- 3785; Chavali et al., 2005, Bioinformatics 21(20):3918-3925).

In some embodiments, the linker is a secondary IgG Fc-specific antibody which is linked to the DNA-based carrier. In some embodiments, the antibody specifically binds to the Fc portion of the primary IgG antibody, e.g., the targeting moiety or an intervening antibody. The secondary antibody is preferably specific for the species source of the primary antibody. For instance, if the targeting moiety is a human IgG antibody, the secondary antibody is antihuman IgG. In another aspect, the secondary antibody recognizes epitopes of the entire primary antibody, not just the Fc portion. Alternatively, the linker is an Fc gamma receptor that binds specifically to the Fc portion of an IgG antibody. In any of these, the carrier may be readily linked to any targeting moiety that is an IgG antibody.

Non-covalent linkages include, but are not limited to, affinity binding pairs, such as biotin-streptavidin and immunoaffinity, having sufficiently high affinity to maintain the linkage during use and are well-known in the art. The art is also replete with conjugation chemistries useful for covalently linking a target moiety to a DNA-based carrier, secondary carrier or cargo, directly or via a linker. Art-recognized covalent coupling techniques are disclosed, for instance, in U.S. Pat. Nos. 5,416,016, 6,335,435, 6,528,631, 6,861,514 and 6,919,439, incorporated herein by reference in their entirety. Other conjugation chemistries are disclosed in U.S. Patent Publication No. 20040249178, incorporated herein by reference in its entirety. Still other conjugation chemistries include: p-hydroxy-benzoic acid linkers (Chang-Po et al., 2002, Bioconjugate Chem. 13(3):525-529) ; native ligation (Stetsenko et al., 2000, J Org. Chem. 65:4900-4908): disulfide bridge conjugates (Oehlke et al., 2002, Eur J. Biochem. 269:4025-4032 and Rogers et al., 2004, Nuc Acids Res. 32(22) 6595-6604); maleimide linkers (Zhu et al., 1993, Antisense Res Dev. 3:265-275); thioester linkers (Ede et al., 1994, Bioconjug Chem. 5:373-378); Diels-Alder cycloaddition (Marchan et al., 2006. Nuc Acids Res. 34(3): e24, 2006 Feb. 14 Epub); U.S. Pat. No. 6,656,730 and the like. For reviews of peptide-oligonucleotide conjugation chemistries, see also Tung et al., 2000, Bioconjugate Chem. 11:605-618; Zatsepin et al., 2005, Curr Pharm Des. 11(28):3639-3654; and luliano, 2005, Curr Opin Mol. Ther. 7(2): 132-136.

C. Cargos

The compositions provided herein can also comprise a cargo. In some embodiments, the cargo is attached directly or indirectly to a DNA dendrimer. A cargo that is attached to the DNA dendrimer is associated with the DNA dendrimer via a linker or other type of molecule (adaptor), and the like. One, two, or more different cargoes may be delivered by a targeted DNA-based carrier. Cargoes that can be delivered in accordance the compositions disclosed herein include, but are not limited to, a variety of agents, including, but not limited to, therapeutic agents, imaging agents, monitoring agents, chemotherapeutic agents, anti- oncogenic agents, anti- angiogenic agents, tumor suppressor agents, anti-microbial agents, enzyme replacement agents, gene expression modulating agents and expression constructs comprising a nucleic acid encoding a therapeutic protein or nucleic acid. In some embodiments, cargo may be an exogenous material or an endogenous material. Cargoes include any molecule that induces an effect in a cell, including any protein, nucleic acid, small molecule, carbohydrate, or lipid. Cargoes may be peptides, proteins (including enzymes, antibodies and peptidic hormones), ligands of cytoskeleton, nucleic acid, small molecules, non-peptidic hormones and the like. Nucleic acids and cargo polynucleotides that may be delivered by the method of the invention include synthetic and natural nucleic acid material, including DNA, RNA, transposon DNA, antisense nucleic acids, dsRNA, siRNAs, transcription RNA, messenger RNA, ribosomal RNA, small nucleolar RNA, microRNA, ribozymes, plasmids, and expression constructs. When the cargo is or comprises a nucleic acid, the nucleic acid may be a separate entity from the DNA-based carrier. In these embodiments, the DNA-based carrier is not itself the cargo.

In some embodiments, the cargo is a polynucleotide comprising at least one promoter and at least one coding sequence that encodes for at least one molecule of interest. For example, the cargo polynucleotide may comprise one promoter and two or more coding sequences that encode for different molecules of interest, or one promoter for each coding sequence present. In some embodiments, the promoter or promoters are tissue specific promoters that transcript or translate the at least one molecule of interest only when the composition comprising the cargo polynucleotide is present within a target cell that matches the tissue specific promoter. Tissue specific promoters are known in the art and any such tissue specific promoter is within the scope of the present disclosure.

In some embodiments, the molecule of interest is a gene product, such as an enzyme, protein, receptor, and the like. In some embodiments, the cargo polynucleotide encodes two or more molecules of interest. In some embodiments, at least one of the molecules of interest is a detectable molecule, such as, but not limited to, proteins are tagged, detectable via bioluminescence, fluorescence, radiation signals, or some combination thereof. In some embodiments, such molecules of interest are useful for biodistribution studies and other quantitative and/or real-time assessments.

In some embodiments, the cargo polynucleotide may have one or more structures or topologies. For example, the cargo polynucleotide may exist as a fully circular, double stranded DNA or RNA with no free 5’ or 3’ ends. In some embodiments, the cargo polynucleotide is a plasmid. In some embodiments, the cargo polynucleotide can be a nicked circular polynucleotide, a linear polynucleotide with a closed 5’ and 3’ end, a linear polynucleotide with open 5’ and 3’ ends, and a linear polynucleotide with one open and one closed end. In some embodiments, a nicked circular polynucleotide is a fully circular, double stranded DNA or RNA that has at least one break in a single strand of the DNA or RNA. As used herein, a nicked circular polynucleotide is understood to be a circular polynucleotide that has a 1-100 base gap as compared to the un- nicked polynucleotide. In some embodiments, the nick results in a 1-100 base gap, a 2-100 base gap, a 3-100 base gap, a 4- 100 base gap, a 5-100 base gap, a 6-100 base gap, a 7-100 base gap, an 8-100 base gap, a 9- 100 base gap, a 10-100 base gap, a 15-100 base gap, a 20-100 base gap, a 25-100 base gap, a 30-100 base gap, a 35-100 base gap, a 40-100 base gap, a 45-100 base gap, a 50-100 base gap, a 60-100 base gap, a 70-100 base gap, an 80-100 base gap, a 90-100 base gap, or a base gap having any value or range in-between. In some embodiments, the nick results in a 1-90 base gap, a 1-80 base gap, a 1-70 base gap, a 1-60 base gap, a 1-50 base gap, a 1-45 base gap, a 1-40 base gap, a 1-35 base gap, a 1-30 base gap, a 1-25 base gap, a 1-20 base gap, a 1-15 base gap, a 1-10 base gap, a 1-9 base gap, a 1-8 base gap, a 1-7 base gap, a 1-6 base gap, a 1- 5 base gap, a 1-4 base gap, a 1-3 base gap, a 1-2 base gap, or any value or range in-between. In some embodiments, the nick can be due to the action of a nicking restriction enzyme. In some embodiments, a linear polynucleotide has defined 5’ and 3’ ends, with either end, or both, being optionally closed via a covalent bond.

The different topologies or structures can be used for different attachments to the DNA dendrimer.

Provided herein is a plasmid comprising a plasmid backbone comprising at least two restriction sites, at least one promoter, and at least one coding sequence encoding for at least one molecule of interest, wherein the plasmid can form a cargo polynucleotide with various topologies, such as those provided for herein. For example, a gene cassette comprising a promoter sequence and a coding sequence encoding for at least one molecule of interest can be inserted into the plasmid backbone. The resulting plasmid can have a fully circular topology, and if such a topology is desired, no further steps are required. However, if a different topology is desired, the plasmid can be contacted with one or more restriction enzymes to break one or both nucleotide strands of the plasmid and create a nicked circular polynucleotide, a linear polynucleotide with a closed 5’ and 3’ end, a linear polynucleotide with open 5’ and 3’ ends, or a linear polynucleotide with one open and one closed end. In some embodiments, closed ends on liner polynucleotides are formed using TelN Protelomerase.

In some embodiments, a cargo polynucleotide is transported to a nucleus of a cell. In some embodiments, a cargo polynucleotide comprises one or more of a DNA targeting sequence (DTS) or a nuclear localization signal (NLS). DTS and NLS are, independently, short amino acid sequences that target proteins and associated nucleic acids for import into the nucleus. In a non- limiting example, an NLS that can be encoded by the cargo polynucleotide has the amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to YPDEVKRKKKP (SEQ ID NO: 1), SLLESPFDKPDEVKRKKKPPTSHQSDATAEDDSSSKKK (SEQ ID NO: 2), PKKKRKV (SEQ ID NO: 15), or KKPNKKKRKE (SEQ ID NO: 46). These are non-limiting examples of NLS sequences and any NLS can be used. In some embodiments, the NLS comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the NLS comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the NLS comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15. In some embodiments, the NLS comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%>, at least 98%), or at least 99% sequence identity to SEQ ID NO: 46. In some embodiments, the NLS comprises an amino acid sequence of any one of SEQ ID NO: 1, 2, 15, or 46. In some embodiments, the NLS comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments, the NLS comprises an amino acid sequence of SEQ ID NO: 2. In some embodiments, the NLS comprises an amino acid sequence of SEQ ID NO: 15. In some embodiments, the NLS comprises an amino acid sequence of SEQ ID NO: 46. In some embodiments, at least one DTS, at least one NLS, or both can be located anywhere within or in respect to the cargo polynucleotide. For example, the DTS, NLS, or both can be located upstream or downstream of the at least one promoter of the cargo polynucleotide. In such a molecule, the polypeptide is linked to the cargo polynucleotide.

In some embodiments, one or more DTS or NLS sequences can be attached or linked to the cargo polynucleotide, instead of being located within the cargo polynucleotide. In some embodiments, the one or more DTS or NLS sequences can be included in any adaptor molecule described herein, wherein the adaptor molecule is linked to the cargo polynucleotide. In some embodiments, the one or more DTS or NLS sequences can be linked directly to the cargo polynucleotide without being associated with a corresponding adaptor molecule. In some embodiments, the DTS or NLS sequence can be attached to a cargo polynucleotide via a modified nucleotide. For example, a DTS or NLS sequence can be attached to a nicked circular cargo polynucleotide at the nick site, optionally where a modified nucleotide has been first inserted at the nick site.

In some embodiments, the cargo, including cargo polynucleotides described herein, can be linked to a DNA dendrimer directly by any covalent or non-covalent interaction or bond described herein, such as a hydrogen bond. Thus, in some embodiments, the cargo polynucleotide comprises a DNA dendrimer binding sequence (DBS), which links the cargo polynucleotide to the DNA dendrimer. In some embodiments, the DBS comprises a nucleic acid sequence that is complimentary to a nucleic acid sequence on the DNA dendrimer. In a non-limiting example, the DBS comprises a nucleic acid sequence that is complimentary to at least one binding arm of the DNA dendrimer. In some embodiments, the DBS binds to the DNA dendrimer via hydrogen bonding.

In some embodiments, the DBS comprises a nucleic acid sequence selected from any one of: TAGAGGTAACAACTAGCGTACAA (SEQ ID NO: 3);

TAGAGGTAACAACTAGCGTACAATTTTTTTTTT (SEQ ID NO: 4);

TCAGCTTGTACTCTAGTTGTTACCTCTAATGCTGGACC (SEQ ID NO: 10); GCACCCTACAGAGTAACCTAGATTGATCAAACACCTCA (SEQ ID NO: 11); CCTCAGCTTGTACTCTAGTTGTTACCTCTAATGCTGGACCTCAGC (SEQ ID NO: 22); or CCTCAGCACCCTACAGAGTAACCTAGATTGATCAAACACCTCAGC (SEQ ID NO: 23). In some embodiments, the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3, 4, 22, or 23. In some embodiments, the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3. In some embodiments, the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. In some embodiments, the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11. In some embodiments, the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 22. In some embodiments, the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23. In some embodiments, the DBS comprises a nucleic acid sequence of any one of SEQ ID NOs: 3, 4, 22, or 23. In some embodiments, the DBS comprises a nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the DBS comprises a nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the DBS comprises a nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the DBS comprises a nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the DBS comprises a nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the DBS comprises a nucleic acid sequence of SEQ ID NO: 23.

In some embodiments, the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, provided that the nucleic acid sequence comprises at least 20 contiguous nucleic acids of the sequence of SEQ ID NO: 10. The at least 20 contiguous nucleic acids of SEQ ID NO: 10 may be any 20 contiguous nucleic acids. Thus, the at least 20 contiguous nucleic acids may comprise nucleic acids 1-20 of SEQ ID NO: 10, nucleic acids 2-21 of SEQ ID NO: 10, nucleic acids 3-22 of SEQ ID NO: 10, nucleic acids 4-23 of SEQ ID NO: 10, nucleic acids 5- 24 of SEQ ID NO: 10, nucleic acids 6-25 of SEQ ID NO: 10, nucleic acids 7-26 of SEQ ID NO: 10, nucleic acids 8-27 of SEQ ID NO: 10, nucleic acids 9-28 of SEQ ID NO: 10, nucleic acids 10-29 of SEQ ID NO: 10, nucleic acids 11-30 of SEQ ID NO: 10, or nucleic acids 12- 31 of SEQ ID NO: 10. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 1-20 of SEQ ID NO: 10. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 2-21 of SEQ ID NO: 10. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 3-22 of SEQ ID NO: 10. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 4-23 of SEQ ID NO: 10. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 5-24 of SEQ ID NO: 10. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 6-25 of SEQ ID NO: 10. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 7-26 of SEQ ID NO: 10. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 8-27 of SEQ ID NO: 10. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 9-28 of SEQ ID NO: 10. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 10-29 of SEQ ID NO: 10. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 11-30 of SEQ ID NO: 10. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 12-31 of SEQ ID NO: 10.

In some non-limiting embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, wherein the nucleic acid sequence comprises at least 20 contiguous nucleic acids of the sequence of SEQ ID NO: 10, comprises 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 contiguous nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, comprises at least 20 contiguous nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, comprises at least 21 contiguous nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, comprises at least 22 contiguous nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, comprises at least 23 contiguous nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, comprises at least 24 contiguous nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, comprises at least 25 contiguous nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, comprises at least 26 contiguous nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, comprises at least 27 contiguous nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, comprises at least 28 contiguous nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, comprises at least 29 contiguous nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, comprises at least 30 contiguous nucleic acids of the sequence of SEQ ID NO: 10. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 10, comprises at least 31 contiguous nucleic acids of the sequence of SEQ ID NO: 10.

In some embodiments, the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11, provided that the nucleic acid sequence comprises at least 20 contiguous nucleic acids of the sequence of SEQ ID NO: 11. The at least 20 contiguous nucleic acids of SEQ ID NO: 11 may be any 20 contiguous nucleic acids. Thus, the at least 20 contiguous nucleic acids may comprise nucleic acids 1-20 of SEQ ID NO: 11, nucleic acids 2-21 of SEQ ID NO: 11, nucleic acids 3-22 of SEQ ID NO: 1 1, nucleic acids 4-23 of SEQ ID NO: 11, nucleic acids 5- 24 of SEQ ID NO: 11, nucleic acids 6-25 of SEQ ID NO: 11, nucleic acids 7-26 of SEQ ID NO: 11, nucleic acids 8-27 of SEQ ID NO: 11, nucleic acids 9-28 of SEQ ID NO: 11, nucleic acids 10-29 of SEQ ID NO: 11, nucleic acids 11-30 of SEQ ID NO: 11, or nucleic acids 12- 31 of SEQ ID NO: 11. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 1-20 of SEQ ID NO: 11. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 2-21 of SEQ ID NO: 11. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 3-22 of SEQ ID NO: 11. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 4-23 of SEQ ID NO: 11. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 5-24 of SEQ ID NO: 11. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 6-25 of SEQ ID NO: 11. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 7-26 of SEQ ID NO: 11. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 8-27 of SEQ ID NO: 11. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 9-28 of SEQ ID NO: 11. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 10-29 of SEQ ID NO: 11. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 11-30 of SEQ ID NO: 11. In some embodiments, the at least 20 contiguous nucleic acids comprises nucleic acids 12-31 of SEQ ID NO: 11.

In some non-limiting embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11, wherein the nucleic acid sequence comprises at least 20 contiguous nucleic acids of the sequence of SEQ ID NO: 11, comprises 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 contiguous nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 1 , comprises at least 20 contiguous nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11, comprises at least 21 contiguous nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11, comprises at least 22 contiguous nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11, comprises at least 23 contiguous nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11, comprises at least 24 contiguous nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11, comprises at least 25 contiguous nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11, comprises at least 26 contiguous nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1 1 , comprises at least 27 contiguous nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11, comprises at least 28 contiguous nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11 , comprises at least 29 contiguous nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11, comprises at least 30 contiguous nucleic acids of the sequence of SEQ ID NO: 11. In some embodiments, the DBS comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11, comprises at least 31 contiguous nucleic acids of the sequence of SEQ ID NO: 11.

In some embodiments, the DBS sequence further comprises a polythymine sequence at either the 5’ or 3’ end of the DBS sequence. These are non- limiting examples of DBS molecules and any DBS sequence can be used that is complimentary to the dendrimer arm. In some embodiments, the DBS and the dendrimer arm have a percent complementarity as provided for herein.

D. Adaptor Molecules

The cargo can also be linked to a DNA dendrimer by an adaptor molecule as provided for herein. In some embodiments, the adaptor molecule comprises a DBS sequence that links the adaptor molecule to the DNA dendrimer, and a cargo binding region that links to the cargo, for example, a cargo polynucleotide as provided for herein.

In some embodiments, the DBS comprises a nucleic acid sequence that is complimentary to a nucleic acid sequence on the DNA dendrimer. For example, the DBS comprises a nucleic acid sequence that is complimentary to at least one binding arm of the DNA dendrimer. In some embodiments, the DBS binds to the DNA dendrimer via hydrogen bonding. In some embodiments, the DBS comprises any DBS sequence disclosed herein. In some embodiments, any DBS sequence can be used that is complimentary to the dendrimer arm.

In some embodiments, the cargo binding region can be linked to the cargo polynucleotide by range of linkers known in the art. For example, the cargo binding region can comprise a nucleic acid sequence that is complimentary to at least one part of a cargo polynucleotide, and/or links to the cargo by DNA ligation. In another example, the cargo binding region can link to a cargo polynucleotide by chemical coupling, such as, but not limited to, click chemistry or EDC cross-linking. In some embodiments, the cargo binding region comprises at least one nucleotide with an amine, azide, or other reactive group. In some embodiments, the cargo binding region comprises at least one cysteine residue. The adaptor molecule can also have other regions or features included. In some embodiments, such regions or features can be located between the DBS and the cargo binding region but may also be located in any order within the adaptor molecule.

In some embodiments, the adaptor molecule further comprises at least one purification region. In some embodiments, purification region comprises at least one purification or affinity tag. The purification or affinity tag can be any such chemical or amino acid tag known in the art, for example, a polyhistidine tag such as, but not limited to, His6, His 12, and the like.

In some embodiments, the adaptor molecule further comprises a DTS, NLS, or both as provided for herein. In some embodiments, the DTS and NLS regions are located between the DBS and the cargo binding region. In some embodiments, the NLS comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1, 2, 15, or 46. In some embodiments, the NLS comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the NLS comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the NLS comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15. In some embodiments, the NLS comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 46. In some embodiments the NLS comprises an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 46. In some embodiments the NLS comprises an amino acid sequence of SEQ ID NO: 1. In some embodiments the NLS has an amino acid sequence of SEQ ID NO: 2. In some embodiments the NLS has an amino acid sequence of SEQ ID NO: 15. In some embodiments the NLS has an amino acid sequence of SEQ ID NO: 46.

In some embodiments, the DTS comprises any sequence that promotes an interaction with DNA. For example, a DTS sequence may comprise a transcription factor binding sequence or the like. Such sequences are known in the art and are within the scope of the present disclosure. Alternatively, the skilled artisan may utilize a DTS that is synthetically derived from a known sequence that promotes an interaction with DNA. Such a DTS is within the scope of the present disclosure. Further, the skilled artisan may screen putative DTS sequences to provide an appropriate and functional DTS sequence, i.e. one that promotes an interaction with DNA. Such a DTS is within the scope of the present disclosure.

In some embodiments, the DTS is an SV40-DTS. In some embodiments, the DTS comprises a nucleic acid sequence that behaves substantially similar to an SV40-DTS. In some embodiments, the SV40-DTS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to GGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATC TCAATTAGTCAGCAACCA (SEQ ID NO: 8). In some embodiments, the SV40-DTS comprises a nucleic acid sequence of SEQ ID NO: 8.

In some embodiments the DTS or NLS regions further comprise at least one spacer located either before or after the DTS or NLS regions. If two spacers are present, they are located immediately before and after the DTS, NLS, or both. Spacers are generally known in the art and comprise a range of flexible or semi-flexible amino acids or molecules. In some embodiments, the spacer or spacers comprise a polyglycine, optionally with alanine and/or serine residues. In some embodiments, the spacer comprises an amino acid sequence selected from any one of GGGG (SEQ ID NO: 5) or GGGGGG (SEQ ID NO: 29). In some embodiments, the spacer comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the spacer or spacers comprise polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), poly lactic-co-glycolic acid (PGLA), or any combination thereof. In some embodiments, the spacer or spacers comprise a saturated or unsaturated hydrocarbon chain comprising 3-6 carbons that can optionally be substituted.

In some embodiments, the adaptor molecule further comprises a cell penetrating peptide sequence (CPP). CPPs are a group of short peptides that have the ability to increase membrane transduction and can be used to assist the transportation of molecule through cell membrane (for review see, Xu et al., 2019, J Control Release, 309: 106-124). A wide range of CPPs may be included in the adaptor molecule to assist the DNA dendrimer-based composition to pass into a target cell or target nucleus. For example, in some embodiments, the CPP has an amino acid sequence of QPRRRPRRKKRG (SEQ ID NO: 6). In some embodiments, the CPP is located between the DBS and the cargo binding region.

In some embodiments, the cargo is separable from the DNA dendrimer. Thus, in some embodiments the adaptor molecule further comprises one or more cleavage sites. Cleavage sites may be located anywhere between the DBS and the cargo binding region. In some embodiments, the cleavage site is located adjacent to, or nearly adjacent to, the DBS. For example, in some embodiments, the cleavage site is a valine-citruline/p-aminobenzyl carbamate (val-cit) cleavable linker. Val-cit linkers are known in the art and are used in drug conjugation to release cargoes from targeting moieties. In the context of the provided exemplary sequences herein, a val-cit linker may be represented by the sequence VCit, wherein Cit is citroline. Other cleavable linkers known in the art may also be included, such as a ribozyme self-cleaving cleavage site, or tunable pH-sensitive linkers (see, e.g., Choy et al., Bioconjugate Chem. (2016) 27(3): 824-830).

In some embodiments, the adaptor molecule further comprises at least one flexible linker. In some embodiments, the flexible linker is located between the DNS and the cargo binding region. In some embodiments, the at least one flexible linker is or comprises polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), poly lactic -co-glycolic acid (PGLA), (GG)n (SEQ ID NO: 31), (GGGGS)n (SEQ ID NO: 7), (GGGGA)n (SEQ ID NO: 27), or any combination thereof, wherein each n is independently, 1-5.

In some embodiments, the adaptor molecule comprises a purification region. In some embodiments, the purification region comprises an amino acid sequence of HHHHHHC (SEQ ID NO: 28).

Adaptor molecules provided for herein may have none, some, or all elements disclosure herein. In a non-limiting example, an adaptor molecule may comprise one or more purification regions, DTS, NLS, CPP, spacers, cleavage sites, flexible linkers, or any combination thereof, in addition to the DBS and cargo binding region. In some embodiments, an adaptor molecule comprises, in any order, any one, or any combination of DBS, flexible linker, CPP, NLS, purification region, spacer, and cargo binding region, such as those provided herein. Non-limiting examples of adaptor molecules are listed below and comprise in 5’ to 3’ direction, the following elements:

It is to be understood that the adaptor molecules recited in the preceding table are contiguous sequences comprising the various specific sequence identifiers. Accordingly, the adaptor molecule represented by the sequence TAGAGGTAACAACTAGCGTACAATTTTTTTTTT(SEQ ID NO: 4)-HHHHHHC(SEQ ID NO: 28) provided above is a contiguous sequence, wherein the 3’ terminal thymine of SEQ ID NO: 4 is conjugated to the N-terminal histidine of SEQ ID NO: 28, wherein the hyphen (-) separating the various portions of the contiguous sequence represent a conjugation.

The adaptor molecule represented by the sequence TAGAGGTAACAACTAGCGTACAATTTTTTTTTT(SEQ ID NO: 4)-GGGGGG(SEQ ID NO: 29)-YPDEVKRKKKP(SEQ ID NO: 1) provided above is a contiguous sequence, wherein the 3’ terminal thymine od SEQ ID NO: 4 is conjugated to the N-terminal glycine of SEQ ID NO: 29, and the C-terminal glycine of SEQ ID NO: 29 is conjugated to the N- terminal tyrosine of SEQ ID NO: 1, wherein the hyphen (-) separating the various portions of the contiguous sequence represent a conjugation. The adaptor molecule represented by the sequence TAGAGGTAACAACTAGCGTACAATTTTTTTTTT(SEQ ID NO: 4)-GGGGGG(SEQ ID NO: 29)-(VCit)-GGGG(SEQ ID NO: 5)-YPDEVKRKKKP(SEQ ID NO: 1)-GGGG(SEQ ID NO: 5)-HHHHHHC(SEQ ID NO: 28) provided above is a contiguous sequence, wherein the 3’ terminal thymine of SEQ ID NO: 4 is conjugated to the N-terminal glycine of SEQ ID NO: 29, the C-terminal glycine of SEQ ID NO: 29 is conjugated to a val-cit linker (VCit) which is in turn conjugated to the N-terminal glycine of SEQ ID NO: 5, the C-terminal glycine of SEQ ID NO: 5 is conjugated to the N-terminal tyrosine of SEQ ID NO: 1, the C-terminal proline of SEQ ID NO: 1 is conjugated to the N-terminal glycine of SEQ ID NO: 5, and the C- terminal glycine of SEQ ID NO: 5 is conjugated to the N-terminal histidine of SEQ ID NO: 28, wherein the hyphen (-) separating the various portions of the contiguous sequence represent a conjugation.

The adaptor molecule represented by the sequence TAGAGGTAACAACTAGCGTACAATTTTTTTTTT(SEQ ID NO: 4)-GGGGGG(SEQ ID NO: 29)-(VCit)-QPRRRPRRKKRG(SEQ ID NO: 6)- SLLESPFDKPDEVKRKKKPPTSHQSDATAEDDSSSKKK(SEQ ID NO: 2)-GGGG(SEQ ID NO: 5)-HHHHHHC(SEQ ID NO: 28) provided above is a contiguous sequence, wherein the 3’ terminal thymine of SEQ ID NO: 4 is conjugated to the N-terminal glycine of SEQ ID NO: 29, the C-terminal glycine of SEQ ID NO: 29 is conjugated to a val-cit linker (VCit) which is in turn conjugated to the N-terminal glutamine of SEQ ID NO: 6, the C-terminal glycine of SEQ ID NO: 6 is conjugated to the N-terminal serine of SEQ ID NO: 2, the C- terminal lysine of SEQ ID NO: 2 is conjugated to the N-terminal glycine of SEQ ID NO: 5, and the C-terminal glycine of SEQ ID NO: 5 is conjugated to the N-terminal histidine of SEQ ID NO: 28, wherein the hyphen (-) separating the various portions of the contiguous sequence represent a conjugation.

As provided for herein, the conjugation of protein-oligonucleotide, i.e. amino acid: - nucleic acid conjugations, are known in the art and any such conjugation technique may be utilized to construct the above mentioned adaptor molecules. Accordingly, the conjugation between the various portions of the contiguous sequence as represented by the hyphen (-) above may be any appropriate conjugation as known in the art and provided for herein

In some embodiments, an adaptor molecule comprises the sequences provided in the table below. In some embodiments, an adaptor molecule comprises, from 5’ to 3’ end of the nucleotide sequence and N-terminus to C-terminus of the amino acid sequence, the sequences provided in the table below.

In some embodiments, an adaptor molecule comprises, from 5’ to 3’ end, the sequences of SEQ ID NO: 4 and SEQ ID NO: 28. In some embodiments, an adaptor molecule comprises, from 5’ to 3’ end, the sequences of SEQ ID NO: 4, SEQ ID NO: 29, and SEQ ID NO: 1. In some embodiments, an adaptor molecule comprises, from 5’ to 3’ end, the sequences of SEQ ID NO: 4, SEQ ID NO: 29, VCit, SEQ ID NO: 5, SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 28. In some embodiments, an adaptor molecule comprises, from 5’ to 3’ end, the sequences of SEQ ID NO: 4, SEQ ID NO: 29, VCit, SEQ ID NO: 6, SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 28. In some embodiments, an adaptor molecule comprises, from 5’ to 3’ end, the sequences of SEQ ID NO: 4 and SEQ ID NO: 28, wherein the sequences are conjugated to each other. In some embodiments, an adaptor molecule comprises, from 5’ to 3’ end, the sequences of SEQ ID NO: 4, SEQ ID NO: 29, and SEQ ID NO: 1, wherein the sequences are conjugated to each other. In some embodiments, an adaptor molecule comprises, from 5’ to 3’ end, the sequences of SEQ ID NO: 4, SEQ ID NO: 29, VCit, SEQ ID NO: 5, SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 28, wherein the sequences are conjugated to each other. In some embodiments, an adaptor molecule comprises, from 5’ to 3’ end, the sequences of SEQ ID NO: 4, SEQ ID NO: 29, VCit, SEQ ID NO: 6, SEQ ID NO: 2, SEQ ID NO: 5, and SEQ ID NO: 28, wherein the sequences are conjugated to each other.

E. Support Molecules

The compositions herein may also comprise one or more support molecules. Support molecules as provided for herein can be used for a range of tasks, including, but not limited to, protecting DNA dendrimers and cargo polynucleotides from nuclease degradation, enhancing transfection efficiency, assisting in nuclear delivery, and condensing the size of the DNA dendrimer, cargo polynucleotide, or both. Support molecules can be covalently or non- covalently linked to the DNA dendrimer in the same or similar manner as the targeting moieties and cargos. However, in some embodiments, one or more support molecules are not linked to the DNA dendrimer and can associate with the composition through other means.

Without wishing to be bound by theory, because many polynucleotide structures have a negatively changed amine backbone, a support molecule with a sufficient positive charge can associate with the composition through charge attraction. Thus, in some embodiments, the one or more support molecules associate with the composition through charge attraction. In some embodiments, the one or more support molecules have a net-positive charge that is high enough to allow for the one or more support molecules to interact with and, optionally, compact the negatively charged DNA dendrimer. In some embodiments, the one or more support molecules have a net-positive charge that is low enough to avoid aggregation or other cytotoxic side effects.

Additionally, such support molecules can also associate with cargo polynucleotides and DNA dendrimers simultaneously. Thus, in some embodiments, compositions provided for herein comprise a cargo polynucleotide, a support molecule, and a DNA dendrimer linked to a targeting moiety, wherein the cargo polynucleotide is not covalently linked to the DNA dendrimer. The support molecule can associate the cargo polynucleotide with the DNA dendrimer in this way regardless of the topology of the cargo polynucleotide. However, when the cargo polynucleotide has a full circular topology, without any nicks, sites, or regions for direct covalent linkage to a DNA dendrimer or an adaptor molecule, the one or more support molecule can associate the cargo polynucleotide and the DNA dendrimer together to form the composition.

Support molecules are provided for herein can be used individually, as repeats, or in combination either within one molecular entity or a complex mixture of individual molecular entities. In some embodiments, support molecules can be added to the composition prior to the association of the cargo, DNA dendrimer, and optionally, the adaptor molecule. In some embodiments, support molecules can be added to the composition after the association of the cargo, DNA dendrimer, and optionally, the adaptor molecule. In some embodiments, and without limitation, support molecules provided herein are stabilizers.

Without wishing to be bound by a particular theory, pore sizes vary from cell type to cell type and only permit materials of the same size to be taken up. To enable delivery to cells with smaller pore sizes, the particle must be reduced or compacted. In addition to size, particle size and particle protection can increase durability of the scaffold and payload. So far, a variety of stabilizer moieties have been developed and tested to both compact nanoparticles and shield them from degrading enzymes. Net-positive charge allows for the stabilizer to interact and compact the negatively charged nucleic acids through electrostatic interactions. This compaction allows for protection against nucleases since the stabilizer is now at the surface of the nanostructure which interacts with the in vivo environment. The charge must be enough to complex the nucleic acids but not high enough to cause aggregation or other cytotoxic side effects brought on due to positive charge. Thus, the stabilizer can be comprised of biocompatible peptides and/or polymers. The term “biocompatible”, as used herein, refers to a substance that has minimal toxicity in vitro/in vivo, is excretable and/or can degrade into processable units by the body. Thus, in some embodiments, the stabilizer comprises peptide portions that enhance cellular uptake and nuclear localization. CPPs sequences and NLS sequences are elements added into the stabilizer sequences if the initial base stabilizer (positively-charged compaction portion only) is successful at compacting the nucleic acids and protects against nucleases.

In some embodiments, a stabilizer is a support molecule. In some embodiments, a support molecule comprises biocompatible peptides, polymers, or both. Thus, in some embodiments, the support molecule is a peptide-based support molecule or a polymer-based support molecule. In some embodiments, a support molecule comprises a CPP sequence, a NLS sequence, or both. In some embodiments, the CPP sequence is any CPP sequence disclosed herein. In some embodiments, the NLS sequence is any NLS sequence disclosed herein. In some embodiments, the stabilizer is a peptide-based stabilizer. In some embodiments, the support molecule is a peptide-based support molecule. In some embodiments, the support molecule comprises an amino acid sequence selected from any one of: ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (histone Hl sequence, SEQ ID NO: 12); WRRRGFGRRR (SEQ ID NO: 13); GRKKRRQRRRPQ (SEQ ID NO: 14); PKKKRKV (SV40 NLS sequence, SEQ ID NO: 15); GLFHAIAHFIHGGWHGLIHGWYG (SEQ ID NO: 16); WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO: 17); HHHHHHHHHH (SEQ ID NO: 18); (KK)n (SEQ ID NO: 32), where n = 2-15; GLFHAIAHFIHGGWHGLIHGWYGP (H5WYG sequence, SEQ ID NO: 9); WSQ (spacer sequence); AEAEAKAKAEAEAKAK (SEQ ID NO: 33), AEAEAEAEAKAKAKAK (SEQ ID NO: 34); GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVATPKKSTKKTPKKAKKATPKKS TKKTPKKAKKATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 35), ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKATPKKSTK KTPKKAKK (SEQ ID NO: 36), THRPPMWSPVWP-(VCit)-

ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSKKPNKKKRKE (SEQ ID NO:

37), THRPPMWSPVWP-(VCit)- ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSPKKKRKVC (SEQ ID NO:

38), THRPPMWSPVWP-(VCit)-ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKC (SEQ ID NO: 39), ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSPKKKRKV (SEQ ID NO: 40), ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSKKPNKKKRKE (SEQ ID NO: 41), THRPPMWSPVWP (SEQ ID NO: 42), or any combination thereof. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one, or any combination of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 32. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 33. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 34. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 35. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 41. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 42. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 13. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 14. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 15. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 16. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 17. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 18. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 32. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 33. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 34. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 35. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 36. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 37. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 38. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 39. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 40. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 41. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 42. In some embodiments, the support molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprises an amino acid sequence selected from any one of: GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVATPKKSTKKTPKKAKKATPKKS TKKTPKKAKK (SEQ ID NO: 19);

HHHHHHHHHHATPKKSTKKTPKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 20); or GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVGRKKRRQRRRPQWRRRGFGRR R (SEQ ID NO: 21). In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 19, 20, or 21. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 19. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 20. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 21.

In some embodiments, the stabilizer is a polymer-based stabilizer. In some embodiments, the support molecule is a polymer-based support molecule. In some embodiments, the support molecule is a lysine-based support molecule. In some embodiments, the support molecule is a lysine polymer support molecule. In some embodiments, a support molecule comprises an amino acid sequence selected from any one of: KKKKKKKKKKKKKKKKKKK (SEQ ID NO: 24); KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK (SEQ ID NO: 25); or KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK KKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK KKKKKKKKKK (SEQ ID NO: 26). In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 24, 25, or 26. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 25. In some embodiments, the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 26.

In some embodiments, any of the support molecules provided for herein may further comprise additional moieties, such as but not limited to visualization moieties or moieties to support further chemical modification. In some non-limiting embodiments, the visualization moiety is Cy3, or any other small molecule fhiorophore known in the art. In some nonlimiting embodiments, the moiety to support further chemical modification is dibenzylcyclooctyne (DBCO) or the like. The visualization moieties may be attached at the N-terminus, C-terminus, or both the N and C-terminus of the support molecule. Likewise, the chemical modification moieties may be attached at the N-terminus, C-terminus, or both the N and C-terminus of the support molecule. The visualization and/or chemical modification moieties may be conjugated directly to the support molecule, or they may be conjugated through a cleavable linker, such as a val-cit linker as provided for herein.

In some embodiments, a support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37 further comprises a visualization moiety on the N-terminus, the C-terminus, or both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on the N-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on the C-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37 and a visualization moiety is given by the sequence: THRPPMWSPVWP-(VCit)- ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSKKPNKKKRKE-Cy3 (SEQ ID NO: 44).

In some embodiments, a support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38 further comprises a visualization moiety on the N-terminus, the C-terminus, or both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on the N-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on the C-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38 and a visualization moiety is given by the sequence: THRPPMWSPVWP-(VCit)- ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSPKKKRKVC-Cy3 (SEQ ID NO: 45).

In some embodiments, a support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39 further comprises a visualization moiety on the N-terminus, the C-terminus, or both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on the N-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on the C-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39 and a visualization moiety is given by the sequence: THRPPMWSPVWP-(VCit)- ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKC-Cy3 (SEQ ID NO: 46).

In some embodiments, a support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40 further comprises a visualization moiety, a chemical modification moiety, or a combination thereof. In some embodiments, the support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40 further comprises a visualization moiety on the N-terminus, the C-terminus, or both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on the N-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on the C-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40 further comprises a chemical modification moiety on the N-terminus, the C-terminus, or both the N- terminus and C-terminus of the amino acid sequence. In some embodiments, the chemical modification moiety is on the N-terminus of the amino acid sequence. In some embodiments, the chemical modification moiety is on the C-terminus of the amino acid sequence. In some embodiments, the chemical modification moiety is on both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40 further comprises both a visualization moiety and a chemical modification moiety. In some embodiments, the visualization moiety is on the N-terminus of the amino acid sequence and the chemical modification moiety is on the C-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on the C-terminus of the amino acid sequence and the chemical modification moiety is on the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40 and both a visualization moiety and a chemical modification moiety is given by the sequence: DBCO-(VCit)-ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSPKKKRKV- Cy3 (SEQ ID NO: 47).

In some embodiments, a support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 41 further comprises a visualization moiety, a chemical modification moiety, or a combination thereof. In some embodiments, the support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 41 further comprises a visualization moiety on the N-terminus, the C-terminus, or both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on the N-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on the C-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 41 further comprises a chemical modification moiety on the N-terminus, the C-terminus, or both the N- terminus and C-terminus of the amino acid sequence. In some embodiments, the chemical modification moiety is on the N-terminus of the amino acid sequence. In some embodiments, the chemical modification moiety is on the C-terminus of the amino acid sequence. In some embodiments, the chemical modification moiety is on both the N-terminus and C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 41 further comprises both a visualization moiety and a chemical modification moiety. In some embodiments, the visualization moiety is on the N-terminus of the amino acid sequence and the chemical modification moiety is on the C-terminus of the amino acid sequence. In some embodiments, the visualization moiety is on the C-terminus of the amino acid sequence and the chemical modification moiety is on the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 41 and both a visualization moiety and a chemical modification moiety is given by the sequence: DBCO-(VCit)-

ATPKKSTKKTPKKAKKATPKKSTKKTPKKAKKGGGGSKKPNKKKRKE-Cy3 (SEQ ID NO: 48).

In some embodiments, polyethylene glycol (PEG) can be linked or conjugated to any support molecule disclosed herein. The PEG molecule can be any known PEG in the art, including a variety of molecular weights. In some embodiments, the PEG is PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000. In some embodiments, the PEG is PEG400. In some embodiments, the PEG is PEG500. In some embodiments, the PEG is PEG 1000. In some embodiments, the PEG is PEG2000. In some embodiments, the PEG is PEG3000. In some embodiments, the PEG is PEG4000. In some embodiments, the PEG is PEG-5000. In some embodiments, the combination of the PEG molecule and the support molecule increases circulation time of the support molecule, shields positive charges, enhances stability of the composition, or any combination thereof. In some embodiments, the PEG molecule is attached to the N-terminus of the support molecule. In some embodiments, the PEG molecule is attached to the C-terminus of the support molecule.

In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 25 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 32 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 33 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 34 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 35 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 41 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 42 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 43 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 44 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 45 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 46 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 47 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 48 further comprises a PEG molecule. In some embodiments, the support molecule comprises an amino acid sequence of WSQ and further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 and a PEG molecule further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 25 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 32 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 33 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 34 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 35 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 41 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 42 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 43 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 44 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 45 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 46 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 47 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 and a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 25 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 32 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 33 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 34 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 35 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 41 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 42 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 43 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 44 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 45 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 46 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 47 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 48 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 and a PEG2000 molecule further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG molecule linked or conjugated at the N-terminus or the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus or the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or

PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 and a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus or the C-terminus of the amino acid sequence further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 25 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N- terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N- terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 32 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 33 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 34 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 35 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 41 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 42 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 43 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 44 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 45 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 46 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 47 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 48 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 and a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%’, at least 96%), at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19 further comprises a PEG2000 molecule linked or conjugated at the N- terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 25 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 32 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 33 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 34 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 35 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 41 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 42 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 43 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 44 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 45 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 46 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 47 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 48 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 and a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 25 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 32 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 33 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 34 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 35 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 41 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 42 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 43 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 44 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 45 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 46 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 47 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 and a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 17 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%’, at least 96%), at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 25 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 32 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 33 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 34 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 35 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 40 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 41 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 42 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 43 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 44 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 45 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 46 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 47 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 48 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, or 48 and a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 9 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 12 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 13 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 14 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 15 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 16 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 17 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 18 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 19 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 20 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 21 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 24 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 25 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 26 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 32 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 33 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 34 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 35 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 36 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 37 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 38 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 39 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 40 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 41 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 42 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 43 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 44 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 45 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 46 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 47 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 48 further comprises a PEG molecule. In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 and a PEG molecule further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 9 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 12 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 13 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 14 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 15 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG- 5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 16 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 17 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 18 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG- 5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 19 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 20 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 21 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG- 5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 24 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 25 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 26 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG- 5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 32 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 33 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 34 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG- 5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 35 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 36 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 37 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG- 5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 38 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 39 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 40 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG- 5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 41 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 42 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 43 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG- 5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 44 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 45 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 46 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG- 5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 47 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 and a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 9 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 12 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 13 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 14 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 15 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 16 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 17 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 18 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 19 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 20 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 21 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 24 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 25 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 26 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 32 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 33 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 34 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 35 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 36 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 37 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 38 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 39 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 40 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 41 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 42 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 43 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 44 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 45 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 46 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 47 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 48 further comprises a PEG2000 molecule. In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 and a PEG2000 molecule further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG molecule linked or conjugated at the N-terminus or the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus or the C- terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence.

In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 9 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 12 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 13 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 14 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support comprising an amino acid sequence of SEQ ID NO: 15 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 16 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 17 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support comprising an amino acid sequence of SEQ ID NO: 18 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 19 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 20 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 21 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 24 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 25 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support comprising an amino acid sequence of SEQ ID NO: 26 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 32 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 33 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 34 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 35 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 36 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 37 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 38 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 39 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 40 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 41 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 42 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 43 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 44 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 45 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 46 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 47 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 48 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 and a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG- 5000 molecule linked or conjugated at the N-terminus of the amino acid sequence further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 9 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 12 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 13 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 14 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 15 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 16 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 17 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 18 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 19 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 20 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 21 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 24 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 25 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 26 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 32 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 33 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 34 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 35 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 36 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 37 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 38 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 39 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 40 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 41 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 42 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 43 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 44 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 45 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 46 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 47 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 48 further comprises a PEG2000 molecule linked or conjugated at the N-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 and a PEG2000 molecule linked or conjugated at the N- terminus of the amino acid sequence further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 9 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 12 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 13 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 14 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 15 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 16 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 17 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 18 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 19 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 20 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 21 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 24 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 25 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 26 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 32 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 33 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 34 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 35 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 36 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 37 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 38 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 39 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 40 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 41 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 42 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 43 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 44 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 45 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 46 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 47 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 48 further comprises a PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000 molecule linked or conjugated at the C-terminus of the amino acid sequence further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 9 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 12 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 13 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 14 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 15 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 16 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 17 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 18 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 19 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 20 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 21 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 24 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 25 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 26 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 32 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 33 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 34 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 35 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 36 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 37 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 38 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 39 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 40 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 41 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 42 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 43 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 44 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 45 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 46 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 47 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of SEQ ID NO: 48 further comprises a PEG2000 molecule linked or conjugated at the C-terminus of the amino acid sequence. In some embodiments, the support molecule comprising an amino acid sequence of any one of SEQ ID NOs: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 and a PEG2000 molecule linked or conjugated at the C- terminus of the amino acid sequence further comprises one or more spacer molecules. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence of SEQ ID NO: 5. In some embodiments, the spacer molecule comprises an amino acid sequence of WSQ.

In some embodiments, the support molecule comprising a PEG2000 molecule linked or conjugated the amino acid sequence comprises, from N- to C-terminal, the formula of:

[PEG2000]GLFHAIAHFIHGGWHGLIHGWYGWSQPPKKKRKVATPKKSTKKT PKKAKKATPKKSTKKTPKKAKK (SEQ ID NO: 30).

In some embodiments, a composition as provided for herein may comprise one or more of the support molecules provided for herein. In compositions comprising more than one support molecule, any of the support molecules provided for herein may be combined with any other support molecule provided for herein. The combination of multiple support molecules into a single composition may enhance the individual attributes of the provided support molecules providing for a synergistic benefit. Accordingly, in some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any variant of the any of the forgoing as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any variant of the any of the forgoing, wherein the first and the at least a second support molecule are not the same.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12, 35, or 36, or any variant of the any of the forgoing as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, 39, 44, 45, or 46, or any variant of the any of the forgoing as provided for herein. In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 35, or any variant of SEQ ID NO: 35 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, 39, 44, 45, or 46, or any variant of the any of the forgoing as provided for herein. In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12, or any variant of SEQ ID NO: 12 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, 39, 44, 45, or 46, or any variant of the any of the forgoing as provided for herein. In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36, or any variant of SEQ ID NO: 36 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, 39, 44, 45, or 46, or any variant of the any of the forgoing as provided for herein.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12, 35, or 36, or any variant of the any of the forgoing as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, or 39, or any variant of the any of the forgoing as provided for herein. In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 35, or any variant of SEQ ID NO: 35 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, or 39, or any variant of the any of the forgoing as provided for herein. In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12, or any variant of SEQ ID NO: 12 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, or 39, or any variant of the any of the forgoing as provided for herein. In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36, or any variant of SEQ ID NO: 36 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, or 39, or any variant of the any of the forgoing as provided for herein.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12, or any variant of SEQ ID NO: 12 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38, or any variant of SEQ ID NO: 38 as provided for herein.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36, or any variant of SEQ ID NO: 36 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38, or any variant of SEQ ID NO: 38 as provided for herein.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36, or any variant of SEQ ID NO: 36 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39, or any variant of SEQ ID NO: 39 as provided for herein.

In some embodiments, compositions comprising multiple support molecules comprise specific ratios of said support molecules. The ratios utilized may be any appropriate ratio to provide the desired benefit. For example, in a composition comprising support molecule A and support molecule B, the molar ratio of A:B may be >10: 1 , 10: 1 , 9:1 , 8: 1 , 7: 1 , 6:1 , 5: 1 , 4:1, 3:1, 2: 1, 1 :1, 1 :2, 1:3, 1 :4, 1:5, 1:6, 1 :7, 1 :8, 1:9, 1 :10, or 1 : < 10, or any appropriate ratio therein. As used herein, molar ratio is understood to have its normal connotation in the art, i.e. based on the molar concentration of the individual support molecules. Accordingly, it is to be understood that a molar ratio of 4:1 of A:B would encompass, for example, [A] = 4M: [B] = IM; [A] = 6M: [B] = 1.5M; [A] = 400pM: [B] = WOpM; [A] = lOOpM: [B] = 25pM, and so on.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, or 48, or any variant of the any of the forgoing as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any variant of the any of the forgoing, wherein the first and the at least a second support molecule are not the same and wherein the molar ratio of the first support molecule to the at least a second support molecule is >10:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1 :2, 1:3, 1:4, 1 :5, 1:6, 1 :7, 1:8, 1 :9, 1:10, or 1 :<10, or any appropriate ration therein. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is > 10: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 9:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 8:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 7 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 6:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 5: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 4: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 3 : 1 . In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 2:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:2. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :3. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:4. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :5. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:6. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :7. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:8. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :9. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:10. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : < 10.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12, 35, or 36, or any variant of the any of the forgoing as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, 39, 44, 45, or 46, or any variant of the any of the forgoing as provided for herein, wherein the molar ratio of the first support molecule to the at least a second support molecule is >10: 1, 10:1, 9:1, 8: 1, 7:1, 6:1, 5: 1, 4:1, 3:1, 2: 1, 1 : 1, 1:2, 1 :3, 1 :4, 1:5, 1 :6, 1 :7, 1:8, 1 :9, 1 :10, or 1 :<10, or any appropriate ration therein. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is >10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 10: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 9: 1 . In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 8:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 7: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 6:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 5: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 4:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 3:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 2:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:2. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:3. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :4. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:5. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :6. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:7. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :8. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:9. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :10. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is l:<10.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 35, or any variant of SEQ ID NO: 35 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, 39, 44, 45, or 46, or any variant of the any of the forgoing as provided for herein, wherein the molar ratio of the first support molecule to the at least a second support molecule is >10:1, 10: 1, 9:1, 8:1, 7: 1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1 :2, 1 :3, 1:4, 1 :5, 1 :6, 1:7, 1:8, 1 :9, 1: 10, or 1 : < 10, or any appropriate ration therein. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is > 10: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 9: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 8: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 7 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 6: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 5: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 4: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 3 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 2: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:2. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :3. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:4. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :5. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:6. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :7. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:8. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :9. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:10. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : < 10.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12, or any variant of SEQ ID NO: 12 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, 39, 44, 45, or 46, or any variant of the any of the forgoing as provided for herein, wherein the molar ratio of the first support molecule to the at least a second support molecule is >10: 1, 10: 1, 9:1 , 8:1, 7: 1, 6: 1, 5:1, 4:1, 3: 1, 2:1, 1:1, 1 :2, 1 :3, 1:4, 1 :5, 1 :6, 1 :7, 1:8, 1 :9, 1: 10, or 1 : < 10, or any appropriate ration therein. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is >10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 9: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 8: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 7 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 6:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 5: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 4:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 3 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 2:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:2. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :3. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:4. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :5. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:6. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :7. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:8. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :9. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:10. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is l:< 10.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36, or any variant of SEQ ID NO: 36 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, 39, 44, 45, or 46, or any variant of the any of the forgoing as provided for herein, wherein the molar ratio of the first support molecule to the at least a second support molecule is >10: 1, 10: 1, 9: 1, 8:1, 7: 1, 6: 1, 5:1, 4:1, 3: 1, 2: 1, 1:1, 1 :2, 1 :3, 1:4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, or 1 : < 10, or any appropriate ration therein. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is >10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 9:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 8:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 7 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 6: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 5:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 4:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 3 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 2:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:2. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :3. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:4. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :5. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:6. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :7. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:8. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :9. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:10. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : < 10.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12, 35, or 36, or any variant of the any of the forgoing as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, or 39, or any variant of the any of the forgoing as provided for herein wherein the molar ratio of the first support molecule to the at least a second support molecule is >10:1, 10:1, 9: 1, 8:1, 7: 1, 6:1, 5:1, 4:1, 3:1, 2:1, 1 :1, 1:2, 1:3, 1:4, 1:5, 1 :6, 1:7, 1 :8, 1 :9, 1: 10, or l:<10, or any appropriate ration therein. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is >10: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 9: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 8:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 7:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 6: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 5: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 4: 1 . In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 3:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 2: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :2. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:3. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :4. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:5. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :6. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:7. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :8. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:9. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 10. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is l:<10.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 35, or any variant of SEQ ID NO: 35 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, or 39, or any variant of the any of the forgoing as provided for herein, wherein the molar ratio of the first support molecule to the at least a second support molecule is >10:1, 10:1, 9:1, 8:1, 7:1, 6: 1, 5:1, 4:1, 3:1, 2:1, 1 :1, 1 :2, 1:3, 1 :4, 1 :5, 1:6, 1 :7, 1 :8, 1:9, 1:10, or 1 : < 10, or any appropriate ration therein. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is > 10: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 10:1 . In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 9:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 8: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 7 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 6:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 5:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 4:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 3 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 2:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:2. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :3. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:4. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :5. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:6. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :7. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:8. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :9. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:10. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is l:< 10.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12, or any variant of SEQ ID NO: 12 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, or 39, or any variant of the any of the forgoing as provided for herein, wherein the molar ratio of the first support molecule to the at least a second support molecule is >10: 1, 10: 1, 9:1, 8:1, 7: 1, 6:1, 5: 1, 4: 1, 3: 1, 2: 1, 1:1, 1 :2, 1:3, 1:4, 1 :5, 1:6, 1 :7, 1 :8, 1:9, 1 : 10, or 1 : < 10, or any appropriate ration therein. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is >10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 9:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 8: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 7 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 6: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 5:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 4:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 3 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 2:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:2. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :3. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:4. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :5. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:6. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :7. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:8. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :9. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 10. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : < 10.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36, or any variant of SEQ ID NO: 36 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37, 38, or 39, or any variant of the any of the forgoing as provided for herein, wherein the molar ratio of the first support molecule to the at least a second support molecule is >10:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1 :2, 1:3, 1:4, 1 :5, 1:6, 1 :7, 1 :8, 1:9, 1:10, or 1 : < 10, or any appropriate ration therein. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is > 10: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 9: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 8: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 7 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 6: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 5:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 4: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 3 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 2: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:2. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :3. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:4. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :5. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:6. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:7. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :8. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :9. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:10. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : < 10.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 12, or any variant of SEQ ID NO: 12 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38, or any variant of SEQ ID NO: 38 as provided for herein, wherein the molar ratio of the first support molecule to the at least a second support molecule is >10: 1, 10: 1, 9:1, 8:1, 7:1 , 6:1, 5: 1, 4:1, 3:1, 2:1, 1 :1, 1 :2, 1:3, 1 :4, 1 :5, 1:6, 1 :7, 1 :8, 1 :9, 1 :10, or l :<10, or any appropriate ration therein. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is >10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 10: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 9:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 8: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 7: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 6:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 5:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 4: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 3: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 2:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :2. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :3. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :4. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:5. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :6. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:7. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :8. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:9. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 10. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : < 10.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36, or any variant of SEQ ID NO: 36 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 38, or any variant of SEQ ID NO: 38 as provided for herein, wherein the molar ratio of the first support molecule to the at least a second support molecule is >10:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5: 1, 4:1, 3:1, 2: 1, 1 :1, 1:2, 1:3, 1 :4, 1 :5, 1:6, 1 :7, 1 :8, 1:9, 1 :10, or l :< 10, or any appropriate ration therein. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is >10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 9:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 8: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 7: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 6:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 5:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 4: 1 . Tn some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 3:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 2:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :2. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:3. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :4. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:5. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :6. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:7. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :8. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:9. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 10. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :< 10.

In some embodiments, a composition as provided for herein comprises a first support molecule and at least a second support molecule, wherein the first support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 36, or any variant of SEQ ID NO: 36 as provided for herein and the at least a second support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 39, or any variant of SEQ ID NO: 39 as provided for herein, wherein the molar ratio of the first support molecule to the at least a second support molecule is >10:1, 10: 1, 9:1, 8:1, 7:1, 6:1, 5: 1, 4:1, 3:1, 2: 1, 1 : 1, 1 :2, 1:3, 1 :4, 1 :5, 1:6, 1 :7, 1 :8, 1 :9, 1 :10, or l :<10, or any appropriate ration therein. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is >10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 10:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 9: 1 . In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 8: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 7: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 6: 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 5:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 4:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 3:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 2:1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 1. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :2. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:3. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :4. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:5. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :6. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:7. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 :8. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1:9. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : 10. In some embodiments, the molar ratio of the first support molecule to the at least a second support molecule is 1 : < 10.

In some embodiments, support molecules are added at a specific concentration to the composition. In some embodiments, a support molecule is added to the composition to bring the ionic balance of the overall composition within an acceptable Nitrogen/Phosphate (N/P) ratio. A N/P ration is the number of nitrogen groups in the support molecule relative to the number of phosphorus groups in any nucleic acid molecule in a composition. In some embodiments, the higher the N/P ratio, the more support molecule is added to a composition. In some embodiments, the N/P ratio is calculated using the following formula:

In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 0.25 and 10. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 0.5 and 10. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 1 and 10. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 2 and 10. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 3 and 10. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 4 and 10. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 5 and 10. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 6 and 10. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 7 and 10. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 8 and 10. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 9 and 10. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 0.25 and 9. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 0.25 and 8. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 0.25 and 7. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 0.25 and 6. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 0.25 and 5. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 0.25 and 4. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 0.25 and 3. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 0.25 and 2. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 0.25 and 1. In some embodiments, the support molecule is added to the composition so that the ionic balance of the composition has a N/P ratio between 0.25 and 5.

F. Modifications

Modified nucleic acids may be used throughout the compositions described herein, including the DNA dendrimers, cargoes, adaptor molecules, and support molecules. Nonlimiting examples of such chemical modifications independently include without limitation phosphate backbone modification (e.g. phosphorothioate internucleotide linkages), nucleotide sugar modification (e.g., 2'-O-methyl nucleotides, 2'-O-allyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxyribonucleotides), nucleotide base modification (e.g., “universal base” containing nucleotides, 5-C-methyl nucleotides), and non-nucleotide modification (e.g., abasic nucleotides, inverted deoxyabasic residue) or a combination of these modifications. In addition, oligonucleotides having morpholino backbone structures (U.S. Pat. No. 5,034,506) or polyamide backbone structures (Nielsen et al., 1991, Science 254: 1497) may also be used. These and other chemical modifications can preserve biological activity of a nucleic acid in vivo while at the same time, dramatically increasing the serum stability, potency, duration of effect and/or specificity of these compounds. Nucleic acids containing modified internucleoside linkages may also be synthesized using reagents and methods that are well known in the art. For example, methods for synthesizing nucleic acids containing phosphonate phosphorothioate, phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate, formacetal, thioformacetal, diisopropylsilyl, acetamidate, carbamate, dimethylene-sulfide ( — CH2 — S — CH2), dimethylene- sulfoxide ( — CH2 — SO — CH2), dimethylene-sulfone ( — CH2 — SO2 — CH2), 2'-O-alkyl, and 2'-deoxy-2'-fluoro phosphorothioate internucleoside linkages are well known in the art (see Uhlmann et al., 1990, Chem. Rev. 90:543-584; Schneider et al., 1990, Tetrahedron Lett. 31:335 and references cited therein).

The examples of oligonucleotide modifications described herein are not exhaustive and it is understood that the compositions includes additional modifications which serve to enhance the therapeutic or other properties of the oligonucleotides without appreciable alteration of the basic sequence of the oligonucleotide. Similarly, protein cargoes may be modified as described elsewhere herein.

Additionally, linkages described herein may be noncovalent or covalent. Covalent linkages include linkages susceptible to cleavage once internalized in a cell. Such linkages include pH-labile, photo-labile and radio-labile bonds and are well known in the art. Cargos may be linked to an oligonucleotide comprising a sequence that is substantially complementary to a binding arm to a sequence present in one of the binding arms on the surface of the DNA dendrimer or to a portion of single stranded sequence of any DNA-based carrier. The oligonucleotide may further comprise a nucleic acid cargo. The binding arm of a DNA dendrimer, or a portion of any DNA-based carrier, may also be designed to comprise a sequence complementary to a sequence in a known nucleic acid molecule (e.g., genomic DNA, cDNA, RNAs, plasmids, etc.) in order to link a nucleic acid cargo directly to the DNA dendrimer (via hydrogen bonding). A binding arm, branch or the body of a DNA dendrimer, or a portion of any DNA-based carrier, may also be designed to comprise a sequence that is a cargo (e.g., a DNA oligonucleotide).

G. Compositions

In some embodiments, a composition is provided, wherein the composition comprises a DNA dendrimer, a targeting moiety, and an adaptor molecule-cargo polynucleotide complex. In some embodiments, the DNA dendrimer is as provided for herein. In some embodiments, the targeting moiety is as provided for herein. In some embodiments, the targeting moiety and adaptor molecule-cargo polynucleotide complex are each, individually, linked to, conjugated to, or associated with the DNA dendrimer. In some non-limiting embodiments, linked to comprises an electrostatic interaction, a covalent bond, a non- covalent bond, or a hydrogen bond. In some embodiments, the targeting moiety is linked to, conjugated to, or associated with the DNA dendrimer through an electrostatic interaction. In some embodiments, the targeting moiety is linked to, conjugated to, or associated with the DNA dendrimer through a covalent bond. In some embodiments, the targeting moiety is linked to, conjugated to, or associated with the DNA dendrimer through a non-covalent bond. In some embodiments, the targeting moiety is linked to, conjugated to, or associated with the DNA dendrimer through a hydrogen bond. In some embodiments, the adaptor moleculecargo polynucleotide complex is linked to, conjugated to, or associated with the DNA dendrimer through an electrostatic interaction. In some embodiments, the adaptor moleculecargo polynucleotide complex is linked to, conjugated to, or associated with the DNA dendrimer through a covalent bond. In some embodiments, the adaptor molecule-cargo polynucleotide complex is linked to, conjugated to, or associated with the DNA dendrimer through a non-covalent bond. In some embodiments, the adaptor molecule-cargo polynucleotide complex is linked to, conjugated to, or associated with the DNA dendrimer through a hydrogen bond.

In some embodiments, the cargo polynucleotide is as provided for herein. In some embodiments, the cargo polynucleotide comprises at least one promoter as provided for herein. In some embodiments, the cargo polynucleotide is a nicked circular polynucleotide as provided for herein. In some embodiments, the cargo polynucleotide is a linear polynucleotide with closed 5’ and 3’ ends as provided for herein. In some embodiments, the cargo polynucleotide is a linear polynucleotide with open 5’ and 3’ ends as provided for herein. In some embodiments, the cargo polynucleotide is a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5’ or 3’ end of the polynucleotide, as provided for herein.

In some embodiments, the cargo polynucleotide comprises at least one DNA targeting sequence (DTS). In some embodiments, the identity of the DTS is as provided for herein. In some embodiments, the at least one DTS is located upstream or downstream of the at least one promoter. In some embodiments, the at least one DTS is located upstream of the at least one promoter. In some embodiments, the at least one DTS is located downstream of the at least one promoter. In some embodiments, the cargo polynucleotide comprises two or more DTS sequences, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 DTS sequences. In some embodiments, the two or more DTS sequences are located next to, or approximately next to each other. In some embodiments, the two or more DTS sequences are not located next to, or approximately next to each other.

In some embodiments, the cargo polynucleotide comprises at least one nuclear localization signal sequence (NLS). In some embodiments, the identity of the NLS is as provided for herein. In some embodiments, the at least one NLS is located upstream or downstream of the at least one promoter. In some embodiments, the at least one NLS is located upstream of the at least one promoter. In some embodiments, the at least one NLS is located downstream of the at least one promoter.

In some embodiments, the cargo polynucleotide comprises at least one DTS and at least one NLS. In some embodiments, the identity of the DTS is as provided for herein. In some embodiments, the identity of the NLS is as provided for herein. In some embodiments, the location of the DTS with respect to the at least one promoter is as provided for herein. In some embodiments, the location of the NLS with respect to the at least one promoter is as provided for herein.

In some embodiments, the adaptor molecule-cargo polynucleotide complex comprises an adaptor molecule, as provided for herein, and a cargo polynucleotide, as provided for herein. In some embodiments, the adaptor molecule comprises a DNA dendrimer binding sequence (DBS) and a cargo binding region (CBR). In some embodiments, the sequence of the DBS is as provided for herein. In some embodiments, the DNA dendrimer comprises a nucleic acid sequence that is complementary to the DBS. Accordingly, in some embodiments, the adaptor molecule is linked to the DNA dendrimer through the DBS. The sequence that is complementary to the DBS may be located on any of the terminal arms of the DNA dendrimer. In some embodiments, the sequence that is complementary to the DBS is a capture sequence, such as those provided for herein.

In some embodiments, the adaptor molecule can further comprise a purification region, as provided for herein. As provided for herein, the purification region can comprise a tag sequence. The tag sequence can be, for example, an affinity tag sequence. Affinity tag sequences are known in the art and any such sequence is within the scope of the present disclosure. In some non-limiting embodiments, the affinity tag is a polyhistidine tag, a FLAG tag, a HA-tag, a Myc-tag, a polyglutamate tag, a polyarginine tag, a strep-tag, a GST tag, and the like. In some embodiments, the affinity tag is a polyhistidine tag. The purification region can be located at any appropriate location within the adaptor molecule. In some embodiments, the purification region is located at the N-terminus of the adaptor molecule. In some embodiments, the purification region is located at the C-terminus of the adaptor molecule. In some embodiments, the purification region is located between the DBS and the CBR regions of the adaptor molecule. In some embodiments, the purification region comprises an affinity tag sequence. In some embodiments, the affinity tag sequence is located at the N-terminus of the adaptor molecule. In some embodiments, the affinity tag sequence is located at the C-terminus of the adaptor molecule. In some embodiments, the affinity tag sequence is between the DBS and the CBR regions of the adaptor molecule. In some embodiments, the purification region comprises a polyhistidine tag. In some embodiments, the polyhistidine tag is located at the N-terminus of the adaptor molecule. In some embodiments, the polyhistidine tag is located at the C-terminus of the adaptor molecule. In some embodiments, the polyhistidine tag is between the DBS and the CBR regions of the adaptor molecule.

In some embodiments, the adaptor molecule further comprises at least one NLS. NLS sequences are known in the art and any such sequence is within the scope of the present disclosure. In some embodiments, the NLS comprises an amino acid sequence as provided for herein. In some embodiments, the location of the NLS within the adaptor molecule is as provided for herein. In some embodiments, the at least one NLS of the adaptor molecule is located between the DBS and CBR, overlaps with the DBS, or overlaps with the CBR.

In some embodiments, the adaptor molecule further comprises at least one DTS. In some embodiments, the DTS comprises a nucleic acid sequence as provided for herein. In some embodiments, the location of the DTS within the adaptor molecule is as provided for herein. In some embodiments, the at least one DTS of the adaptor molecule is located between the DBS and CBR, overlaps with the DBS, or overlaps with the CBR. In some embodiments, the at least one DTS of the adaptor molecule is located between the DBS and CBR. In some embodiments, the at least one DTS of the adaptor molecule overlaps with the DBS. In some embodiments, the at least one DTS of the adaptor molecule overlaps with the CBR.

In some embodiments, the adaptor molecule further comprises at least one spacer molecule. In some embodiments, the adaptor molecule comprises more than one spacer molecule, such as 2, 3, 4, 5 or more than 5 spacer molecules. In some embodiments, the location of the spacer molecule within the adaptor molecule is as provided for herein. In some embodiments, the at least one spacer molecule is located upstream, downstream, or both upstream and downstream of the NLS sequence. In some embodiments, the at least one spacer molecule is located immediately upstream, immediately downstream, or both immediately upstream and immediately downstream of the NLS sequence. In some embodiments, the at least one spacer molecule is located upstream, downstream, or both upstream and downstream of the DTS sequence. In some embodiments, the at least one spacer molecule is located immediately upstream, immediately downstream, or both immediately upstream and immediately downstream of the DTS sequence. In some embodiments, the spacer molecule is as provided for herein. In some embodiments, the spacer molecule comprises an amino acid sequence as provided for herein.

In some embodiments, the adaptor molecule further comprises a cell penetrating peptide sequence (CPP). In some embodiments, the location of the CPP within the adaptor molecule is as provided for herein. In some embodiments, the CPP is located between the DBS and the CBR, overlaps with the DBS, or overlaps with the CBR. In some embodiments, the CPP is located between the DBS and the CBR. In some embodiments, the CPP overlaps with the DBS. In some embodiments, the CPP overlaps with the CBR. In some embodiments, the CPP is as provided for herein. In some embodiments, the amino acid sequence of the CPP is as provided for herein.

In some embodiments, the adaptor molecule further comprises at least one cleavage site. The cleavage site may be useful to, for example, cleave the adaptor molecule-cargo polynucleotide complex from the DNA dendrimer provided for herein. In some embodiments, the cleavage site is located within the adaptor molecule at a position as provided for herein. In some embodiments, the cleavage site is located at the N-terminus or 5’ end of the adaptor molecule. In some embodiments, the cleavage site is located at the C- terminus or 3’ end of the adaptor molecule. In some embodiments, the cleavage site is located between the DBS and the CBR, overlaps with the DBS, or overlaps with the CBR. In some embodiments, the cleavage site is located between the DBS and the CBR. In some embodiments, the cleavage site overlaps with the DBS. In some embodiments, the cleavage site overlaps with the CBR. In some embodiments, the cleavage site is located adjacent to the DBS, wherein the adjacent location can be on the 5’ end of the DBS or on the 3’ end of the DBS. In some embodiments, the cleavage site is located adjacent to the CBR, wherein the adjacent location can be on the 5’ end of the CBR or on the 3’ end of the CBR. In some embodiments, the at least one cleavage site is as provided for herein.

In some embodiments, the adaptor molecule further comprises at least one flexible linker. In some embodiments, the location of the flexible linker within the adaptor molecule is as provided for herein. In some embodiments, the flexible linker is located between the DBS and the CBR. In some embodiments, the flexible linker is as provided for herein. In some embodiments, the cargo polynucleotide is linked or conjugated to the adaptor molecule through the CBR located on the adaptor molecule, thereby forming the adaptor molecule-cargo polynucleotide complex. In some embodiments, and as provided for herein, the CBR is wholly or partially complementary to a sequence in the cargo polynucleotide. The cargo polynucleotide may be linked or conjugated to the CBR through any appropriate mechanism known in the art, which are within the scope of the present disclosure. Nonlimiting examples of such linkages or conjugations include, but are not limited to, covalent bonds, such as but not limited to a phosphodiester bond or a disulfide bond.

In some embodiments, the composition comprising a DNA dendrimer, a targeting moiety, and an adaptor molecule-cargo polynucleotide complex further comprises a support molecule. In some embodiments, the composition comprising a DNA dendrimer, a targeting moiety, and an adaptor molecule-cargo polynucleotide complex further comprises at least one support molecule. As provided for herein, the support molecule may, or may not, be linked or conjugated to the DNA dendrimer. As provided for herein, the support molecule may serve a variety of functions within the composition. In some embodiments, the support molecule condenses the size of the cargo polynucleotide, the DNA dendrimer, or both. In some embodiments, the support molecule condenses the size of the cargo polynucleotide. In some embodiments, the support molecule condenses the size of the DNA dendrimer. In some embodiments, the support molecule condenses the size of both the cargo polynucleotide and the DNA dendrimer. In some embodiments, the support molecule assists in nuclear delivery. In some embodiments, the support molecule protects the cargo polynucleotide, the DNA dendrimer, or both from nuclease degradation. In some embodiments, the support molecule protects the cargo polynucleotide from nuclease degradation. In some embodiments, the support molecule protects the DNA dendrimer from nuclease degradation. In some embodiments, the support molecule protects both the cargo polynucleotide and the DNA dendrimer from nuclease degradation. In some embodiments, the support molecule enhances the transfection efficiency of the composition. In some embodiments, the support molecule provides any number or all of the functions provided for herein.

In some embodiments, the support molecule is as provided for herein. In some embodiments, the amino acid sequence of the support molecule is as provided for herein. In some embodiments, the support molecule further comprises a PEG molecule as provided for herein. In some embodiments, the support molecule comprises an amino acid sequence as provided for herein and a PEG molecule as provided for herein. In some embodiments, a composition is provided, wherein the composition comprises a cargo polynucleotide, a support molecule, and a DNA dendrimer linked to a targeting moiety. In some embodiments, the targeting moiety is as provided for herein.

In some embodiments, the cargo polynucleotide is as provided for herein. In some embodiments, the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for a molecule of interest. In some embodiments, the promoter is as provided for herein. In some embodiments, the molecule of interest is as provided for herein. In some embodiments, the cargo polynucleotide optionally comprises a DBS. In some embodiments, the cargo polynucleotide does not comprise a DBS. In some embodiments, the cargo polynucleotide comprises a DBS. In some embodiments, the DBS is as provided for herein. In some embodiments, the cargo polynucleotide is a nicked circular polynucleotide as provided for herein. In some embodiments, the cargo polynucleotide is a linear polynucleotide with closed 5’ and 3’ ends as provided for herein. In some embodiments, the cargo polynucleotide is a linear polynucleotide with open 5’ and 3’ ends as provided for herein. In some embodiments, the cargo polynucleotide is a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5’ or 3’ end of the polynucleotide, as provided for herein.

In some embodiments, the cargo polynucleotide is, or is not, covalently linked to the DNA dendrimer. In some embodiments, the cargo polynucleotide is not covalently linked to the DNA dendrimer. In some embodiments, the cargo polynucleotide is linked to the DNA dendrimer.

In some embodiments, the cargo polynucleotide comprises at least one DNA targeting sequence (DTS). In some embodiments, the identity of the DTS is as provided for herein. In some embodiments, the location of the at least one DTS as provided for herein. In some embodiments, the at least one DTS is located within the cargo polynucleotide as provided for herein. In some embodiments, the location of the DTS within the cargo polynucleotide is as provided for herein.

In some embodiments, the at least one DTS is linked to the cargo polynucleotide. The linkage may be by any means known in the art. In one non-limiting embodiment, the cargo polynucleotide is a nicked circular polynucleotide and the DTS is linked to the cargo polynucleotide at the single stranded DNA break, i.e. at the location of the nick.

In some embodiments, the cargo polynucleotide comprises at least one NLS. In some embodiments, the identity of the NLS is as provided for herein. In some embodiments, the location of the at least one NLS is as provided for herein. In some embodiments, the cargo polynucleotide comprises at least one DTS and at least one NLS. In some embodiments, the identity of the DTS is as provided for herein. In some embodiments, the identity of the NLS is as provided for herein. In some embodiments, the location of the DTS is as provided for herein. In some embodiments, the location of the NLS is as provided for herein.

In some embodiments, the sequence of the DBS is as provided for herein. In some embodiments, a DBS sequence as provided for herein may further comprise a poly thymine sequence at the 5’ end of the DBS. In some embodiments, a DBS sequence as provided for herein may further comprise a polythymine sequence at the 3’ end of the DBS. In some embodiments, the DBS comprises a nucleic acid sequence of SEQ ID NO: 3, 4, 22 or 23, as provided for herein.

As provided for herein, the support molecule may, or may not, be linked or conjugated to the DNA dendrimer. In some embodiments, the support molecule is linked or conjugated to the DNA dendrimer. In some embodiments, the support molecule is not linked or conjugated to the DNA dendrimer. As provided for herein, the support molecule may serve a variety of functions within the composition. In some embodiments, the support molecule condenses the size of the cargo polynucleotide, the DNA dendrimer, or both. In some embodiments, the support molecule condenses the size of the cargo polynucleotide. In some embodiments, the support molecule condenses the size of the DNA dendrimer. In some embodiments, the support molecule condenses the size of both the cargo polynucleotide and the DNA dendrimer. In some embodiments, the support molecule assists in nuclear delivery. In some embodiments, the support molecule protects the cargo polynucleotide, the DNA dendrimer, or both from nuclease degradation. In some embodiments, the support molecule protects the cargo polynucleotide from nuclease degradation. In some embodiments, the support molecule protects the DNA dendrimer from nuclease degradation. In some embodiments, the support molecule protects both the cargo polynucleotide and the DNA dendrimer from nuclease degradation. In some embodiments, the support molecule enhances the transfection efficiency of the composition. In some embodiments, the presence of the support molecule facilitates the association of the cargo polynucleotide and the DNA dendrimer to each other. In some embodiments, the support molecule provides any number or all of the functions provided for herein.

In some embodiments, the support molecule has a positive charge. In some embodiments, the support molecule is as provided for herein. In some embodiments, the amino acid sequence of the support molecule is as provided for herein. In some embodiments, the support molecule further comprises a PEG molecule as provided for herein. In some embodiments, the support molecule comprises an amino acid sequence as provided for herein and a PEG molecule as provided for herein.

In any of the embodiments provided for herein, the cargo polynucleotide encodes for a cargo as provided for herein. In some embodiments, the coding sequence of the cargo polynucleotide encodes for a cargo as provided for herein.

In embodiments provided for herein wherein a support molecule is present, the composition may have a defined nitrogen/phosphate (N/P) ratio. In some embodiments, the N/P ratio of the composition is as provided for herein.

In some embodiments, a plasmid is provided, the plasmid comprising a plasmid backbone comprising at least two enzyme restriction recognition sites, at least one promoter, at least one coding sequence encoding for at least one molecule of interest, and, optionally, a DBS, wherein the plasmid is capable of forming a cargo polynucleotide with various structures depending on whether the plasmid has one or more or none or a 5’ end and a 3’ end. In some embodiments, the plasmid comprises a DBS. In some embodiments, the plasmid does not comprise a DBS. In some embodiments, the plasmid is a full circular polynucleotide, a nicked circular polynucleotide, a linear nucleotide with a closed 5’ and 3’ end, a linear nucleotide with open 5’ and 3’ ends, or a linear nucleotide with one open and one closed end, wherein the open or closed end may be the 5’ end or the 3’ end. In some embodiments, the plasmid is a full circular polynucleotide. In some embodiments, the plasmid is a nicked circular polynucleotide. In some embodiments, the plasmid is a linear nucleotide with a closed 5’ and 3’ end. In some embodiments, the plasmid is a linear nucleotide with open 5’ and 3’ ends. In some embodiments, the plasmid is a linear nucleotide with one open and one closed end, wherein the open or closed end may be the 5’ end or the 3’ end. In some embodiments, the plasmid comprising a nicked circular polynucleotide, a linear nucleotide with a closed 5’ and 3’ end, a linear nucleotide with open 5’ and 3’ ends, or a linear nucleotide with one open and one closed end is formed by cutting the a plasmid with one or more restriction enzymes.

In some embodiments, the plasmid further comprises at least one DTS. In some embodiments, the DTS is as provided for herein. In some embodiments, the location of the DTS within the plasmid is as provided for herein. In some embodiments, the at least one DTS is located between or overlapping with the at least two enzyme restriction recognition sites. In some embodiments, the DTS is located upstream of the at least one promoter. In some embodiments, the DTS is located downstream of the at least one promoter. In some embodiments, the plasmid further comprises at least one NLS. In some embodiments, the NLS is as provided for herein. In some embodiments, the location of the at least one NLS within the plasmid is as provided for herein. In some embodiments, the at least one NLS is located upstream of the at least one promoter. In some embodiments, the NLS is located downstream of the at least one promoter.

In some embodiments, the plasmid comprises at least one DTS and at least one NLS. In some embodiments the DTS is as provided for herein. In some embodiments, the location of the DTS is as provided for herein. In some embodiments the NLS is as provided for herein. In some embodiments, the location of the NLS is as provided for herein.

In some embodiments, the plasmid comprises a DBS. In some embodiments, the DBS is as provided for herein. In some embodiments, a DBS as provided for herein further comprises a polythymine sequence at either the 5’ end or the 3’ end of the DBS. In some embodiments, the DBS comprises a nucleic acid sequence of SEQ ID NO: 3, 4, 22 or 23, as provided for herein.

In some embodiments, the at least one promoter of the plasmid is any promoter provided for herein. In some embodiments, the at least one promoter is a tissue specific promoter. As provided for herein, tissue specific promoters are known in the art, and any such promoter is within the scope of the present disclosure.

In some embodiments, the plasmid comprises one polynucleotide sequence encoding for a molecule of interest. In some embodiments, the plasmid comprises more than one polynucleotide sequence encoding for a molecule of interest, such as 1 , 2, 3, 4, 5 or more than 5 polynucleotide sequences. In some embodiments, the plasmid comprises two polynucleotide sequences encoding for a molecule of interest. In some embodiments, the plasmid comprises three polynucleotide sequences encoding for a molecule of interest. In some embodiments, the plasmid comprises four polynucleotide sequences encoding for a molecule of interest. In some embodiments, the plasmid comprises five polynucleotide sequences encoding for a molecule of interest. In some embodiments, the plasmid comprises more than two polynucleotide sequences encoding for a molecule of interest. In some embodiments, the plasmid comprises more than five polynucleotide sequences encoding for a molecule of interest. It is to be understood that each instance of a more than one polynucleotide sequence may be the same (i.e. increase expression copy number) or unique (i.e. encode multiple unique molecules of interest).

Exemplary Compositions In some embodiments, a composition is provided, the composition comprising a DNA dendrimer, a targeting moiety, and an adaptor molecule-cargo polynucleotide complex comprising an adaptor molecule and a cargo polynucleotide; wherein the cargo polynucleotide comprises at least one DTS, at least one NLS, or a combination thereof; wherein the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest, wherein the cargo polynucleotide is a nicked circular polynucleotide, a linear polynucleotide with a closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the polynucleotide; and wherein the adaptor molecule comprises a DBS and a CBR. In some embodiments, the adaptor molecule further comprises at least one NLS, at least one DTS, or a combination thereof. In some embodiments, the adaptor molecule further comprises a CPP. In some embodiments, the adaptor molecule further comprises at least one cleavage site. In some embodiments, the composition further comprises a support molecule.

In some embodiments, a composition is provided, the composition comprising a DNA dendrimer, a targeting moiety, an adaptor molecule-cargo polynucleotide complex comprising an adaptor molecule and a cargo polynucleotide, and a support molecule; wherein the cargo polynucleotide comprises at least one DTS, at least one NLS, or a combination thereof; wherein the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest, wherein the cargo polynucleotide is a nicked circular polynucleotide, a linear polynucleotide with a closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the polynucleotide; and wherein the adaptor molecule comprises a DBS and a CBR. In some embodiments, the adaptor molecule further comprises at least one NLS, at least one DTS, or a combination thereof. In some embodiments, the adaptor molecule further comprises a CPP. In some embodiments, the adaptor molecule further comprises at least one cleavage site. In some embodiments, the DTS of the cargo polynucleotide, the adaptor molecule, or both the cargo polynucleotide and the adaptor molecule are as provided for herein. In some embodiments, the DTS of the cargo polynucleotide, the adaptor molecule, or both the cargo polynucleotide and the adaptor molecule comprise a nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the NLS of the cargo polynucleotide, the adaptor molecule, or both the cargo polynucleotide and the adaptor molecule are as provided for herein. In some embodiments, the NLS of the cargo polynucleotide, the adaptor molecule, or both the cargo polynucleotide and the adaptor molecule comprise an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 46. In some embodiments, the DBS is as provided for herein. In some embodiments, the DBS comprises a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 22, or SEQ ID NO: 23. In some embodiments, the DBS comprises a nucleic acid sequence comprising at least 20 contiguous nucleic acids of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the CPP is as provided for herein. In some embodiments, the CPP comprises an amino acid sequence of SEQ ID NO: 6. In some embodiments, the cleavage site is as provided for herein. In some embodiments, the cleavage site is a val-cit linker. In some embodiments, the support molecule is as provided for herein. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48. In some embodiments, the support molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C-terminus of the support molecule. In some embodiments, the support molecule comprises the sequence of SEQ ID NO: 30. In some embodiments, the adaptor molecule-cargo polynucleotide complex is linked or conjugated to the DNA dendrimer through the DBS. In some embodiments, the DNA dendrimer comprises terminal peripheral arms having sequence complementarity to the DBS sequences provided for herein.

In some embodiments, a composition is provided, the composition comprising a DNA dendrimer, a targeting moiety, an adaptor molecule-cargo polynucleotide complex comprising an adaptor molecule and a cargo polynucleotide, and a support molecule; wherein the cargo polynucleotide comprises at least one DTS having a nucleic acid sequence of SEQ ID NO: 8, at least one NLS having an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 46, or a combination thereof; wherein the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest, wherein the cargo polynucleotide is a nicked circular polynucleotide, a linear polynucleotide with a closed 5' and 3’ end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the polynucleotide; and wherein the adaptor molecule comprises a DBS having a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 22 or SEQ ID NO: 23, and a CBR. In some embodiments, the DBS comprises a nucleic acid sequence comprising at least 20 contiguous nucleic acids of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the adaptor molecule further comprises at least one NLS, at least one DTS, or a combination thereof. In some embodiments, the adaptor molecule further comprises a CPP. In some embodiments, the adaptor molecule further comprises at least one cleavage site. In some embodiments, the DTS of the adaptor molecule is as provided for herein. In some embodiments, the DTS of the adaptor molecule comprises a nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the NLS of the adaptor molecule is provided for herein. In some embodiments, the NLS of the adaptor molecule comprises an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 46. In some embodiments, the CPP comprises an amino acid sequence of SEQ ID NO: 6. In some embodiments, the cleavage site is a val-cit linker. In some embodiments, the support molecule is as provided for herein. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48. In some embodiments, the support molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C-terminus of the support molecule. In some embodiments, the support molecule comprises the sequence of SEQ ID NO: 30. In some embodiments, the adaptor molecule-cargo polynucleotide complex is linked or conjugated to the DNA dendrimer through the DBS. In some embodiments, the DNA dendrimer comprises terminal peripheral arms having sequence complementarity to the DBS sequences provided for herein.

In some embodiments, a composition is provided, the composition comprising a DNA dendrimer, a targeting moiety, an adaptor molecule-cargo polynucleotide complex comprising an adaptor molecule and a cargo polynucleotide, and a support molecule; wherein the cargo polynucleotide comprises at least one DTS having a nucleic acid sequence of SEQ ID NO: 8, at least one NLS having an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 46, or a combination thereof; wherein the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest, wherein the cargo polynucleotide is a nicked circular polynucleotide, a linear polynucleotide with a closed 5' and 3’ end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the polynucleotide; and wherein the adaptor molecule comprises a DBS having a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 22 or SEQ ID NO: 23, and a CBR. In some embodiments, the DBS comprises a nucleic acid sequence comprising at least 20 contiguous nucleic acids of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the adaptor molecule further comprises at least one NLS, at least one DTS, or a combination thereof. In some embodiments, the adaptor molecule further comprises a CPP comprising an amino acid sequence of SEQ ID NO: 6. In some embodiments, the adaptor molecule further comprises at least one cleavage site, wherein the at least one cleavage site is a val-cit linker. In some embodiments, the DTS of the adaptor molecule is as provided for herein. In some embodiments, the DTS of the adaptor molecule comprises a nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the NLS of the adaptor molecule is provided for herein. In some embodiments, the NLS of the adaptor molecule comprises an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 46. In some embodiments, the support molecule is as provided for herein. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26. In some embodiments, the support molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C-terminus of the support molecule. In some embodiments, the support molecule comprises the sequence of SEQ ID NO: 30. In some embodiments, the adaptor molecule-cargo polynucleotide complex is linked or conjugated to the DNA dendrimer through the DBS. In some embodiments, the DNA dendrimer comprises terminal peripheral arms having sequence complementarity to the DBS sequences provided for herein.

In some embodiments, a composition is provided, the composition comprising a DNA dendrimer, a targeting moiety, an adaptor molecule-cargo polynucleotide complex comprising an adaptor molecule and a cargo polynucleotide, and a support molecule; wherein the cargo polynucleotide comprises at least one DTS having a nucleic acid sequence of SEQ ID NO: 8, at least one NLS having an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 46, or a combination thereof; wherein the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest, wherein the cargo polynucleotide is a nicked circular polynucleotide, a linear polynucleotide with a closed 5' and 3’ end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the polynucleotide; and wherein the adaptor molecule comprises a DBS having a nucleic acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23, and a CBR. In some embodiments, the DBS comprises a nucleic acid sequence comprising at least 20 contiguous nucleic acids of SEQ ID NO: 10 or SEQ ID NO: 1 1. In some embodiments, the adaptor molecule further comprises at least one NLS, at least one DTS, or a combination thereof. In some embodiments, the adaptor molecule further comprises a CPP comprising an amino acid sequence of SEQ ID NO: 6. In some embodiments, the adaptor molecule further comprises at least one cleavage site, wherein the at least one cleavage site is a val-cit linker. In some embodiments, the DTS of the adaptor molecule is as provided for herein. In some embodiments, the DTS of the adaptor molecule comprises a nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the NLS of the adaptor molecule is provided for herein. In some embodiments, the NLS of the adaptor molecule comprises an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 46. In some embodiments, the support molecule is as provided for herein. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 19. In some embodiments, the support molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C-terminus of the support molecule. In some embodiments, the support molecule comprises the sequence of SEQ ID NO: 30. In some embodiments, the adaptor molecule-cargo polynucleotide complex is linked or conjugated to the DNA dendrimer through the DBS. In some embodiments, the DNA dendrimer comprises terminal peripheral arms having sequence complementarity to the DBS sequences provided for herein.

In some embodiments, a composition is provided, the composition comprising a cargo polynucleotide, a support molecule, and a DNA dendrimer linked to a targeting moiety, wherein the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest, and, optionally, a DBS; wherein the cargo polynucleotide is full circular polynucleotide (e.g., plasmid), nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the cargo polynucleotide; and wherein the cargo polynucleotide comprises at least one DTS, at least one NLS, or a combination thereof. In some embodiments, the DTS is as provided for herein. In some embodiments, the DTS comprises a nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the NLS is as provided for herein. In some embodiments, the NLS comprises an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 46. In some embodiments, the DBS is present and is as provided for herein. In some embodiments, the DBS is present and comprises a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 22, or SEQ ID NO: 23. In some embodiments, the DBS comprises a nucleic acid sequence comprising at least 20 contiguous nucleic acids of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the support molecule is as provided for herein. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48. In some embodiments, the support molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C-terminus of the support molecule. In some embodiments, the support molecule comprises the sequence of SEQ ID NO: 30. In some embodiments, the adaptor molecule-cargo polynucleotide complex is linked or conjugated to the DNA dendrimer through the DBS. In some embodiments, the DNA dendrimer comprises terminal peripheral arms having sequence complementarity to the DBS sequences provided for herein.

In some embodiments, a composition is provided, the composition comprising a cargo polynucleotide, a support molecule, and a DNA dendrimer linked to a targeting moiety, wherein the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest, and, optionally, a DBS; wherein the cargo polynucleotide is full circular polynucleotide (e.g., plasmid), nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the cargo polynucleotide; and wherein the cargo polynucleotide comprises at least one DTS, at least one NLS, or a combination thereof. In some embodiments, the DTS comprises a nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the NLS comprises an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 46. In some embodiments, the DBS is present and comprises a nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 22, or SEQ ID NO: 23. In some embodiments, the DBS comprises a nucleic acid sequence comprising at least 20 contiguous nucleic acids of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO: 48. In some embodiments, the support molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C-terminus of the support molecule. In some embodiments, the support molecule comprises the sequence of SEQ ID NO: 30. In some embodiments, the adaptor molecule-cargo polynucleotide complex is linked or conjugated to the DNA dendrimer through the DBS. In some embodiments, the DNA dendrimer comprises terminal peripheral arms having sequence complementarity to the DBS sequences provided for herein.

In some embodiments, a composition is provided, the composition comprising a cargo polynucleotide, a support molecule, and a DNA dendrimer linked to a targeting moiety, wherein the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest, and, optionally, a DBS; wherein the cargo polynucleotide is full circular polynucleotide (e.g., plasmid), nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the cargo polynucleotide; and wherein the cargo polynucleotide comprises at least one DTS, at least one NLS, or a combination thereof. In some embodiments, the DTS comprises a nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the NLS comprises an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 46. In some embodiments, the DBS is present and comprises a nucleic acid sequence of SEQ ID NO: 22, or SEQ ID NO: 23. In some embodiments, the DBS comprises a nucleic acid sequence comprising at least 20 contiguous nucleic acids of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26. In some embodiments, the support molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C-terminus of the support molecule. In some embodiments, the support molecule comprises the sequence of SEQ ID NO: 30. In some embodiments, the adaptor molecule-cargo polynucleotide complex is linked or conjugated to the DNA dendrimer through the DBS. In some embodiments, the DNA dendrimer comprises terminal peripheral arms having sequence complementarity to the DBS sequences provided for herein.

In some embodiments, a composition is provided, the composition comprising a cargo polynucleotide, a support molecule, and a DNA dendrimer linked to a targeting moiety, wherein the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest, and, optionally, a DBS; wherein the cargo polynucleotide is full circular polynucleotide (e.g., plasmid), nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the cargo polynucleotide; and wherein the cargo polynucleotide comprises at least one DTS, at least one NLS, or a combination thereof. In some embodiments, the DTS comprises a nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the NLS comprises an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 15, or SEQ ID NO: 46. In some embodiments, the DBS is present and comprises a nucleic acid sequence of SEQ ID NO: 22, or SEQ ID NO: 23. In some embodiments, the DBS comprises a nucleic acid sequence comprising at least 20 contiguous nucleic acids of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the support molecule comprises an amino acid sequence of SEQ ID NO: 19. In some embodiments, the support molecule further comprises a PEG molecule linked or conjugated at the N-terminus or C-terminus of the support molecule. In some embodiments, the support molecule comprises the sequence of SEQ ID NO: 30. In some embodiments, the adaptor molecule-cargo polynucleotide complex is linked or conjugated to the DNA dendrimer through the DBS. In some embodiments, the DNA dendrimer comprises terminal peripheral arms having sequence complementarity to the DBS sequences provided for herein.

Pharmaceutical Compositions and Kits

In some embodiments, pharmaceutical compositions of the compositions are provided. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. For therapeutic use, the compositions may be prepared as pharmaceutical compositions containing an effective amount of the cargo, composition, domain, or molecule as an active ingredient in a pharmaceutically acceptable carrier. A “pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered. The pharmaceutically acceptable carrier is separate from a DNA dendrimer or other components as provided for herein. In some embodiments, the pharmaceutical composition does not have any additional pharmaceutically acceptable carriers. Such vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well- known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating, and coloring agents, etc. The concentration of the molecules disclosed herein in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691 -1092, See especially pp. 958-989.

In some embodiments, any composition disclosed herein may also comprise suitable formulation agents known in the art, such as stabilizers, buffers, excipients, and the like that are different from a support molecule or other components as provided for herein. For example, suitable formulation agents include, but are not limited to purified water, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, polymers such as polyethylene glycols, propylene glycol, PEG 400, glycerin, DMA, ethanol, benzyl alcohol, citric acid/sodium citrate (pH3), citric acid/sodium citrate (pH5), tris(hydroxymethyl)amino methane HC1 (pH7.0), 0.9% saline, 1.2% saline, silicone, waxes, petroleum jelly, polyethylene glycol, propylene glycol, liposomes, sugars such as mannitol and lactose, and other materials depending on the specific type of formulation used.

The mode of administration for therapeutic use of the compositions disclosed herein may be any suitable route that delivers the agent to the host, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary; transmucosal (oral, intranasal, intravaginal, rectal), using a formulation in a tablet, capsule, solution, powder, gel, particle; and contained in a syringe, an implanted device, osmotic pump, cartridge, micropump; or other means appreciated by the skilled artisan, as well known in the art. Site specific administration may be achieved by for example intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intracardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.

Pharmaceutical compositions can be supplied as a kit comprising a container that comprises the pharmaceutical composition as described herein. A pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection. Such a kit can further comprise written information on indications and usage of the pharmaceutical composition. Accordingly, in some embodiments, a kit is provided. In some embodiments, the kit comprises a composition as provided for herein, a pharmaceutical composition as provided for herein, a plasmid as provided for herein, or any combination thereof.

Methods

In some embodiments, methods of delivering a molecule of interest to a target cell is provided. In some embodiments, the molecule of interest is delivered to the nucleus of the target cell. In some embodiments, the methods comprise contacting the target cell with any of the compositions or pharmaceutical compositions disclosed herein. Without being bound to any theory, the targeting moiety can bind to the target cell to facilitate the composition to enter into the target cell, and wherein the cargo polynucleotide, can be transported to the nucleus of the target cell. In some embodiments, the cargo polynucleotide is linked to an adaptor molecule, such as any adaptor molecule disclosed herein. In some embodiments, the cargo polynucleotide, the adaptor molecule, or both, further comprise a DTS, a NLS, or both, to assist with transport into the nucleus of the target cell. In some embodiments, the adaptor molecule comprises a CPP. In some embodiments, the cargo polynucleotide expresses the molecule of interest inside the nucleus of the target cell. In some embodiments, the composition of pharmaceutical composition can express more than one molecule of interest, such as 1, 2, 3, 4, 5 or more molecules of interest. In some embodiments, the composition of pharmaceutical composition expresses a molecule of interest in the target cell. In some embodiments, the composition of pharmaceutical composition expresses two molecules of interest in the target cell. In some embodiments, the composition of pharmaceutical composition expresses three molecules of interest in the target cell. In some embodiments, the composition of pharmaceutical composition expresses four molecules of interest in the target cell. In some embodiments, the composition of pharmaceutical composition expresses five molecules of interest in the target cell. In some embodiments, the composition of pharmaceutical composition expresses more than five molecules of interest in the target cell. In some embodiments, the promoter or promoters of the cargo polynucleotide are tissue specific promoters. In some embodiments, the targeting moiety is a tissue specific targeting moiety. In some embodiments, the composition or pharmaceutical composition comprises two or more tissue specific targeting moieties, such as 2, 3, 4, 5 or more targeting tissue specific targeting moieties. It is to be understood that in the context of the composition or pharmaceutical composition, the reference to number of targeting moieties is a reference to number of unique targeting moieties, i.e. not a reference to the number of targeting moieties conjugated to the DNA dendrimer of the composition. As provided for herein, the DNA dendrimer may have any number of targeting moieties conjugated to the terminal peripheral arms in order to provide effective delivery of the composition to the intended target. Accordingly, in some embodiments, the composition or pharmaceutical composition comprises two or more unique tissue specific targeting moieties, such as 2, 3, 4, 5, or more than 5 unique tissue specific targeting moieties. In some embodiments, the composition or pharmaceutical composition comprises two unique tissue specific targeting moieties. In some embodiments, the composition or pharmaceutical composition comprises three unique tissue specific targeting moieties. In some embodiments, the composition or pharmaceutical composition comprises four unique tissue specific targeting moieties. In some embodiments, the composition or pharmaceutical composition comprises five unique tissue specific targeting moieties. In some embodiments, the composition or pharmaceutical composition comprises more than five unique tissue specific targeting moieties. In some embodiments, the targeting moieties can be of any type disclosed herein.

In some embodiments, methods of treating a disease are provided. In some embodiments, the methods comprise contacting the target cell with any of the compositions or pharmaceutical compositions disclosed herein. In some embodiments, the targeting moiety binds to the target cell to facilitate the composition to enter into the target cell. In some embodiments, he cargo polynucleotide can enter into the nucleus of the target cell. In some embodiments, the cargo polynucleotide is linked to the DNA dendrimer. In some embodiments, the cargo polynucleotide is linked to an adaptor molecule, such as any adaptor molecule disclosed herein. In some embodiments, the cargo polynucleotide, the adaptor molecule, or both further comprise a DTS, a NLS, or both, to assist with transport into the nucleus of the target cell. In some embodiments, the adaptor molecule comprises a CPP. In some embodiments, the cargo polynucleotide expresses the molecule of interest inside the nucleus of the target cell. In some embodiments, the composition of pharmaceutical composition can express more than one molecule of interest, as provided for herein. In some embodiments, the promoter or promoters of the cargo polynucleotide are tissue specific promoters, such as those provided for herein. In some embodiments, the targeting moiety is a tissue specific targeting moiety, as provided for herein. In some embodiments, the composition or pharmaceutical composition comprises two or more tissue specific targeting moieties, as provided for herein. In some embodiments, the targeting moieties can be of any type disclosed herein.

In some embodiments, the composition is administered to a subject according to a route of administration as provided for herein. In some embodiments, the composition is administered parenterally, such as intravenously.

In some embodiments, the cargo of the composition is a therapeutic agent, and the method is used to alleviate a disorder or disease or provide a prophylactic treatment for a disorder or disease. The method is carried out by administering any composition or pharmaceutical provided herein to an individual in need thereof. Without being bound to any theory, by avoiding or limiting lysosomal delivery and degradation of a cargo and successfully delivering the cargo to the nucleus of the cell, the compositions and pharmaceutical compositions provided for herein may enable the reduction in dose of a therapeutic, compared to prior art delivery methods. Reducing the dose also advantageously reduces the risk of potential side effects. In some embodiments, the therapeutic agent is a polypeptide or a small molecule drug. In other embodiments, the therapeutic agent comprises nucleic acid. The therapeutic molecule may be any therapeutic molecule that can be encoded in a polynucleotide. Non-limiting examples of types of therapeutic molecules that can be encoded in an expression cassette in the instant invention include, but are not limited to, polypeptide enzymes, cytokines, hormones, antibodies, such as intrabodies or scFvs, a suicide gene, such as HSV-TK, a molecule that inhibits vascularization, a molecule that increases vascularization, tumor suppressors, such as p53 and p21, pro-apoptotic molecules, such as TRAIL, transcription factors, receptors, ligands, immunogenic molecules, anti-proliferative molecules, agonists, antagonists, anti-inflammatory molecules, antibiotics, antidepressants, prodrugs, anti-hypertensives, anti-oxidants, and the like. The therapeutic molecule comprising a nucleic acid may be a nucleic acid that modulates the expression in vivo of a gene. Such nucleic acids include antisense molecules, siRNA and ribozymes.

Thus, the methods herein provides a novel therapeutic approach to a broad spectrum of diseases and conditions, including cancer or cancerous disease, infectious disease, ocular disease, cardiovascular disease, neurological disease, prion disease, inflammatory disease, autoimmune disease, metabolic disease, genetic disease, pulmonary disease, renal disease, liver disease, mitochondrial disease, endocrine disease, reproduction related diseases and conditions, graft vs host disease, and any other indications that can respond to the level of an expressed gene product in a cell or individual. In some embodiments, the disease is a genetic disease or disorder, including, but not limited to, spinocerebelar ataxia, dentatorubral- pallidoluysian atrophy, Huntington disease, muscular atrophy, machado joseph disease, choreoacanthocytosis, spastic paraplegia, dystrophia moytonica, fragile X syndrome, fragile X ataxia syndrome, spinocerebellar ataxia, frontotemporal dementia, amyotrophic lateral sclerosis, myotonic dystrophy, cystic fibrosis, and down syndrome. In some embodiments, the disease has been shown to be treatable or potentially treatable by gene therapy, including, but not limited to, multiple myeloma, B-cell lymphoma, melanoma, Leber congenital amaurosis, spinal muscular atrophy, lipoprotein lipase deficiency, metachromatic leukodystrophy, mantel cell lymphoma, large B-cell lymphoma, beta thalassemia, vascular endothelial growth factor peripheral artery disease, cerebral adrenoleukodystrophy, head and neck squamous cell carcinoma, adenosine deaminase deficiency, and B cell lymphoblastic leukemia.

The art is replete with exemplary molecules and associated diseases or disorders where a patient may benefit from the expression or inhibition of expression of one or more molecules. For instance, an assessment of expression changes in gene families in a variety of human cancers has been pursued (U.S. Pat. Appl. Pub. No. 20060168670). In addition, tissuespecific expression levels have been mapped for thousands of genes through expression profiling (Alon et al., 1999, Proc. Natl. Acad. Sci. USA 96:6745-50; Iyer et al., 1999, Science 283: 83-87; Khan et al., 1998, Cancer Res. 58: 5009-13; Lee et al., 1999, Science 285: 1390- 93; Wang et al., 1999, Gene 229:101-08; and Whitney et al., 1999, Ann. Neurol. 46:42). Thus, the skilled artisan is able to select molecules useful in the practice of the present invention without undue experimentation. In some embodiments, a method of manufacturing a cargo polynucleotide is provided, the method comprising adding at least one promoter and at least one coding sequence encoding for at least one molecule of interest to a plasmid backbone to form a plasmid, then optionally contacting the plasmid with one or more restriction enzymes. In some embodiments, the plasmid is contacted with one or more restriction enzymes to form various topologies, including a nicked circular nucleotide, a linear nucleotide with a closed 5’ and 3’ end, a linear nucleotide with open 5’ and 3’ ends, or a linear nucleotide with one open and one closed end topology. In some embodiments, the cargo polynucleotide is then linked to an adaptor molecule, which is in turn linked to a DNA dendrimer to form the composition. In some embodiments, the plasmid is not contacted with one or more restriction enzymes to form a full circular nucleotide topology. In some embodiments, the cargo polynucleotide is then contacted with a DNA dendrimer linked to a targeting moiety in the presence of a support molecule capable, such that the cargo polynucleotide and the DNA dendrimer associated together to form the composition. In some embodiments, the support molecule is any support molecule described herein. In some embodiments, the support molecule condenses the size of the cargo polynucleotide, the DNA dendrimer, or both.

In some embodiments, the method of manufacturing a composition as provided for herein comprises contacting a plasmid as provided for herein with one or more restriction enzymes that cut at the restriction enzyme recognition site to form a nicked circular polynucleotide, a linear polynucleotide with a close 5’ and 3’ end, a linear polynucleotide with open 5’ and 3’ ends, or a linear polynucleotide with one open and one closed end, linking the cargo polynucleotide to the adaptor molecule to form an adaptor molecule-cargo polynucleotide complex, and linking the adaptor molecule-cargo polynucleotide complex and a targeting moiety to a DNA dendrimer to form the composition.

In some embodiments, the method of manufacturing a composition as provided for herein comprises contacting a plasmid as provided for herein, wherein the plasmid comprises a DBS, with one or more restriction enzymes that cut at the restriction enzyme recognition site to form a nicked circular polynucleotide, a linear polynucleotide with a close 5’ and 3’ end, a linear polynucleotide with open 5’ and 3’ ends, or a linear polynucleotide with one open and one closed end, and linking the adaptor molecule-cargo polynucleotide complex and a targeting moiety to a DNA dendrimer to form the composition.

In some embodiments, the method of manufacturing a composition as provided for herein comprises contacting i) a circular uncut plasmid as provided for herein, ii) a DNA dendrimer linked to a targeting moiety and iii) a support molecule as provided for herein to form the composition, wherein the support molecule is capable of condensing the size of the cargo polynucleotide and the DNA dendrimer, wherein the support molecule facilitates the association of the DNA dendrimer and the cargo polynucleotide.

In some embodiments, the plasmid containing the at least one coding sequence is amplified by traditional bacterial methods, for example, E. coli. In some embodiments, the plasmid containing the at least one coding sequence is amplified by rolling circle amplification, or other non-bacterial methods. For example, amplification can occur using enzymatic synthesis of covalently closed linear DNA, as described in U.S. Patent No. 11,149,302 and U.S. Patent Publication No. US2019/0185924, both of which are incorporated by reference in their entirety.

Embodiments

Embodiments provided herein also include, but are not limited to, the following:

1. A composition comprising: a DNA dendrimer; a targeting moiety; and an adaptor molecule-cargo polynucleotide complex.

2. The composition of embodiment 1, wherein the DNA dendrimer is linked to, conjugated to, or associated with the targeting moiety and the adaptor molecule-cargo polynucleotide complex.

3. The composition of embodiment 1, wherein the adaptor molecule-cargo polynucleotide complex comprises an adaptor molecule and a cargo polynucleotide.

4. The composition of embodiment 3, wherein the DNA dendrimer is linked to the adaptor molecule-cargo polynucleotide complex through an electrostatic interaction, a covalent bond, a non-covalent bond, or a hydrogen bond with the adaptor molecule.

5. The composition of embodiment 3, wherein the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest, wherein the cargo polynucleotide is a nicked circular polynucleotide, a linear polynucleotide with a closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the polynucleotide.

6. The composition of embodiment 3, wherein the cargo polynucleotide further comprises at least one DNA targeting sequence (DTS).

7. The composition of embodiment 6, wherein the at least one DTS is located upstream of the at least one promoter.

8. The composition of embodiment 6, wherein the at least one DTS is located downstream of the at least one promoter.

9. The composition of any one of embodiments 6-8, wherein the cargo polynucleotide comprises two or more DTSs.

10. The composition of embodiment 9, wherein the two or more DTSs are located next to, or approximately next to, each other.

11. The composition of embodiment 9, wherein the two or more DTSs are not located next to, or approximately next to, each other.

12. The composition of any one of embodiments 3-11, wherein the cargo polynucleotide further comprises at least one nuclear localization signal sequence (NLS).

13. The composition of embodiment 12, wherein the at least one NLS is located upstream of the at least one promoter.

14. The composition of embodiment 12, wherein the at least one NLS is located downstream of the at least one promoter.

15. The composition of any one of embodiments 6-14, wherein the cargo polynucleotide comprises at least one DTS and at least one NLS. 16. The composition of any one of embodiments 3-15, wherein the adaptor molecule comprises a DNA dendrimer binding sequence (DBS) and a cargo binding region (CBR).

17. The composition of embodiment 16, wherein the adaptor is linked to the DNA dendrimer through the DBS.

18. The composition of embodiments 16 or 17, wherein the DBS comprises a nucleic acid sequence that is complimentary to a nucleic acid sequence on the DNA dendrimer.

19. The composition of any one of embodiments 16-18, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3, 10 , 11, 22, or 23.

20. The composition of any one of embodiments 16-19, wherein the DBS comprises a nucleic acid sequence of any one of SEQ ID NOs: 3, 10 , 11, 22, or 23.

21. The composition of any one of embodiments 16-20, wherein the DBS further comprises a polythymine sequence at either the 5’ or 3’ end of the DBS.

22. The composition of embodiment 21, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4.

23. The composition of embodiment 21 or 22, wherein the DBS comprises a nucleic acid sequence of SEQ ID NO: 4.

24. The composition of any one of embodiments 16-23, wherein the CBR is linked or conjugated to the cargo polynucleotide by DNA ligation, i.e., is linked or conjugated to the polynucleotide through a covalent bond, such as a phosphodiester bond. 25. The composition of any one of embodiments 16-24, wherein the CBR is linked or conjugated to the cargo polynucleotide by chemical coupling, i.e. is linked or conjugated to the polynucleotide through a covalent bond, such as a disulfide bond.

26. The composition of any one of embodiments 16-25, wherein the cargo binding region comprises at least one cysteine.

27. The composition of any one of embodiments 16-26, wherein the adaptor molecule further comprises a tag, such as, for example, a tag that can be used as an affinity tag to isolate/purify the composition complex.

28. The composition of embodiment 27, wherein the tag is located between the DBS and the CBR.

29. The composition of embodiment 28, wherein the tag is a polyhistidine tag.

30. The composition of any one of embodiments 16-29, wherein the adaptor molecule further comprises at least one NLS.

31. The composition of embodiment 30, wherein the at least one NLS is located between or overlaps with the DBS and the cargo binding region.

32. The composition of embodiments 30 or 31, wherein the at least one NLS comprises one or more NLS sequences disclosed herein.

33. The composition of any one of embodiments 30-32, wherein the at least one NLS comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 1, 2, 15, or 43.

34. The composition of any one of embodiments 30-33, wherein the at least one NLS comprises an amino acid sequence of SEQ ID NO: 1, 2, 15, or 43. 35. The composition of any one of embodiments 30-34, wherein the at least one NLS further comprises at least one spacer located before or after the at least one NLS.

36. The composition of embodiment 35, wherein the at least one NLS comprises two spacers located before or after the at least one NLS, such as one before the NLS and one after the NLS, or both spacers before or after the NLS.

37. The composition of embodiment 35 or 36, wherein the spacer comprises polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), poly lactic-co- gly colic acid (PGLA), or any combination thereof.

38. The composition of embodiment 35 or 36, wherein spacer is a glycine/alanine or glycine/serine spacer.

39. The composition of embodiment 38, wherein the spacer comprises the amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 5 or 29.

40. The composition of any one of embodiments 8 or 39, wherein the spacer comprises the amino acid sequence of any one of SEQ ID NO: 5 or 29.

41. The composition of any one of embodiments 16-40, wherein the adaptor molecule further comprises at least one DTS.

42. The composition of embodiment 41 , wherein the at least one DTS is located between or overlaps with the DBS and the CBR.

43. The composition of any one of embodiments 16-42, wherein the adaptor molecule further comprises a cell penetrating peptide sequence (CPP).

44. The composition of embodiment 43, wherein the CPP is located between or overlapping with the DBS and the CBR. 45. The composition of embodiments 43 or 44, wherein the CPP comprises a CPP disclosed herein.

46. The composition of any one of embodiments 43-45, wherein the CPP comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6.

47. The composition of any one of embodiments 43-46, wherein the CPP comprises an amino acid sequence of SEQ ID NO: 6.

48. The composition of any one of embodiments 16-47, wherein the adaptor molecule further comprises at least one cleavage site.

49. The composition of embodiment 48, wherein the at least one cleavage site is located between, or overlaps with, the DBS and the CBR.

50. The composition of embodiments 48 or 49, wherein the cleavage site is located adjacent to the DBS.

51. The composition of any one of embodiments 48-50, wherein the cleavage site is a val- cit linker.

52. The composition of any one of embodiments 16-51, wherein the adaptor molecule further comprises at least one flexible linker.

53. The composition of embodiment 52, wherein the at least one flexible linker is located between the DBS and the CBR.

54. The composition of embodiments 52 or 53, wherein the at least one flexible linker is selected from the group consisting of polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG -poly lactic acid (PLA), poly lactic-co-glycolic acid (PGLA), (GG)n, (GGGGS)n, or (GGGGA)n, wherein each n is, independently, 1-5.

55. The composition of any one of embodiments 1-47, wherein the composition further comprises a support molecule.

56. The composition of embodiment 55, wherein the support molecule is linked or conjugated to the DNA dendrimer.

57. The composition of embodiment 55, wherein the support molecule is not linked or conjugated to the DNA dendrimer.

58. The composition of any one of embodiments 55-57, wherein the support molecule condenses the size of the cargo polynucleotide, the DNA dendrimer, or both.

59. The composition of any one of embodiments 55-58, wherein the support molecule assists in nuclear delivery.

60. The composition of any one of embodiments 55-59, wherein the support molecule protects the cargo polynucleotide, the DNA dendrimer, or both, from nuclease degradation.

61. The composition of any one of embodiments 55-60, wherein the support molecule enhances the transfection efficiency of the composition.

62. The composition of any one of embodiments 55-61, wherein the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

63. The composition of any one of embodiments 55-62, wherein the support molecule comprises an amino acid sequence of any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

64. The composition of any one of embodiments 55-63, wherein the support molecule further comprises a PEG molecule linked or conjugated at N-terminus or C-terminus of the amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

65. The composition of any one of embodiments 55-64, wherein the support molecule further comprises a PEG molecule linked or conjugated at N-terminus or C-terminus of the amino acid sequence of any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

66. The composition of any one of embodiments 64 or 65, wherein the PEG molecule is selected from any one of PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000.

67. The composition of any one of embodiments 1-66, wherein the targeting moiety is selected from the group consisting of an antibody, a naturally-occurring ligand for the receptor or a functional derivative thereof, a vitamin, a hormone, a small molecule mimetic of a naturally-occurring ligand, a peptide, a polypeptide, a peptidomimetic, a carbohydrate, a lipid, an aptamer, a nucleic acid, a toxin, a component of a microorganism, any other molecule provided it binds specifically to the cell surface molecule and induces endocytosis of the bound moiety, or any combination thereof.

68. A composition comprising a cargo polynucleotide, a support molecule, and a DNA dendrimer linked to a targeting moiety. 69. The composition of embodiment 68, wherein the targeting moiety is selected from the group consisting of an antibody, a naturally-occurring ligand for the receptor or a functional derivative thereof, a vitamin, a hormone, a small molecule mimetic of a naturally-occurring ligand, a peptide, a polypeptide, a peptidomimetic, a carbohydrate, a lipid, an aptamer, a nucleic acid, a toxin, a component of a microorganism, any other molecule provided it binds specifically to the cell surface molecule and induces endocytosis of the bound moiety, or any combination thereof.

70. The composition of embodiment 68 or 69, wherein: the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest, and, optionally, a DBS, wherein the cargo polynucleotide is full circular polynucleotide (e.g., plasmid), nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the cargo polynucleotide.

71. The composition of embodiment 70, wherein the cargo polynucleotide a closed circle polynucleotide.

72. The composition of embodiment 70, wherein the cargo polynucleotide is a nicked circular polynucleotide.

73. The composition of any one of embodiments 68-72, wherein the cargo polynucleotide is not covalently linked to the DNA dendrimer.

74. The composition of any one of embodiments 68-72, wherein the cargo polynucleotide further comprises at least one DTS.

75. The composition of embodiment 74, wherein the DTS is located within the cargo polynucleotide. 76. The composition of embodiment 74, wherein the DTS is linked to the cargo polynucleotide.

77. The composition of embodiment 76, wherein the cargo polynucleotide is a nicked circular polynucleotide, and the DTS is linked to the cargo polynucleotide at the single stranded DNA break (i.e., the location of the nick).

78. The composition of any one of embodiments 68-77, wherein the cargo polynucleotide further comprises at least one NLS.

79. The composition of any one of embodiments 68-78, wherein the cargo polynucleotide comprises at least one DTS and at least one NLS.

80. The composition of any one of embodiments 70-79, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 3, 10, 11, 22, or 23.

81. The composition of any one of embodiments 70-80, wherein the DBS comprises a nucleic acid sequence of any one of SEQ ID NO: 3, 10, 1 1 , 22, or 23.

82. The composition of any one of embodiments 80 or 81, wherein the DBS further comprises a poly thy mine sequence at either the 5’ or 3’ end of the DBS.

83. The composition of embodiment 82, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4.

84. The composition of any one of embodiments 82 or 83, wherein the DBS comprises a nucleic acid sequence of SEQ ID NO: 4. 85. The composition of any one of embodiments 68-84, wherein the support molecule is linked or conjugated to the DNA dendrimer.

86. The composition of any one of embodiments 68-84, wherein the support molecule is not linked or conjugated to the DNA dendrimer.

87. The composition of any one of embodiments 68-86, wherein the support molecule condenses the size of the cargo polynucleotide, the DNA dendrimer, or both.

88. The composition of any one of embodiments 68-87, wherein the support molecule assists in nuclear delivery.

89. The composition of any one of embodiments 68-88, wherein the support molecule protects the cargo polynucleotide, the DNA dendrimer, or both from nuclease degradation.

90. The composition of any one of embodiments 68-89, wherein the support molecule enhances the transfection efficiency of the composition.

91. The composition of any one of embodiments 68-90, wherein the presence of the support molecule facilitates the association of the cargo polynucleotide and the DNA dendrimer to each other.

92. The composition of any one of embodiments 68-91, wherein the support molecule has a positive charge.

93. The composition of any one of embodiments 68-92, wherein the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof. 94. The composition of any one of embodiments 68-93, wherein the support molecule comprises an amino acid sequence of any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

95. The composition of any one of embodiments 68-94, wherein the support molecule further comprises a PEG molecule linked or conjugated at N-terminus or C-terminus of the amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

96. The composition of any one of embodiments 68-95, wherein the support molecule further comprises a PEG molecule linked or conjugated at N-terminus or C-terminus of the amino acid sequence of any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

97. The composition of any one of embodiments 95 or 96, wherein the PEG molecule is selected from any one of PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000.

98. The composition of embodiment 97, wherein the polyethylene molecule is PEG2000.

99. The composition of any one of embodiments 1-98, wherein the coding sequence of the cargo polynucleotide encodes for an antibody, an enzyme, a protein, a miRNA, a siRNA, an antisense RNA, and the like.

100. The compositions of any one of embodiments 55 or 68, wherein the nitrogen/phosphate (N/P) ratio of the support molecule is between 0.5 and 10.

101. The composition of embodiment 100, wherein the N/P ratio is between 2 and 5. 102. A pharmaceutical composition comprising a composition of any one of embodiments 1 -99 and a pharmaceutically acceptable carrier.

103. A method of delivering a molecule of interest to the nucleus of a target cell, the method comprising contacting the target cell with the composition of any one of embodiments 1-101 or the pharmaceutical composition of embodiment 102, wherein the targeting moiety binds to the target cell.

104. The method of embodiment 103, wherein the cargo polynucleotide expresses the molecule of interest in the nucleus of the target cell.

105. The method of embodiments 103 or 104, wherein the cargo polynucleotide expresses two or more molecules of interest in the nucleus of the target cell.

106. The method of any one of embodiments 103-105, wherein the cargo polynucleotide comprises a tissue specific promoter.

107. The method of any one of embodiments 103-106, wherein the targeting moiety is a tissue specific targeting moiety.

108. The method of any one of embodiments 103-107, wherein the composition or pharmaceutical composition comprises two or more targeting moieties.

109. A method of treating a disease, the method comprising administering the composition of any one of embodiments 1-101 or the pharmaceutical composition of embodiment 102 to a subject to treat the disease, wherein the targeting moiety binds to the target cell.

110. The method of embodiment 109, wherein the cargo polynucleotide expresses the molecule of interest in the nucleus of the target cell.

111. The method of embodiments 109 or 110, wherein the cargo polynucleotide expresses two or more molecules of interest in the nucleus of the target cell. 112. The method of any one of embodiments 109- 111, wherein the cargo polynucleotide comprises a tissue specific promoter.

113. The method of any one of embodiments 109-112, wherein the targeting moiety is a tissue specific targeting moiety.

114. The method of any one of embodiments 109-113, wherein the composition or pharmaceutical composition comprises two or more targeting moieties.

115. A plasmid comprising a plasmid backbone comprising at least two enzyme restriction recognition sites, at least one promoter, at least one coding sequence encoding for at least one molecule of interest, and, optionally, a DBS, wherein the plasmid is capable of forming a cargo polynucleotide with various structures depending on whether the plasmid has one or more, or none of, a 5' end and a 3' end.

116. The plasmid of embodiment 115, wherein the plasmid is a full circular polynucleotide, a nicked circular nucleotide, a linear nucleotide with a closed 5’ and 3’ end, a linear nucleotide with open 5’ and 3’ ends, or a linear nucleotide with one open and one closed end.

1 17. The plasmid of embodiment 1 16, wherein the nicked circular nucleotide, linear nucleotide with a closed 5’ and 3’ end, linear nucleotide with open 5’ and 3’ ends, and linear nucleotide with one open and one closed end are formed by cutting the plasmid with one or more restriction enzymes.

118. The plasmid of any one of embodiments 115-117, wherein the plasmid further comprises at least one DTS.

119. The plasmid of embodiment 118, wherein the at least one DTS is located in the plasmid backbone, i.e., between, or overlapping with, the at least two enzyme restriction recognition sites.

120. The plasmid of embodiments 118 or 119, wherein the at least one DTS is located upstream of the at least one promoter. 121. The plasmid of embodiments 118 or 119, wherein the at least one DTS is located downstream of the at least one promoter.

122. The plasmid of any one of embodiments 115-121, wherein the plasmid further comprises at least one NLS.

123. The plasmid of embodiment 122, wherein the at least one NLS is located upstream of the at least one promoter.

124. The plasmid of embodiment 122, wherein the at least one NLS is located downstream of the at least one promoter.

125. The plasmid of any one of embodiments 115-124, wherein the plasmid comprises at least one DTS and at least one NLS.

126. The plasmid of any one of embodiments 115-125, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 3, 10, 1 1 , 22, or 23.

127. The plasmid of any one of embodiments 115-126, wherein the DBS comprises a nucleic acid sequence of any one of SEQ ID NO: 3, 10, 11, 22, or 23.

128. The plasmid of any one of embodiments 126 or 127, wherein the DBS further comprises a poly thy mine sequence at either the 5’ or 3’ end of the DBS.

129. The plasmid of embodiment 128, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. 130. The plasmid of any one of embodiments 128 or 129, wherein the DBS comprises a nucleic acid sequence of SEQ ID NO: 4.

131. The plasmid of any one of embodiments 115-130, wherein the at least one promoter is a tissue specific promoter.

132. The plasmid of any one of embodiments 115-131, wherein the plasmid comprises polynucleotide sequences encoding for two or more molecules of interest.

133. A method of manufacturing the composition of any one of embodiments 1-67, the method comprising: contacting the plasmid of any one of embodiments 115-132 with one or more restriction enzymes that cut at the restriction enzyme recognition sites to form a nicked circular polynucleotide, a linear polynucleotide with a closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, linking the cargo polynucleotide to the adaptor molecule to form an adaptor moleculecargo polynucleotide complex, and linking the adaptor molecule- cargo polynucleotide complex and a targeting moiety to a DNA dendrimer to form the composition.

134. A method of manufacturing the composition of any one of embodiments 68-101, the method comprising: contacting the plasmid of any one of embodiments 115-132 that comprise a DNA dendrimer binding sequence (DBS) with one or more restriction enzymes that cut at the restriction enzyme recognition sites to form a nicked circular polynucleotide, a linear polynucleotide with a closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, and linking the cargo polynucleotide and a targeting moiety to a DNA dendrimer to form the composition.

135. A method of manufacturing the composition of any one of embodiments 68-101, the method comprising contacting a circular uncut plasmid of any one of embodiments 115-132 with a DNA dendrimer linked to a targeting moiety with a support molecule capable of condensing the size of the cargo polynucleotide and the DNA dendrimer, such that the cargo polynucleotide and the DNA dendrimer are associated together to form the composition.

136. A kit comprising a composition of embodiments 1-101, a pharmaceutical composition of embodiment 102, a plasmid of any one of embodiments 115-132, or some combination thereof.

137. The composition of any one of embodiments 16-67, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 10, provided that the DBS comprises at least 20 contiguous nucleic acids of SEQ ID NO: 10.

138. The composition of any one of embodiments 16-67 or 99-101, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 1 1 , provided that the DBS comprises at least 20 contiguous nucleic acids of SEQ ID NO: 11.

139. The composition of any one of embodiments 70-101, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 10, provided that the DBS comprises at least 20 contiguous nucleic acids of SEQ ID NO: 10.

140. The composition of any one of embodiments 70-101, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 11, provided that the DBS comprises at least 20 contiguous nucleic acids of SEQ ID NO: 11.

141. The plasmid of any one of embodiments 115-132, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 10, provided that the DBS comprises at least 20 contiguous nucleic acids of SEQ ID NO: 10.

142. The plasmid of any one of embodiments 115-132, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 11, provided that the DBS comprises at least 20 contiguous nucleic acids of SEQ ID NO: 11.

EXAMPLES

The following examples are provided for the purpose of illustration only and the claims should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Those of skill in the art will readily recognize a variety of non- critical parameters that could be changed or modified to yield essentially similar results.

Example 1: Characterization of Multiple Topologies for Polynucleotide Cargo

To determine how the topology structures of various polynucleotide cargo can affect expression efficiency, a gene cassette containing luciferase and eGFP driven by a CMV promoter was inserted into a plasmid backbone comprising multiple restriction enzyme recognition sites, including Sall, BbcCI, and Tel. To generate various topologies, the resulting plasmid was cut with the following restriction enzymes as listed in Table 1. A depiction of the various topologies is shown in FIG. 1.

Table 1: Plasmid Topologies

using equal molar amount of each topological variation. GFP expression was measured oved time using cell imaging and quantitative fluorescence measurement. The percent of GFP positive cells and the overall fluorescent intensity were compared. Results shown in FIG. 2 and FIG. 3. The unmodified full circular (i.e., plasmid) and the linear with 2 closed ends topologies generally had the best overall fluorescence activity, however all the topologies had active fluorescence, which is useful if a change in overall activity of a specific therapeutic is needed.

Example 2: Nuclear Localization for Non-Dividing Cells

In many cases, it is critical that a cargo polynucleotide be delivered to the nucleus of a target cell for expression of the encoded molecules of interest. When cells divide, the nuclear membrane naturally breaks down, allowing direct access from the cytoplasm. However, in the absence of mitosis, the nuclear membrane remains intact and largely impermeable to plasmids. A potential strategy is to include at least one copy of a DNA targeting sequence (DTS), a nuclear localization sequence (NLS), or both in the cargo polynucleotide, an adaptor molecule that can be associated with the cargo polynucleotide, or both. In a healthy cytoplasm, transcription factors, including NLS, can bind to a DTS and recruit the cargo for nuclear transport. Likewise, a NLS can recruit the necessary importins directly, reading the cargo for nuclear transport. To test this general strategy, a DTS (SV40 DTS, SEQ ID NO: 8) was included in a polynucleotide plasmid expressing eGFP and compared to a plasmid expressing eGFP without DTS in three different cell lines: fast growing CH0-K1, medium growth A427, and relatively slow dividing C2C12 differentiated myoblasts.

In general, both the no DTS plasmid (Pl A) and the DTS plasmid (P1B) were able to express GFP in the CH0-K1 (FIG. 4) and the A427 cell lines (FIG. 5). This was expected, as both cell lines undergo enough mitosis to allow ready access to the cell nuclei. However, in the C2C12 cell line, where the no-to-slow growth rates limit access to the otherwise intact nuclei, the DTS containing plasmid cargo had significantly better expression than the control plasmid in relative mean fluorescence (FIG. 6), relative integral fluorescence (FIG. 7), and relative peak fluorescence (FIG. 8). For FIGs. 4-8, roughly 5,000 cells per well were plated, and imaging and quantification of fluorescence was measured 24 hours after initial transfection. Significance was determined using one-way ANOVA, and Tukey’s multiple comparison’s test between groups; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Example 3: Additional Characterization of Multiple Topologies for Polynucleotide Cargo

Additional polynucleotide cargo plasmids were made and tested.

Reporter Constructs

All reporter constructs contain a bacterial selection sequence (antibiotic resistance, origin of replication (ORI)), SV40-DTS, codon-optimized firefly luciferase (Luc2_C0), codon-optimized enhanced green fluorescent protein (eGFP_CO), 2A self-cleaving peptide (P2A), and the bovine growth hormone poly adenylate ion (bgh-PolyA) signal. In addition, a TelN protelomerase sequence is used in conjunction with one of two DBS sequences (SEQ ID NO: 22 or SEQ ID NO: 23) that allows for plasmid attachment to the DNA dendrimer nanoparticle via complementary hybridization. Two nickase enzyme sites flanking the DBS enzyme are used to expose the single-stranded DNA (ssDNA) sequence to the chosen attachment arm of the synthetic scaffold.

Three reporter constructs were made. Reporter constructs 1 and 2 share a cytomegalovirus (CMV) enhancer and promoter which is known for robust and ubiquitous expression in mammalian cell lines. Both reporter constructs also share a Kozak sequence for initiation of eukaryotic translation. Reporter construct 1 has a DBS sequence of SEQ ID NO: 22 while reporter construct 2 has the DBS sequence of SEQ ID NO: 23. Reporter construct 3 has a DBS sequence of SEQ ID NO: 22, but a different promoter: the muscle specific promoter mCK8e. All three reporter constructs have various cut sites for restriction enzymes and double nickase sites were placed for plasmid manipulation. The area containing the selected enzyme cut sites and attachment sequences is designated as the “CB Backbone Elements” region. An additional feature in this region is the TelN protelomerase recognition site. This sequence is a palindrome and upon recognition by the enzyme forms two covalently closed ends (denoted “TelR” and “TelL”) at the site of cleavage.

Additionally, a molecule of interest construct was made, where the DNA insert encoding the construct is more than 10K base pairs. This construct has many of the same sequences found in the reporter constructs: the bacterial selection portion, SV40-DTS, mCK8e promoter, Kozak sequence, and TelN protelomerase site. An additional promoter, the T7 promoter, is incorporated before the coding sequence for enhanced bacterial expression. Similar restriction enzymes and double nickase sites are present near DBS sequence (SEQ ID NO: 22).

Methods

Mfel (20,000 U/mL, New England Biolabs, R3589L, Lot # 10150327) restriction enzyme was used only with reporter constructs, and EagI (100,000 U/mL, New England Biolabs, R3505M, Lot # 10157687) was used only with the 10K insert construct. A nickase denoted as “Nt” is only used with constructs with the SEQ ID NO: 22 DBS site. A nickase denoted as “Nb” is only used with constructs with the SEQ ID NO: 23 DBS site. Any enzymes or nickases without symbols are universal and can be used for any construct listed.

1. Open Nicked Circle (OpC)

Constructs with the SEQ ID NO: 22 DBS site were incubated with Nt.BbvCI in a ratio of 1 pg of plasmid to 1 unit (U) of enzyme (10,000 U/mL, New England Biolabs, R0632L, Lot # 10141032). Those with the SEQ ID NO: 23 DBS site were incubated with Nb.BbvCI in a ratio of 1 pg of plasmid to 1 U of enzyme (10,000 U/mL, New England Biolabs, R0631L, Lot # 10150965). The reaction was then incubated for 1 hour at 50°C at 300 revolutions per minute (rpm).

The appropriate capture sequence for the DBS site was then added in a 20 pmol excess to the plasmid. The reactions were then heated to 90°C for 2 minutes to allow for the nicked strand to dissociate from the plasmid. After, reactions were cooled slowly to room temperature over the course of 3.5 hours for the capture sequence to bind to the nicked sequence and leave the attachment site open.

Ion-exchange chromatography (IEX) was then utilized to separate the nicked plasmid from the captured nicked sequence. Fractions containing the peaks of interest were combined and the OpC plasmid was extracted using ethanol (EtOH) precipitation. Pellets were dried and reconstituted in the buffer of choice.

2. Linear Nicked (LN)

Constructs were incubated with TelN Protelomerase in a ratio of 1 pg of plasmid to 1 U of enzyme (20,000 U/mL, New England Biolabs, M0651B-HC2, Lot # 10151691). Reactions were incubated for 4 hours at 30°C and 300 rpm. A buffer swap into nuclease-free water was carried out to remove the Triton-X from the initial buffer.

TelN linearized constructs with the SEQ ID NO: 22 DBS site were incubated with Nt.BbvCI in a ratio of 1 pg of plasmid to 1 unit (U) of enzyme (10,000 U/mL, New England Biolabs, R0632L, Lot # 10141032). Those with the SEQ ID NO: 23 DBS site were incubated with Nb.BbvCI in a ratio of 1 pg of plasmid to 1 U of enzyme (10,000 U/mL, New England Biolabs, R0631L, Lot # 10150965). The reaction was then incubated for 1 hour at 50°C at 300 rpm.

The appropriate capture sequence for the DBS site was then added in a 20 pmol excess to the plasmid. The reactions were heated to 90°C for 2 minutes to allow for the nicked strand to dissociate from the plasmid. Next, reactions were cooled slowly to room temperature over the course of 3.5 hours for the capture sequence to bind to the nicked sequence and leave the attachment site open.

IEX was then utilized to separate the nicked plasmid from the captured nicked sequence. Fractions containing the peaks of interest were combined and the LN plasmid was extracted using EtOH precipitation. Pellets were dried and reconstituted in the buffer of choice.

3. 1-open, 1 -closed (Ico)

Constructs were incubated with TelN Protelomerase at a ratio of 1 pg of plasmid to 1 U of enzyme (20,000 U/mL, New England Biolabs, M0651B-HC2, Lot # 10151691). Reactions were incubated for 4 hours at 30°C and 300 rpm. A buffer swap into nuclease free water was carried out to remove the Triton X from the initial buffer.

Mfel (20,000 U/mL, New England Biolabs, R3589L, Lot # 10150327) or EagI (100,000 U/mL, New England Biolabs, R35O5M, Lot # 10157687) restriction enzymes were incubated with the TelN linearized constructs at a ratio of 1 pg plasmid to 1 U of enzyme. Reactions were incubated for 1 hour at 37°C at 300 rpm. An EtOH precipitation was used to precipitate the 1-open ended, linearized plasmid before the nickase reaction.

Constructs with the SEQ ID NO: 22 DBS site were incubated with Nt.BbvCI at a ratio of 1 pg of plasmid to 1 unit (U) of enzyme (10,000 U/mL, New England Biolabs, R0632L, Lot # 10141032). Those with the SEQ ID NO: 23 DBS site were incubated with Nb.BbvCI at a ratio of 1 pg of plasmid to 1 U of enzyme (10,000 U/mL, New England Biolabs, R0631L, Lot # 10150965). The reaction was then incubated for 1 hour at 50°C at 300 rpm.

IEX was then utilized to separate the nicked plasmid from the captured nicked sequence. Fractions containing the peaks of interest were combined and the Ico plasmid was extracted using ethanol EtOH precipitation. Pellets were dried and reconstituted in the buffer of choice.

4. 2-open ends (2oe)

Mfel (20,000 U/mL, New England Biolabs, R3589L, Lot # 10150327) or EagI (100,000 U/mL, New England Biolabs, R35O5M, Lot # 10157687) were incubated with the constructs in a ratio of 1 pg plasmid to 1 U of enzyme. Reactions were incubated for 1 hour at 37°C at 300 rpm. An EtOH precipitation was used to precipitate the 2oe linearized plasmid before reconstitution in its chosen buffer.

Constructs with the SEQ ID NO: 22 DBS site were incubated with Nt.BbvCI at a ratio of 1 pg of plasmid to 1 unit (U) of enzyme (10,000 U/mL, New England Biolabs, R0632L, Lot # 10141032). Constructs with the SEQ ID NO: 23 DBS site were incubated with Nb.BbvCI at a ratio of 1 pg of plasmid to 1 U of enzyme (10,000 U/mL, New England Biolabs, R0631L, Lot # 10150965). The reaction was then incubated for 1 hour at 50°C at 300 revolutions per minute rpm.

The appropriate capture sequence for the DBS site was added in a 20 pmol excess to the plasmid. The reactions were then heated to 90°C for 2 minutes to allow for the nicked strand to dissociate from the plasmid. Next, reactions were cooled slowly to room temperature over the course of 3.5 hours for the capture sequence to bind to the nicked sequence and leave the attachment site open.

IEX was utilized to separate the nicked plasmid from the captured nicked sequence. Fractions containing the peaks of interest were combined and the 1 co plasmid is extracted using ethanol EtOH precipitation. Pellets were dried and reconstituted in the buffer of choice.

Confirmation Results

Reporter Construct 1 was used for all experiments unless otherwise noted.

1. Confirmation of OpC plasmid.

Using a restriction fragment length polymorphism (RFLP) analysis the efficacy of the double nickase was assessed on a 15% TBE-Urea denaturing polyacrylamide gel. The plasmid pre- modification showed no gel bands, while the post-nickase plasmid showed a 38 bp band, which correlates with expected band size from the cut. For the RFLP, TelN was used to open the plasmid before an additional restriction enzyme cut further up from the nicked site. This resulted in 2 smaller fragments (47 and 58 bp) from the top nicked strand, and one longer fragment (209 bp) from the bottom strand. Additionally, the 38 bp band from the nickase was present since this analysis was run before the IEX purification. These bands correlated with the expected sizes from the cuts. To observe if the modified plasmids were still functional HepG2 cells were transfected with Reporter Construct 1 and Reporter Construct 2 in the OpC topology with Lipofectamine2000 (Catalog#l 1668019, ThermoFisher Scientific, Waltham, MA). Cells were seeded at ~2.5xl0⁴ cells/well one day prior to treatment. Generation 1 reporter constructs (Genl, A and B) transfected with Lipofectamine2000 were included as positive controls. Cells were imaged after 48 hours for fluorescence (excitation/emission 488/509 nm) due to eGFP expression. The fluorescence signal was quantitated using the sum of integrated fluorescent intensity which allows for the individual pixels to be represented without bias. Genl construct B had a DNA nuclear Targeting Sequence (DTS) upstream the CMV promoter. These plasmids were not codon-optimized, but still exhibited some fluorescence when transfected using lipofectamine (FIG. 9A and B). The Gen3 OpC plasmids with the codon-optimized sequences also exhibited fluorescence (FIG. 9C and D). Gen3 reporters displayed a higher distribution of fluorescence signal (FIG. 9C and D) with a statistical difference of higher average observed fluorescence between generations of reporters (FIG. 10).

2. Confirmation of Linear Nicked (LN) plasmid.

For the RFLP of the LN plasmid, only an additional restriction enzyme upstream from the nickase site was necessary since the plasmid was already treated with TelN. The 38 bp band is present from the nickase as well as the 47, 58, and 209 bp bands, as expected.

3. Confirmation of the 1-open, 1 -closed (Ico) plasmid

The RFLP performed on the 1 co Plasmid showed that prior to the addition of the nickase, the 132 bp band for the removed closed end and 30 bp for the check cut were present as expected. Post-addition of the nickase RFLP with 132, 48, 46, and 38 bp bands produced from the removal of the closed end, smaller fragments of the nicked strand, and the excised piece of DNA, all as expected.

Example 4: Characterization of Support Molecules for Complexation and Protection of Polynucleotide Cargo

The amount of support molecule used to complex and protect the plasmid constructs is determined by the N/P ratio, the formula for which is disclosed herein. The higher the N/P ratio, the more support molecule is present in the final composition. Once the N/P ratio is determined, the support molecule and plasmid solutions can be combined. The solutions were mixed with lOx phosphate buffered saline (PBS) providing salt to assist in complexation and nuclease free water (NFW) to make up the total volume. Support molecules can be used with DNA concentrations from about 0.05 pg/pL to about 0.5 pg/pL. For example, to achieve an N/P ratio of 3 with 300 ug of a plasmid solution with a concentration of 1.0 g/ L and 1300 pg of a support molecule solution of 5.0 pg/pL with a final DNA concentration of 0.5 pg/pL, 300 pL of plasmid solution, 260 pL of support molecule solution, 6 pL of lOx PBS, and 34 pL of NFW were combined to a total volume of 600 pL. This solution was then mixed at 300 rpm for 30 minutes at room temperature.

To determine if the support molecules were successful at complexing the plasmid nucleic acids, final products were run on 1.1% agarose gels to observe if free plasmid was still present after the reaction. Results showed that Exc 1 showed partial complexation at a N/P ratio of 1 or 2, but complete complexation at a N/P ratio of 5 and 8. Exc 2 showed no complexation at a N/P ration of 1 and a partial complexation at a N/P ratio of 5, while Exc 3 showed no complexation at a N/P ratio of 1 or 5. Polymer-based support molecules perform at lower N/P ratios due to their uniformly positive sequences. PLL Pl, PLL P2, and PLL P3 all showed complete complexation at N/P ratios as low as 0.5. The hybrid PEG-Exc 1 support molecule shown complexation at similar N/P ratios to Exc 1 by itself.

Nuclease protection was determined using a DNase I assay. Samples were subjected to 5 U of DNase I (M030S, New England Biolabs) for 30 minutes at 37°C. This amount was determined by titrating the amount of DNase I was required to fully degrade a 0.5 ug of plasmid, the maximum amount of DNA able to be handled by the cleanup kits. Samples were then purified with a commercially available kit from New England Biolabs (T1030L). Following cleanup, samples were run on 1.1 % agarose gels to observe if complexes, plasmid, or both were remaining. Experiments were run with Exc 1 complexed with plasmid DNA at N/P rations 5 and 8. As a control, plasmid complexed with a transfection agent TurboFecf™ (R0531, ThermoFisher Scientific), was also included as it has demonstrated ability to complex as well. While all three solutions complexed with the DNA plasmid, TurboFect™ was unable to protect the plasmid from degradation. Exc 1, at both N/P ratios shows little to no degradation. Additional experiments with the PPL support molecules show that they have weaker DNase protection. The PEG-Exc 1 hybrid (SEQ ID NO: 30) protects equivalently with Exc 1 (SEQ ID NO: 19), indicating that PEG does not impact the stability of the support molecule. The results of these examples demonstrate that while most of the peptide and polymer support molecules tested were able to complex the plasmid DNA, only the support molecules comprising the sequence of Exc 1 (i.e., those of SEQ ID NO: 19 and SEQ ID NO: 30) were able to provide adequate protection from degradation by DNase I. To determine if the PEG-Exc 1 hybrid support molecule would cause any cytotoxicity, a CellTiter-Glo® Luminescent Cell Viability Assay (G7570, Promega) was performed on treated murine cells (C2C12, CRL-1772, ATCC). Cells were incubated with support molecule encapsulate conjugations (nanocarrier, plasmid, targeting moieties, and support molecule) with the dose based on the plasmid mass (0.2 pg and 1.0 pg per well). Samples were tested in quintuplet. At 0.2 pg per well, there was no statistical loss in viability from either PEG-Exc 1 or TurboFect™ as compared the control cells (FIG. 11). At 1.0 pg per well both PEG-Exc 1 and TurboFect™ impacted viability compared with control cells (FIG. 12).

Based on the above results, support molecules containing the sequence of SEQ ID NO: 12 appear to have superior capability to complex the polynucleotide cargo molecule. Accordingly, additional support molecules were designed and tested, with all support molecules tested at least containing the amino acid sequence of SEQ ID NO: 12. The additional molecules tested are recited in the table below:

Sequence Complexation and Protection Results

The sequences of SEQ ID NOs: 44-48 were used for visualization purposes. It is expected that use of sequences that do not contain the DBCO or Cy3 moieties would behave in a similar manner to those utilized in the present example. Accordingly, it is expected that a support molecule of SEQ ID NO: 37 would behave similar to the results for SEQ ID NO: 44, a support molecule of SEQ ID NO: 38 would behave similar to the results for SEQ ID NO: 45, a support molecule of SEQ ID NO: 39 would behave similar to the results for SEQ ID NO: 46.

The support molecules given by SEQ ID NOs: 12, 35, and 36 were able to complex supercoiled and modified plasmids at N/P ratios of 5, 7, and 10 (FIG. 13a). The DNAse 1 protection results (FIG. 13b) show that each of SEQ ID NOs: 12, 35, and 36 were able to afford a level of protection of the complexed plasmid. The support molecules given by SEQ ID NO: 12 and SEQ ID NO: 36 appear to have a slight advantage over the support molecule of SEQ ID NO: 35, but all support molecules afforded some protection. When support molecules given by SEQ ID NOs: 44, 45, and 46 were used at N/P ratios greater than or equal to 1, the sample retrophored when run through the agarose gel. This may be due to the additional positive charge each of these support molecules carry due to inclusion of an NLS sequence (FIG. 14a, representative figure showing results for SEQ ID NO: 44 only). Retrophoresis was not observed at N/P ratios around 0.75 (FIG. 14a). The DNAse 1 protection results (FIG. 14b) show that neither support molecule of SEQ ID NO: 45 or SEQ ID NO: 46 were able to protect the plasmid cargo when utilized at an N/P ratio of 3. However, follow up examination of SEQ ID NOs: 44, 45, and 46 at lower N/P ratios showed that the support molecules were able to provide protection at N/P ratios less than 1 (FIG. 15a). Use of the molecules at an N/P ratio of 1 appear to provide minimal protection from DNAse 1 (FIG. 15b).

Additional support molecules can be designed. Modifications to the number of Hl sequences may compact the nucleic acids and the N/P ratio, and overall stabilizer content, may be reduced through their addition. Different CPPs, such as arginine- 8, may be added in alter transfection both in vitro and in vivo models. Likewise, alternative NLS sequences can be investigated to enhance nuclear delivery. Additionally, peptide-targeting sequences can be added to the base stabilizer during synthesis or can be chemically added through functional groups (e.g., Click chemistry, amides, cross-linking). Finally, specific areas of the support molecules could be made neutral or positive for zonal segregation of charge for complexation and new functional groups could be added at the termini or distributed throughout the chain. For example, these could include binding sites for small molecules, antibodies, and peptide sequences.

Example 5: Characterization of Support Molecules Blends for Complexation and Protection of Polynucleotide Cargo

In an effort to improve on the complexation and protection properties of various support molecules provided for herein, support molecule blends were designed and tested. The blends combined various support molecules at specific molar ratios to determine if the combination enhanced the support molecules ability to complex payload plasmids and to protect the plasmids from nuclease degradation. The specific blends tested in the present example are provided in the table below: Blend Component Component Blend Ratio Complexation and Protection Results

Name 1 (C1) 2 (C2) (C1 :C2)

Compositions were prepared and complexation and protection assays were performed largely as in Example 4 above. To synthesize the blend-payload conjugates 10X PBS was diluted to IX in the required amount of nuclease free water. The plasmid payload was then added before the addition of the Cl support molecule followed by the C2 support molecule in a 4:1 molar ratio. The solution was then incubated at room temperature for 30 minutes before checking for aggregation and performing a gel analysis for complexation.

As shown in FIG. 16a and 16b, the blend of SEQ ID NO: 12 and SEQ ID NO: 38 was able to complex the plasmid DNA and provide protection from DNAse 1. Similar results were obtained for the other blends tested.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising: a DNA dendrimer; a targeting moiety; and an adaptor molecule-cargo polynucleotide complex, wherein the DNA dendrimer is linked to, conjugated to, or associated with the targeting moiety and the adaptor molecule-cargo polynucleotide complex.

2. The composition of claim 1 , wherein the adaptor molecule-cargo polynucleotide complex comprises an adaptor molecule and a cargo polynucleotide.

3. The composition of claim 2, wherein the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest, wherein the cargo polynucleotide is a nicked circular polynucleotide, a linear polynucleotide with a closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the polynucleotide.

4. The composition of claim 2, wherein the cargo polynucleotide further comprises at least one DNA targeting sequence (DTS).

5. The composition of claim 4, wherein the cargo polynucleotide comprises two or more DTSs.

6. The composition of claim 2, wherein the cargo polynucleotide further comprises at least one nuclear localization signal sequence (NLS).

7. The composition of claim 4, wherein the cargo polynucleotide comprises at least one DTS and at least one NLS.

8. The composition of claim 2, wherein the adaptor molecule comprises a DNA dendrimer binding sequence (DBS) and a cargo binding region (CBR).

9. The composition of claim 8, wherein the adaptor is linked to the DNA dendrimer through the DBS, wherein the DBS comprises a nucleic acid sequence that is complimentary to a nucleic acid sequence on the DNA dendrimer.

10. The composition of claim 8, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3, 4, 10 , 11, 22, or 23.

11. The composition of claim 8, wherein the adaptor molecule further comprises a tag, such as, for example, a tag that can be used as an affinity tag to isolate/purify the composition complex.

12. The composition of claim 8, wherein the adaptor molecule further comprises at least one NLS, wherein the at least one NLS is located between or overlaps with the DBS and the cargo binding region.

13. The composition of claim 12, wherein the at least one NLS comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 1, 2, 15, or 43.

14. The composition of claims 12, wherein the at least one NLS further comprises at least one spacer located before or after the at least one NLS, wherein the at least one spacer comprises polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PL A), poly lactic -co-glycolic acid (PGLA), a glycine/alanine spacer, a glycine/serine spacer, or any combination thereof.

15. The composition of claim 14, wherein the at least one spacer comprises the amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 5 or 29.

16. The composition of claim 8, wherein the adaptor molecule further comprises at least one DTS, wherein the at least one DTS is located between or overlaps with the DBS and the CBR.

17. The composition of claim 8, wherein the adaptor molecule further comprises a cell penetrating peptide sequence (CPP), wherein the CPP is located between or overlapping with the DBS and the CBR.

18. The composition of claim 17, wherein the CPP comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 6.

19. The composition of claim 8, wherein the adaptor molecule further comprises at least one cleavage site.

20. The composition of claim 19, wherein the cleavage site is a val-cit linker.

21. The composition of claim 8, wherein the adaptor molecule further comprises at least one flexible linker selected from the group consisting of polyethylene glycol (PEG), propylene glycol alginate (PGA), PEG-polylactic acid (PLA), poly lactic-co-glycolic acid (PGLA), (GG)n, (GGGGS)n, or (GGGGA)n, wherein each n is, independently, 1-5.

22. The composition of claim 1, wherein the composition further comprises a support molecule.

23. The composition of claim 22, wherein the support molecule is or is not linked or conjugated to the DNA dendrimer.

24. The composition of claim 22, wherein the support molecule: i) condenses the size of the cargo polynucleotide, the DNA dendrimer, or both; ii) molecule assists in nuclear delivery; iii) protects the cargo polynucleotide, the DNA dendrimer, or both, from nuclease degradation; and/or iv) enhances the transfection efficiency of the composition.

25. The composition of claim 22, wherein the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

26. The composition of claims 22, wherein the support molecule further comprises a PEG molecule linked or conjugated at N-terminus or C-terminus of the amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

27. The composition of claim 26, wherein the PEG molecule is selected from any one of PEG400, PEG500, PEG 1000, PEG2000, PEG3000, PEG4000, or PEG-5000.

28. The composition of any one of claims 1-27, wherein the targeting moiety is selected from the group consisting of an antibody, a naturally-occurring ligand for the receptor or a functional derivative thereof, a vitamin, a hormone, a small molecule mimetic of a naturally- occurring ligand, a peptide, a polypeptide, a peptidomimetic, a carbohydrate, a lipid, an aptamer, a nucleic acid, a toxin, a component of a microorganism, any other molecule provided it binds specifically to the cell surface molecule and induces endocytosis of the bound moiety, or any combination thereof.

29. A composition comprising a cargo polynucleotide, a support molecule, and a DNA dendrimer linked to a targeting moiety.

30. The composition of claim 29, wherein the targeting moiety is selected from the group consisting of an antibody, a naturally-occurring ligand for the receptor or a functional derivative thereof, a vitamin, a hormone, a small molecule mimetic of a naturally-occurring ligand, a peptide, a polypeptide, a peptidomimetic, a carbohydrate, a lipid, an aptamer, a nucleic acid, a toxin, a component of a microorganism, any other molecule provided it binds specifically to the cell surface molecule and induces endocytosis of the bound moiety, or any combination thereof.

31. The composition of claim 29, wherein: the cargo polynucleotide comprises at least one promoter and at least one coding sequence encoding for at least one molecule of interest, and, optionally, a DBS, wherein the cargo polynucleotide is full circular polynucleotide (e.g., plasmid), nicked circular polynucleotide, a linear polynucleotide with closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, wherein the one open and one closed end can be at either the 5' and 3' of the cargo polynucleotide.

32. The composition of claim 31 , wherein the cargo polynucleotide a closed circle polynucleotide or a nicked circular polynucleotide.

33. The composition of claim 29, wherein the cargo polynucleotide further comprises at least one DTS.

34. The composition of claim 33, wherein the cargo polynucleotide is a nicked circular polynucleotide, and the DTS is linked to the cargo polynucleotide at the single stranded DNA break (i.e., the location of the nick).

35. The composition of claim 29, wherein the cargo polynucleotide further comprises at least one NLS.

36. The composition of claim 29, wherein the cargo polynucleotide comprises at least one DTS and at least one NLS.

37. The composition of claim 31, wherein the DBS comprises a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 3, 4, 10, 11, 22, or 23.

38. The composition of claim 29, wherein the support molecule is or is not linked or conjugated to the DNA dendrimer.

39. The composition of claim 29, wherein the support molecule: i) condenses the size of the cargo polynucleotide, the DNA dendrimer, or both; ii) assists in nuclear delivery; iii) protects the cargo polynucleotide, the DNA dendrimer, or both from nuclease degradation; and/or iv) enhances the transfection efficiency of the composition.

40. The composition of claim 29, wherein the presence of the support molecule facilitates the association of the cargo polynucleotide and the DNA dendrimer to each other.

41. The composition of claim 29, wherein the support molecule has a positive charge.

42. The composition of claim 29, wherein the support molecule comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

43. The composition of claim 29, wherein the support molecule further comprises a PEG molecule linked or conjugated at N-terminus or C-terminus of the amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 24, 25, 26, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48, or any combination thereof.

44. The composition of claim 43, wherein the PEG molecule is selected from any one of PEG400, PEG500, PEG1000, PEG2000, PEG3000, PEG4000, or PEG-5000.

45. The composition of any one of claims 1-44, wherein the coding sequence of the cargo polynucleotide encodes for an antibody, an enzyme, a protein, a miRNA, a siRNA, an antisense RNA, and the like.

46. The compositions of claim 22 or 29, wherein the nitrogen/phosphate (N/P) ratio of the support molecule is between 0.5 and 10.

47. A pharmaceutical composition comprising a composition of any one of claims 1-46 and a pharmaceutically acceptable carrier.

48. A method of delivering a molecule of interest to the nucleus of a target cell, the method comprising contacting the target cell with the composition of any one of claims 1-46 or the pharmaceutical composition of claim 47, wherein the targeting moiety binds to the target cell.

49. A method of treating a disease, the method comprising administering the composition of any one of claims 1-46 or the pharmaceutical composition of claim 47 to a subject to treat the disease, wherein the targeting moiety binds to the target cell.

50. A plasmid comprising a plasmid backbone comprising at least two enzyme restriction recognition sites, at least one promoter, at least one coding sequence encoding for at least one molecule of interest, and, optionally, a DBS, wherein the plasmid is capable of forming a cargo polynucleotide with various structures depending on whether the plasmid has one or more, or none of, a 5' end and a 3' end.

51. A method of manufacturing the composition of any one of claims 1-28, the method comprising: contacting the plasmid of claim 50 with one or more restriction enzymes that cut at the restriction enzyme recognition sites to form a nicked circular polynucleotide, a linear polynucleotide with a closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, linking the cargo polynucleotide to the adaptor molecule to form an adaptor moleculecargo polynucleotide complex, and linking the adaptor molecule- cargo polynucleotide complex and a targeting moiety to a DNA dendrimer to form the composition.

52. A method of manufacturing the composition of any one of claims 29-46, the method comprising: contacting the plasmid of claim 50 that comprise a DNA dendrimer binding sequence (DBS) with one or more restriction enzymes that cut at the restriction enzyme recognition sites to form a nicked circular polynucleotide, a linear polynucleotide with a closed 5' and 3' end, a linear polynucleotide with open 5' and 3' ends, or a linear polynucleotide with one open and one closed end, and linking the cargo polynucleotide and a targeting moiety to a DNA dendrimer to form the composition.

53. A method of manufacturing the composition of any one of claims 29-46, the method comprising contacting a circular uncut plasmid of claim 50 with a DNA dendrimer linked to a targeting moiety with a support molecule capable of condensing the size of the cargo polynucleotide and the DNA dendrimer, such that the cargo polynucleotide and the DNA dendrimer are associated together to form the composition.

54. A kit comprising a composition of claims 1-46, a pharmaceutical composition of claim 47, a plasmid of claim 50, or some combination thereof.