WO2025010185A1

WO2025010185A1 - Methods and systems for characterizing entities on a substrate

Info

Publication number: WO2025010185A1
Application number: PCT/US2024/035740
Authority: WO
Inventors: Paul Wilhelm Karl ROTHEMUND; Bhavik NATHWANI
Original assignee: Somalogic Operating Co Inc
Current assignee: Somalogic Operating Co Inc
Priority date: 2023-07-03
Filing date: 2024-06-27
Publication date: 2025-01-09
Anticipated expiration: 2026-01-03

Abstract

Provided herein are techniques for characterizing entities immobilized on a substrate. In an embodiment, the entities can be randomly immobilized on the substrate. Each entity is associated with one or more identification oligonucleotides comprising a plurality of modular sequences that, in unique combination, form a unique identification sequence that is specific to a particular entity or type of entity. In embodiments, an entity and its associated one or more identification oligonucleotides are both linked to a core structure of a supramolecular structure, which facilitates co-location and single molecule binding to a binding site of the substrate. Hybridization-based detection of the modular sequences in the one or more identification oligonucleotides yields efficient characterization and mapping of randomly ordered entities on the substrate.

Description

METHODS AND SYSTEMS FOR CHARACTERIZING ENTITIES ON A SUBSTRATE

BACKGROUND

[0001] The current state of personalized healthcare is overwhelmingly genome-centric, predominantly focused on quantifying the genes present within an individual. While such an approach has proven to be extremely powerful, it does not provide a clinician with the complete picture of an individual’s health. This is because genes are the “blueprints” of an individual, and it merely informs the likelihood of developing an ailment. Within an individual, these “blueprints” first need to be transcribed into RNA and then translated into various protein molecules, “actors” in the cell, to have effect on the health of an individual.

[0002] The concentration of proteins, the interaction between the proteins (protein-protein interactions or PPI), as well as the interaction between proteins and other molecules, are intricately linked to the health of different organs, homeostatic regulatory mechanism as well as the interaction of these systems with the external environment. Hence, quantitative information about proteins and protein interactions such as PPIs is vital to create a complete picture of an individual’s health at a given time point as well as to predict any emerging health issues. Presence and interactions between these proteins are also essential for drug development and are increasingly becoming a highly sought-after dataset to capture an individual proteome and changes to the proteome in response to environmental or other systemic events. The ability to detect and quantify proteins and protein interaction with other molecules within a given sample is an integral component of such healthcare development.

SUMMARY

[0003] The present disclosure generally relates to substrates that may be used for detection and quantification of analyte molecules (e.g., proteins) in a sample. The substrates may include entities distributed on a surface of the substrate in a random or unknown arrangement such that, at the time of applying the entities to the substrate, the particular location of each entity applied on the substrate is unknown. For example, the substrate may be prepared by contacting the substrate with a pool of different entities in a concentration and/or configuration that is generally associated with a single binding site on the substrate coupling to a single entity. In one example, the entities can be affinity binders having affinities for different analytes. Thus, each entity of the binding site may have affinity for a single type of analyte. However, because the substrate is prepared from pooled entities, the location of the different entities is generally random on the substrate, and is not reproducible between different substrates. While preparing substrates using pooled entities to generate a random arrangement is less complex than printing or other mapped preparation techniques, the resultant substrate may need to be characterized and mapped so that subsequent assays using the substrate can provide useful information about entity-analyte binding. The entities at different binding sites on the substrates can be used to detect binding of an analyte. Thus, binding of an analyte can be linked to a positive binding event signal from an identified entity.

[0004] The present disclosure relates to techniques for mapping or identifying the location of entities applied on the substrate by characterizing a unique identification sequence of one or more oligonucleotides associated with each entity. The oligonucleotides having unique identification sequences are coupled to and co-located with the entity at each binding site. Accordingly, characterization of the co-located nucleic acid sequence at a particular binding site permits identification, e g., mapping, of the associated entity to a particular capture site location on the substrate. The substrate may be ordered, with each available binding site being part of an ordered arrangement or pattern. The entities can become associated with respective binding sites in a random fashion to create a randomly ordered substrate. That is, the entity location is random, but the binding sites are ordered. In another example, the locations of the binding sites can also be unpredictable or random.

[0005] In contrast to mapping techniques that rely on sequencing or amplification of a unique sequence associated with an entity, the disclosed techniques permit hybridization-based characterization using modular nucleic acids (e.g., single-stranded nucleic acids) that are assembled in unique combinations to create a corresponding number of oligonucleotides having unique identification sequences. Hybridization of tagged complementary probes to portions of the oligonucleotides permits modular detection, and the presence of particular identification sequences is based on positive signals of probe binding for the appropriate modules of the identification sequence as well as, in embodiments, negative signals for probes specific for modules not associated with the identification sequence.

[0006] The modular approach of the disclosed techniques reduces complexity requirements for providing detection probes for relatively higher numbers of unique identification sequences. That is, rather than providing at least one tagged complementary probe corresponding to each complete identification sequence, the present techniques involve a lower number of tagged complementary probes. In a specific example, in the context of 16 samples, 16 different identification sequences can be created using a first set of four nucleic acid modules having different sequences that are differently combined with another set of four nucleic acid modules having different sequences. Accordingly, eight different modules (divided as two separate sets) in different combinations scale up to 16 unique sequences. Thus, detection can involve using only 8 different tagged probes specific for the eight different modules rather than 16 different tagged probes. The relationship between the potential number of different sequences and number of modules can be varied depending on the number of different detectable tags used, a number of binding stages for hybridization, as well as via use of common sequences that act as landing pads for variable modules, as generally provided herein. In certain cases, where duplication of a particular modular sequence is permitted (rather than requiring a single module to appear only once in the unique identification sequence), a set of n different modules can scale up to even more unique identification sequences in combination. Modular combination of nucleic acid sequences for entity characterization reduces the associated mapping costs, as tags, e.g., fluorescent tag, are relatively costly.

[0007] As part of mapping the entities on the substrate, the identification sequences may be detected in sequential stages of probe hybridization to the identification oligonucleotide. If the detection modality has a limited number of detection channels for tagged probes, adding additional stages permits more different probes to be detected. Detection using additional stages involves applying a first set of tagged detection probes to the substrate, detecting hybridization of the tagged probes to complementary nucleic acid modules, removal of the hybridized probes, applying a different set of tagged detection probes, and so on. Serial binding and removal of probes, e.g., via heat or chemical denaturation, may impact the integrity of the oligonucleotides or other components of the substrate, thus potentially reducing data quality for mapping and/or subsequent analysis steps. Also provided herein are improved techniques for sequential hybridization to permit removal of a hybridized tagged detection probe from a particular nucleic acid sequence module. The disclosed techniques may use a toehold-mediated displacement of hybridized probes, thus reducing the need for chemical and/or heat denaturation. Thus, in embodiments, disclosed tagged probes for mapping as provided herein may include one or more toehold sequences that do not hybridize to the identification oligonucleotide but that serve as toehold that facilitate dislodgement when contacted with a complement.

[0008] The disclosed embodiments may be implemented using entities and associated unique identification sequence coupled to supramolecular structures that are randomly ordered on the substrate. The supramolecular structure may include one or more of 1) an entity; 2) a unique identification oligonucleotide and/or a set of identification oligonucleotides with a unique combination of identification sequences associated entity of an individual supramolecular structure, and 3) a physical scaffold that promotes single entity placement at an individual binding site.

[0009] An advantage of using supramolecular structures to create a randomly ordered array on a substrate is that each supramolecular structure is sized and shaped to promote single molecule binding at each active binding site on the substrate. Thus, the supramolecular structures prevent mixing of different types of entities at each binding site. In some cases, each supramolecular structure, when associated with a particular binding site, physically blocks other supramolecular structures from coupling to the same binding site. Thus, an individual binding site will have only one supramolecular structure and, as a result only one entity and one unique identification oligonucleotide. A positive binding result at a binding site is a robust indicator of the presence of specific binding for a particular entity. The identity of the particular entity is known based on the mapping discussed herein. Further, the supramolecular structure physically links the entity and an individual identification oligonucleotide so that placement of both on the substrate is simultaneous. [00010] An additional benefit is that the modular and customizable structure of the components of the supramolecular structure permits providing bulk or common core structures that are generally the same and modifying individual supramolecular structures with unique identification oligonucleotides during linkage of a particular entity.

[00011] As provided herein, an individual supramolecular structure may be immobilized on a respective binding site of a substrate. An individual supramolecular structure includes a core structure comprising a plurality of core molecules. In some embodiments, each core structure is a nanostructure. In some embodiments, for any method disclosed herein, the plurality of core molecules for each core structure are arranged into a pre-defined shape and/or have a prescribed molecular weight. In some embodiments, the pre-defined shape is configured to limit or prevent cross-reactivity with another supramolecular structure. In some embodiments, for any method disclosed herein, the plurality of core molecules for each core structure comprises one or more nucleic acid strands, one or more branched nucleic acids, one or more peptides, one or more small molecules, or combinations thereof. In some embodiments, for any method disclosed herein, each core structure independently comprises a scaffolded deoxyribonucleic acid (DNA) origami, a scaffolded ribonucleic acid (RNA) origami, a scaffolded hybrid DNA:RNA origami, a singlestranded DNA tile structure, a multi-stranded DNA tile structure, a single-stranded RNA origami, a multi-stranded RNA tile structure, hierarchically composed DNA or RNA origami with multiple scaffolds, a peptide structure, or combinations thereof. The core structure may include protein nanostructures, protein cages, self-assembling protein structures, protein origami, biopolymers, biopolymer origami (protein-DNA, protein-RNA, protein-protein, etc combinations thereof) and/or canonical/noncanonical amino acids.

[00012J In an embodiment, the entity and the identification oligonucleotide may be co-located on a bead-based core structure (e.g., silica, gold, hydrogel beads). The beads may be functionalized beads or magnetic beads.

[00013] In an embodiment, the structure linking the entity and the identification oligonucleotide (e.g., the core structure) may be removed or degraded after placement of the entity and the identification oligonucleotide on the substrate. Thus, the bead or core structure may be enzymatically or photodegradable.

[00014] The entity of a supramolecular structure can be linked via chemical bond to the core structure. In some embodiments, the entity of the solution-based supramolecular structure and/or immobilized entities of the binding sites independently comprise a protein, a peptide, an antibody, an aptamer (RNA and DNA), a fluorophore, a darpin, a catalyst, a polymerization initiator, a polymer like PEG, or combinations thereof.

[00015] In some embodiments, for any method comprising using a plurality or pool of supramolecular structures disclosed herein, the core structures of the plurality of supramolecular structures are identical to each other. However, the coupled entities can vary for the plurality. Thus, in an embodiment, a pool of (e.g., a plurality of) supramolecular structures is identical except for a coupled entity and the unique identification oligonucleotide that uniquely identifies the coupled entity co-located on the supramolecular structure.

[00016] In some embodiments, each supramolecular structure comprises a prescribed shape, size, molecular weight, or combinations thereof, which may reduce or eliminate cross-reactions between a plurality of supramolecular structures. In some embodiments, each supramol ecul ar structure comprises only one entity or multiple entities. Where a supramolecular structure includes multiple entities on a single core structure, the multiple entities all have a same binding specificity, e.g., all specifically bind a same analyte.

[00017] In some embodiments, the substrate comprises a solid support, solid substrate, a polymer matrix, or one or more beads. In some embodiments, a plurality of supramolecular structures are disposed on a substrate, such as a shaped or planar substrate, wherein the substrate comprises a plurality of binding sites, wherein each individual binding site is coupled to one or more entities configured to bind to the same analyte molecule, e.g., such that an individual binding site is specific for an individual analyte molecule and different binding sites of the substrate have specificity for different analyte molecules. The disclosed embodiments also include sample preparation reagents, substrates, and detection systems for performing the disclosed methods. [00018] In some embodiments, for any method disclosed herein, the disclosed substrates are suitable for detecting analytes in a biological sample. In some embodiments, for any method disclosed herein, the analyte molecule or molecules of a sample can include a protein, a peptide, a peptide fragment, a lipid, a DNA, a RNA, an organic molecule, an inorganic molecule, complexes thereof, or any combinations thereof. In some embodiments, for any method disclosed herein, the sample includes a biological particle or a biomolecule. In some embodiments, for any method disclosed herein, the sample includes an aqueous solution comprising a protein, a peptide, a fragment of a peptide, a lipid, DNA, RNA, an organic molecule, a viral particle, an exosome, an organelle, or any complexes thereof. In some embodiments, for any method disclosed herein, the sample c includes a tissue biopsy, blood, blood plasma, Urine, Saliva, Tear, Cerebrospinal fluid, extracellular fluid, cultures cells, culture media, discarded tissue, plant matter, a synthetic protein, prions, a bacterial and/or viral sample or fungal tissue, or combinations thereof. The sample may be an environmental sample, such as a wastewater or soil sample. The sample may also be a nonbiological sample. In an embodiment, the sample may be a sample from a chemical process step or steps, a sample of food or nutritional components, or packaging components.

[00019] The sample may be processed to release the analytes from cells or to otherwise prepare the sample for analysis prior to contacting the sample with the supramolecular structures in solution as provided herein. [00020] In some embodiments, disclosed techniques include providing a randomly ordered substrate with mapped entities, and using the substrate for detecting a presence of, identifying, and/or quantifying the concentration of the analyte molecule in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[00021] Specific embodiments of the disclosed devices, delivery systems, or methods will now be described with reference to the drawings. Nothing in this detailed description is intended to imply that any particular component, feature, or step is essential to the invention.

[00022] FIG. 1 shows an overview of preparation and mapping of a substrate according to embodiments of the disclosure;

[00023] FIG. 2A shows an example supramolecular structure that can be associated with the substrate of FIG. 1 according to embodiments of the disclosure.

[00024] FIG. 2B shows an identification oligonucleotide and entity of FIG. 2A after removal or degradation of the core structure according to embodiments of the disclosure.

[00025] FIG. 3 shows an example supramolecular structure including a unique identification oligonucleotide and that can be associated with the substrate of FIG. 1 according to embodiments of the disclosure.

[00026] FIG. 4 shows tagged detection probes hybridized to different modules or regions of the unique identification oligonucleotide according to embodiments of the disclosure.

[00027] FIG. 5 shows an example workflow for sequential application of tagged probes to a randomly ordered substrate for characterization and mapping of entities via hybridization to an associated unique identification oligonucleotide according to embodiments of the disclosure.

[00028] FIG. 6 shows an example supramolecular structure including separate oligonucleotides that form a unique identification sequence and that can be associated with the substrate of FIG. 1 according to embodiments of the disclosure.

[00029] FIG. 7 shows an example supramolecular structure in which separate oligonucleotides that form a unique identification sequence are distributed in different quadrants or regions of the supramolecular structure according to embodiments of the disclosure.

[00030] FIG. 8 shows an example supramolecular structure including a universal oligonucleotide that is a linker to modules of a unique identification sequence and that can be associated with the substrate of FIG. 1 according to embodiments of the disclosure. [00031] FIG. 9 shows the supramolecular structure of FIG. 8 in which the modules having separate regions of the unique identification sequence are assembled via complementary regions on the universal oligonucleotide according to embodiments of the disclosure.

[00032] FIG. 10 shows the supramolecular structure of FIG. 9 having a hybridized tagged probe complementary to the particular linked module 1 sequence according to embodiments of the disclosure.

[00033] FIG. 11 A shows an example supramolecular structure including a rolling circle amplification template that generates an amplification product including multiple copies of the unique identification sequence and that can be associated with the substrate of FIG. 1 according to embodiments of the disclosure.

[00034] FIG. 1 IB shows an alternate example supramolecular structure including a rolling circle amplification template that generates an amplification product including multiple copies of the unique identification sequence and that can be associated with the substrate of FIG. 1 according to embodiments of the disclosure.

[00035] FIG. 12 shows an example workflow for toehold-based removal of tagged probes from a unique identification oligonucleotide according to embodiments of the disclosure.

[00036] FIG. 13 shows an example workflow for toehold-based removal of a tagged probe hybridized to a linker that is in turn hybridized to a universal oligonucleotide according to embodiments of the disclosure.

[00037] FIG. 14 shows an example polymerase-based displacement according to embodiments of the disclosure.

[00038] FIG. 15 shows an example photocleavable tagged probe according to embodiments of the disclosure.

[00039] FIG. 16 shows an example chemically labile tagged probe according to embodiments of the disclosure.

[00040] FIG. 17 shows a block diagram of an example analyte detection system according to embodiments of the disclosure.

[00041] FIG. 18 shows a flow diagram of an example analyte detection method according to embodiments of the disclosure. DETAILED DESCRIPTION

[00042] Disclosed herein are techniques for characterization of entities immobilized on a substrate. In an embodiment, the entities can be immobilized on the substrate randomly or in an undirected manner, and the disclosed techniques permit characterization, such as mapping or identification of a particular entity to a particular location on the substrate. The information regarding the mapping of locations of each entity on the substrate can be used to resolve detection data collected from contacting the substrate with a sample of interest such that positive binding or detection events can be associated with a particular entity. The disclosed techniques permit substrate manufacturing and preparation using less complex immobilization techniques while also retaining the ability to characterize the entity at each binding site.

[00043] FIG. 1 is an example workflow of random entity immobilization on a substrate that can be used in the disclosed characterization techniques. In the workflow, different individual entities 2 have respective different affinities to different analytes. A pool 6 of the different entities 2 (and associated structures, such as supramolecular structures as generally discussed herein) can be applied to and immobilized on a prepared substrate surface 7 that includes different active binding sites 8. The pool 6 may be implemented as a solution of entities 2 and associated structures. [00044] In an embodiment, the prepared substrate surface 7 and immobilization may be performed as generally discussed in U.S. Provisional Application No. 63/119,316, filed on November 30, 2020, and incorporated herein by reference for all purposes. In one example, a substrate base layer is provided. A passivation layer is grown, assembled, or deposited on the base layer that can be selectively passivated. The passivation layer or layers may include silicon nitride, graphene, quartz, metal, gold, silver, platinum, palladium, PDMS, polymer film, or combinations thereof. The passivation layer may be graphene, aluminum oxide, HfCh, CnCh (Chromium oxide), Titanium oxide, Tantalum oxide, metal oxides, silicon dioxide (SiCh) or combinations thereof. The passivation layer may be a self-assembled polymer, such as a polyacrylamide.

[00045] The passivation layer is patterned, e.g., by removing portions of the top layer, to expose locations of the base layer that will correspond to binding sites 8 of the substrate 60. The patterning may be photolithography, e-beam lithography, nanoimprinting, polymer spin coating, optical patterning, plasma activation, acid/base treatment, or other patterning modalities. The exposed locations may be activated by chemical or plasma treatment to yield different reactive groups, depending on the individual chemistry of these layers. The activated exposed locations can receive or be coupled to individual entity structures to form the substrate 9.

[00046] A substrate 9 may include a defined set of micropatterned binding sites 8. In some embodiments, the binding sites 8 on the surface 7 are in a periodic or regular pattern, e.g., are ordered. In some embodiments, the binding sites 8 on the surface 7 are in a non-periodic pattern (e.g., random). In some embodiments, a minimum distance is specified between any two binding sites 8. In some embodiments, the minimum distance between any two binding sites 8 is at least about 200 nm. In some embodiments, the minimum distance between any two binding sites 8 is from at least about 40 nm to about 5000 nm. In some embodiments, the geometric shape of the binding sites 8 comprises a circle, square, triangle or other polygon shapes. In some embodiments, an individual binding site 8 is 20-200nm in diameter.

[00047J The substrate 9 may include a glass or silicon wafer having one or more silicon dioxide, silicon nitride, graphene or silicon carbide layers. In some embodiments, the substrate 9 may include fiduciary markers (not shown) having geometric features defined on a surface to be used as reference features for other features on the substrate 9. In some embodiments, the substrate 9 comprises structures that facilitate detection, such as optical or electrical devices like FET, ring resonators, photonic crystals or microelectrode, to be defined prior to the formation of the binding sites 8.

[00048] A substrate 9 is generated that includes entities 2 randomly immobilized at different binding sites 8. The binding sites 8 can be formed in a regular pattern or array in embodiments. However, the identity of the particular entity 2 at a particular binding site 8 is not predictable or generally repeatable between different substrate preparations. The pool 6 is contacted with the prepared substrate surface, and the concentration and arrangement of different entities 2 is selected such that each binding site 8 receives no more than one entity 2 (or one type of entity 2 in a complex). Applying the pool 6 of different entities 2 in bulk or as a batch to the prepared substrate surface 7 is faster and easier than individually placing each different entity 2 at a predetermined binding site location. Further, pooling permits greater flexibility in substrate preparation, as the identities of the different entities 2 in the pool 6 can be easily customized depending on end user preferences. Preparation of a custom pool 6 can be based on selecting the appropriate entities 2 (and associated structures). Once prepared, the pool 6 is immobilized on the prepared surface 7 to generate the substrate 9. The substrate 9 undergoes detection and mapping as disclosed herein to associate individual binding sites 8 with identities of individual entities 2.

[00049] In certain embodiments, each individual entity 2 that is pooled (e.g., in pool 6, see FIG. 1) is coupled to or part of a supramolecular structure 10. FIG. 2 provides an exemplary embodiment of a supramolecular structure 10 comprising a core structure 13 and an individual entity 2. In the depicted example, the supramolecular structure 10 includes a unique identification oligonucleotide 16 that has a unique identifying sequence associated with the entity. Thus, a pooled supramolecular structure 10 of the pool 6 can have different entities 2 with different associated identification oligonucleotides 16. Accordingly, provided herein are one or more supramolecular structures 10 that can be provided to form the pool 6 that is randomly immobilized on the substrate 9. The identification oligonucleotide 16 is used to map the substrate 9 as generally discussed herein.

[00050] In some embodiments, the supramolecular structure 10 is a programmable structure that can spatially organize molecules. In some embodiments, the supramolecular structure 10 comprises a plurality of molecules linked together. In some embodiments, the plurality of molecules of the supramolecular structure 10 interact with at least some of each other. In some embodiments, the supramolecular structure 10 comprises a specific shape, e.g., a substantially planar shape that has a longest dimension in an x-y plane. In some embodiments, the supramolecular structure 10 is a nanostructure. In some embodiments, the supramolecular structure 10 is a nanostructure that comprises a prescribed molecular weight based on the plurality of molecules of the supramolecular structure 10. In some embodiments the plurality of molecules are linked together through a bond, a chemical bond, a physical attachment, or combinations thereof. In some embodiments, the supramolecular structure 10 comprises a large molecular entity, of specific shape and molecular weight, formed from a well-defined number of smaller molecules interacting specifically with each other. In some embodiments, the structural, chemical, and physical properties of the supramolecular structure 10 are explicitly designed. In some embodiments, the supramolecular structure 10 comprises a plurality of subcomponents that are spaced apart according to a prescribed distance. In some embodiments, at least a portion of the supramolecular structure 10 is rigid. In some embodiments, at least a portion of the supramolecular structure 10 is semi-rigid. In some embodiments, at least a portion of the supramolecular structure is flexible. In an embodiment, the supramolecular structure 10 is at least 50-200nm in one dimension. . In an embodiment, the supram olecul ar structure 10 is at least 20nm long in any dimension.

[00051] In some embodiments, the core structure 13 is a polynucleotide structure, a protein structure, a polymer structure, or a combination thereof. In some embodiments, the core structure 13 comprises one or more core molecules linked together. In some embodiments, the one or more core molecules comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200 or 500 unique molecules that are linked together. In some embodiments, the one or more core molecules comprises from about 2 unique molecules to about 1000 unique molecules. In some embodiments, the one or more core molecules interact with each other and define the specific shape of the supramolecular structure. In some embodiments, the plurality of core molecules interact with each other through reversible non-covalent interactions. The core structure may include protein nanostructures, protein cages, self-assembling protein structures, protein origami, biopolymers, biopolymer origami (protein- DNA, protein-RNA, protein-protein, etc combinations thereof) and/or canonical/noncanonical amino acids.

[00052] In an embodiment, core structure 13 is a bead-based core structure (e.g., silica, gold, hydrogel beads). The beads may be functionalized beads or magnetic beads.

[00053] In an embodiment, the structure linking the entity and the identification oligonucleotide (e.g., the core structure) may be removed or degraded after placement of the entity and the identification oligonucleotide on the substrate. Thus, the bead or core structure may be enzymatically or photodegradable. Fig. 2B shows an example in which the core structure 13 is removed after placement at an example binding site 8. For example, the core structure 13 can be enzymatically or photochemically removed. However, the identification oligonucleotide 16 and the entity 2 are nonetheless retained at the binding site 8 via a separate covalent linking to the substrate 9. Accordingly, the core structure 13 can be removed after linking of the identification oligonucleotide 16 and the entity 2 to the substrate 9.

[00054] In some embodiments, the specific shape of the core structure 13 is a three-dimensional (3D) configuration. In some embodiments, the one or more core molecules provide a specific molecular weight. For example, all core structures 13 of supramolecular structures 10 of a plurality may have a same configuration, size, and/or weight, but may different in their attached linker sequences and attached entities 2. However, excluding different linkers 20 and entities 2, the supramolecular structures 10 of a plurality may be otherwise identical. In some embodiments, the core structure 13 is a nanostructure. In some cases, the one or more core molecules comprise one or more nucleic acid strands (e.g., DNA, RNA, unnatural nucleic acids), one or more branched nucleic acids, one or more peptides, one or more small molecules, or combinations thereof. In some embodiments, the core structure 13 comprises an entirely polynucleotide structure. In some embodiments, at least a portion of the core structure 13 is rigid. In some embodiments, at least a portion of the core structure 13 is semi-rigid. In some embodiments, at least a portion of the core structure 13 is flexible. In some embodiments, the core structure 13 comprises a scaffolded deoxyribonucleic acid (DNA) origami, a scaffolded ribonucleic acid (RNA) origami, a scaffolded hybrid DNA / RNA origami, a single-stranded DNA tile structure, a multi-stranded DNA tile structure, a single-stranded DNA origami, a single- stranded RNA origami, a single-stranded RNA tile structure, a multi-stranded RNA tile structures, a hierarchically composed DNA and/or RNA origami with multiple scaffolds, a peptide structure, or combinations thereof. In some embodiments, the DNA origami is scaffolded. In some embodiments, the RNA origami is scaffolded. In some embodiments, the hybrid DNA/RNA origami is scaffolded. In some embodiments, the core structure 13 comprising a DNA origami, RNA origami, or hybrid DNA/RNA origami that comprises a prescribed two-dimensional (2D) or 3D shape. In an embodiment, nucleic acid core molecules of the core structure 13 act as molecular staples that provide structural and/or linking support.

[00055] In an embodiment, the core structure 13 is larger than an individual binding site 8 in at least one dimension. In an embodiment, the core structure 13, when immobilized on the binding site 8, covers at least a majority of a surface area of the binding site 8. In an embodiment, the core structure 13 is a nucleic acid origami that has at least one lateral dimension between about 50nm to about Ip. In an embodiment, the nucleic acid origami has at least one lateral dimension between about 50nm to about 200nm, about 50nm to about 400nm, about 50nm to about 600nm, about 50nm to about 800nm, about lOOnm to about 200nm, about lOOnm to about 300nm, about lOOnm to about 400nm, about lOOnm to about 500nm, about 200nm to about 400nm by way of example. In an embodiment, the nucleic acid origami has at least a first lateral dimension between about 50nm to about Ip and a second lateral dimension, orthogonal to the first, between about 50nm to about Ip. In an embodiment, the nucleic acid origami has a planar footprint having an area of about 200nm² to about Ip². [00056] As shown in FIG 2, in some embodiments, the core structure 13 is configured to be linked to an entity 2 and one or more identification oligonucleotides 16. The identification oligonucleotide may be a double or single-stranded DNA or RNA. While certain detection and mapping processes are performed using a single-stranded identification oligonucleotide 16, the identification oligonucleotide 16 can be provided as a protected and/or end-capped oligonucleotide that is denatured prior to detection and mapping. In embodiments, the identification oligonucleotide 16 is at least 30 bases long, at least 50 bases long, at least 100 bases long, at least 200 bases long, or at least 300 bases long. In embodiments, the identification oligonucleotide 16 is 30-50 bases, 30-100 bases, 30-150 bases, or 50-200 bases.

[00057] In some embodiments, the entity 2 is immobilized with respect to the core nanostructure 13 when linked thereto. In some embodiments, the entity 2 is linked to the core structure 13 through the linker 20. In some embodiments, the linker 20 comprises a polymer that comprises a nucleic acid (double or single-stranded DNA or RNA) of a specific sequence that is associated with the linked entity 2. In some embodiments, the linker 20 includes a barcode sequence that uniquely identifies the entity 2. Thus, the identification nucleotide 2 and the linker 20 may carry identification information. In an embodiment, the linker 20 and the identification oligonucleotide 16 may be combined, or the linker 20 may serve as all or part of the identification oligonucleotide 16. That is, in certain embodiments, the entity 2 may be directly coupled to the identification oligonucleotide 16.

[00058] In some embodiments, any number of the one or more core molecules comprises one or more linkers 20 configured to form a linkage with the entity 2. In some embodiments, the linker 20 is linked to one or more core molecules of the core structure 13 through a chemical bond. In some embodiments, the linker 20 may include a core reactive molecule. In some embodiments, each core reactive molecule independently comprises an amine, a thiol, a DBCO, a NHS ester, a maleimide, biotin, an azide, an acrydite, a single stranded nucleic acid (e.g., RNA or DNA) of specific sequence, or a polymer (e g., polyethylene glycol (PEG) or one or more polymerization initiators). In some embodiments, at least one of the one or more core linkers comprises a DNA sequence domain. In some embodiments, the core structure 13 and/or the identification oligonucleotide 16 are positioned at prescribed locations on the core structure 13.

[00059] In some embodiments, the entity 2 comprises a protein, a peptide, an antibody, antibody- derived reagents, an aptamer (RNA and DNA), a fluorophore, a nanobody or nanostructure, a darpin, a catalyst, a polymerization initiator, a polymer like PEG, an organic molecule, small molecule, a pharmaceutical compound, a candidate pharmaceutical compound, a synthetic molecule, or combinations thereof. In some embodiments, a single entity 2 is linked to the core structure 13. In some embodiments, a plurality of entities 2 are linked to a core structure 13. For example, different entities 2 on a same core structure 13 may represent different binding sites for a same analyte molecule or may bind different analyte molecules of a multi-molecule complex, e.g., of a protein-protein complex. In another example, multiple same entities 2 may be present on a core structure 13. The entity 2 may be capture molecule or an affinity binder.

[00060] In some embodiments, each component of the supram olecular structure 10 may be independently modified or tuned. In some embodiments, modifying one or more of the components of the supram olecular structure 10 may modify the 2D and 3D geometry of the supramolecular structure itself. In some embodiments, modifying one or more of the components of the supramolecular structure may modify the 2D and 3D geometry of the core structure 13. In some embodiments, such capability for independently modifying the components of the supramolecular nanostructure enables precise control over the organization of one or more supramolecular structures.

[00061] In certain embodiments, each supramolecular structure 10 in the pool 6 has generally a same core structure 13 but different entities 2 (and, in embodiments, different linkers 20) and different identification oligonucleotide 16 (e.g., having different sequences). Thus, to form a supramolecular structure 10 as provided herein may include starting from a same base core structure 13 and modifying the core structure 13 with an individual entity 2 and an identification oligonucleotide 16 of a known sequence that is associated with the individual entity 2. The supramolecular structure 10 can be prepared and stored separately (e.g., unpooled) once modified to include a desired entity 2. In an embodiment, the supramolecular structure 10 can include the entity 2 but not the identification oligonucleotide 16 in a stored configuration. The identification oligonucleotide 16 can be added prior to pooling once the total size and plexity of the desired substrate is known.

[00062] As generally disclosed herein, different sets of identification oligonucleotides 16 can support different plexities based on the number of modular sequences used. FIG. 3 shows an example supramolecular structure 10 carrying the entity 2 and its associated identification oligonucleotide 16, illustrated as a contiguous nucleic acid strand that extends away from the core structure 13 in a single-stranded region 17. The identification oligonucleotide 16 includes a linker sequence 18 at a first end that is hybridized to a linker complement 21, and the linker complement 21 is fixed to the core structure 13. Thus, the identification oligonucleotide 16 can be coupled to the supram olecul ar structure 10 by complementary binding or, in embodiments, via direct covalent binding to the core structure 13. The linker 18 can be 8-30 bases in length in an embodiment. While only a single supramolecular structure 10 is illustrated, the linker sequence of the linker 18 (and, therefore, the linker complement 21) can be a common or universal sequence that is a same sequence between all identification oligonucleotides 16 in the pool 6. The single-stranded region 17 carrying the identification sequence is unique to a particular entity 2 and varies between different identification oligonucleotides 16 in the pool 6.

[00063] The identification oligonucleotide 16 is formed from a plurality of modules having different modular sequences. Each module is selected from fixed sets of available modules that are nonoverlapping such that all of the modules have sequences that are distinguishable from one another. For example, to the form the particular identification oligonucleotide 16 of FIG. 3, module 1 can be selected from two or more modules (e.g., module la, module lb) forming a set for module 1 with different modular sequences. Similarly, module 2 can be selected from two or more modules (e.g., module 2a, module 2b) forming a set for module 2 with different modular sequences; module 3 can be selected from two or more modules (e.g., module 3a, module 3b) forming a set for module 3 with different modular sequences; and module 4 can be selected from two or more modules (e g., module 4a, module 4b) forming a set for module 4 with different modular sequences. Different combinations of the modules generates a unique identification sequence of the identification oligonucleotide 16. The identification oligonucleotide 16 associated with the entity 2 can be formed from module la, module 2a, module 3a, and module 4a. A different identification oligonucleotide 16 associated with a different entity 2 can be formed from module la, module 2b, module 3b, and module 4a. Thus, while different identification oligonucleotides 16 of the pool 6 can have common modules between them, the total assembled identification sequences are different. Further, each identification oligonucleotide 16 includes only one module of the module 1 set, only one module of the module 2 set, only one module of the module 3 set, only one module of the module 4 set, and so on.

[00064J The different modules can be distinguished from one another by specific hybridization (or lack thereof) of complementary probes. Thus, the modules that form each identification oligonucleotide 16 on the substrate 9 can be identified (and mapped to the binding site 8) by applying complementary probes with associated tags (e.g., detectable moieties such as fluorescent labels), permitting the complementary probes to hybridize, washing unbound probes, and detecting the remaining bound probes. FIG. 4 shows the supramolecular structure 10 of FIG. 3 with hybridized tagged probes (with tags illustrated schematically as *) that are specifically bound to particular modular sequences. While the tagged probes are shown all simultaneously bound by way of example, it should be understood that the binding of the tagged probes may be in stages or sequential as discussed herein.

[00065] If the identification oligonucleotide 16 is formed from module la, module 2a, module 3a, and module 4a, then tagged probe 1 is complementary to module la, tagged probe 2 is complementary to module 2a, tagged probe 3 is complementary to module 3a, and tagged probe 2 is complementary to module 2a. In the example in which each of four modules can be selected from a total of two different modular sequences, 16 different identification oligonucleotides 16 can be assembled from different combinations of modules 1, 2, 3, and 4. That is, the number of possible identification oligonucleotides 16 in the illustrated contiguous strand example is scaled up from the number of total modules.

[00066] This provides cost and complexity advantages for detection and mapping. In FIG. 4, four different tagged probes are used to detect the illustrated identification oligonucleotide 16. Four different unbound tagged probes 30, 32, 34,36 are also shown that are specific for modular sequences (e.g., module lb, 2b, 2c, 2d) not represented in the identification oligonucleotide 16. These modular sequences are, however, present in other identification oligonucleotides 16 at different binding sites 8 on the substrate 9. Instead of having to manufacture 16 different tagged probes, each specific for a different identification oligonucleotide 16, only eight tagged probes are used to detect all possible combinations of identification oligonucleotides 16. Because tagged probes are relatively costly, a reduction in manufacturing provides cost advantages. Further, in another embodiment, the detection between two different sequences at each module can be based on presence or absence of a detectable signal. That is, modular sequence la can be detected based on the detected presence of tagged probe 1 while the presence of modular sequence lb can be detected based on an absence of any signal from tagged probe 1. Thus, only four different tagged probes can be used to detect all 16 possible combinations, providing additional cost savings. [00067] In an embodiment, the linker complements 21 of the linkers of identification oligonucleotides 16 discussed herein can be CT-rich sequences, and the linkers can be AG-rich sequences. In an embodiment, the modular sequences can use only AGT nucleotides, such that the tagged probes use only ACT nucleotides. The use of CT sequences can reduce cross-talk. Further, the use of a reduced nucleotide set for the identification sequence can eliminate cross-talk between different modules.

[00068] While the depicted embodiment shows four modules assembled in a combinatorial strand, it should be understood that the identification oligonucleotide 16 can be formed from two, three, four, five, six, seven, eight, nine, ten, or more modules. Each different module can be selected from a fixed set of two, three, four, five, six, seven, eight, nine, ten, or more modules with different modular sequences. In one embodiment, module 1 is selected from 20 different available modules. Thus, the module 1 can have a modular sequence la and not 19 other modular sequences in the available set for module 1. Further, while the depicted embodiment shows each module being present in only one instance on the individual identification oligonucleotide 16, modules can be duplicated or repeated to increase detection intensity.

[00069] Additional examples of combinatorial arrangements of the identification oligonucleotide 16 of FIGS. 3-4 can include the following:

Table 1 : Combinatorial Arrangements

[00070] The total number of possible combinations represents the available plexity for the substrate. It should be understood that all possible combinations may not be used on a particular substrate 9. For example, if a substrate 9 has 50000 available binding sites 8, using an arrangement with 65536 possible combinations would require using a majority (e.g., more than half) of all possible identification sequences, but not all possible identification sequences. In an embodiment, at least 25%, at least 50%, or at least 75% of all possible identification sequences are represented in immobilized supramolecular structures 10 on the substrate 9. Thus, certain sequences may be excluded. The exclusion may, in an embodiment, be based on a quality check. For example, some sequences may have poor hybridization specificity with tagged probes or may be susceptible to cross-reactivity, and those sequences can be excluded based on actual or predicted specificity or cross-reactivity. Additional considerations include the detection capabilities of the detection system (see FIG. 13) to distinguish between different tags of the tagged probes. For example, in one embodiment, all tagged probes have distinguishable tags based on their binding specificity, such that the tag complementary to module la has a different tag than the tags on probes specific for modules lb, 2a, 2b, 3a, 3b, 4a, and 4b. Thus, all eight tags can be simultaneously detected.

[00071] However, in other embodiments, the detection system may be a one channel system capable of only detecting one tag. The detection system may alternatively be a two, three, four, eight, or ten channel system.

[00072] If the number of total tagged probes is greater than the channels available in the system, the detection can occur in stages, as shown in FIG. 5. FIG. 5 depicts a single binding site 8 having immobilized thereon a single supramolecular structure 10. Features of the supramolecular structure such as the entity 2 are present but not shown for purposes of illustration. FIG. 5 shows a four stage detection process in which each of the modules 1-4 is detected at a different stage. It should be understood that the number of stages may be increased or decreased depending on the number of modules and/or the number of modular sequences detected at each module. The example of FIG. 5 may be used in conjunction with a four channel detection system that can differentiate between four different fluorescent labels. While the probes are different at each stage, the fluorescent labels can be repeated between stages.

[00073] The process is initiated with the identification oligonucleotide 16 in a single-stranded configuration. At stage 1, tagged probes 50 are provided that, as a group, are complementary to the entire set of modular sequences of a first module. For a tagged probe in embodiments discussed herein, the tagged probe may be provided as a single- stranded oligonucleotide. Binding of a tagged probe to a complementary generates an at least partially double-stranded structure. An individual tagged probe 50a that is complementary to the sequence present on the identification oligonucleotide 16 is permitted to bind, and unbound probes 50 can be washed away. The remaining bound probe 50a is detected to generate detection data. The detection data can indicate fluorescence in a particular wavelength band that is different from detected fluorescence associated with the unbound probes. Thus, detection of, for example, green fluorescence yields a positive signal for a particular 15-50 base modular sequence. Detection of other fluorescent colors would be positive for the particular probes associated with those colors.

[00074] The bound probe 50a is removed by stripping or toehold-mediated removal as discussed herein, and the single-stranded identification oligonucleotide 16 is available for detection in a second stage. As illustrated, different sets of tagged probes 50, 52, 54, 56 are applied and detected in stages. Resolving the detection data to a particular identification sequence can be based on identifying one identification sequence that correlates to the detection data.

Table 2: Detection Data for Determining an Identification Sequence

[00075] Other unique identification sequences present at different binding sites 8 are associated with different combination of detected probes at each stage (e.g., detection of 50b, 50c, or 50d at stage 1 and so on). The depicted detection process can occur simultaneously at all binding sites 8 on a substrate 9 to generate detection data used to map the substrate 9. Thus, steps of applying tagged probes to the binding site 8 refers to a batch application across the substrate 9 such that the tagged probes are available for complementary binding at appropriate modules, where present. [00076] FIGS.3-5 show an example of the identification oligonucleotide 16 being formed by a single combinatorial strand. Additionally or alternatively, the identification oligonucleotide 16 can be a path or set of separate or noncontiguous oligonucleotides that are all coupled to a same supramol ecular structure 10 and that, in total, contain modules that form a noncontiguous unique identification sequence. [00077] FIG. 6 shows an example simplified detection workflow to characterize the identification oligonucleotide 16 implemented as a patch 60, and depicted as including four separate singlestranded modules 62, 64, 66, 68. It should be understood that the patch 60 may include more or fewer modules. The simplified detection steps include sequential binding of tagged probes 70, 72, 74, 76 to respective complementary modules 62, 64, 66, 68. The detection process works similarly to that shown in FIG. 5. However, the use of the patch 60 or noncontiguous identification oligonucleotide 16 provides additional manufacturing advantages and reductions in complexity over a single-stranded arrangement.

Table 3: Combinatorial Arrangements of a Patch

[00078] In the single-stranded arrangement, each possible individual identification oligonucleotide 16 is generated, and the total possible number of combinations equals the total number of generated oligonucleotides for full plexity. However, in a patch or noncontiguous arrangement, a much smaller number of these separate strands are generated, and separately applying the different modules to the surface in different combinations yields the full total possible combinations. Thus, rather than having to generate separate 65536 identification oligonucleotides 16 for full plexity in a four module/sixteen sequence arrangement, it is possible to generate the full set of 64 possible modules, representing each of the four module position and all sixteen modular sequence possibilities at each module, and apply a module having one of the sixteen modular sequences at each module position in the patch 60. In embodiments, certain modules can be added as batch steps for supramolecular structures carrying different entities 2 to further increase manufacturing efficiency. Thus, module 62 can be one of sixteen possibilities, module 64 can be one of sixteen possibilities, module 66 can be one of sixteen possibilities, and module 68 can be one of sixteen possibilities. To yield the full set of 65536, different combination of the 64 modular sequences (of the modules 62, 64, 66, 68) can be applied in different combinations. Thus, generating only 64 modules yields 65536 plexity. Further, the patch modules are shorter and cheaper to generate relative to a single strand that contains all of the modules. In an embodiment, the single-strand identification oligonucleotide 16 is between 80-150 bases, while in the patch embodiment the different modules are between 30-50 bases in length. In embodiments, the modules include linker sequences that couple to complementary sequences extending from the core structure 13. However, the modules may be covalently linked to the core structure 13. [00079] The core structure 13 can be tuned to couple to the modules at predetermined locations on the supramolecular structure 10. FIG. 7 shows a quadrant or region based coupling of the modules 62, 64, 66, 68 to respective linker complements 80, 82, 84, 88. Spatial specificity can be achieve by having different linker sequences at a terminus of each of the modules 62, 64, 66, 68 that hybridize to only one of the linker complements 80, 82, 84, 88. However, other arrangements are also contemplated. In one example, a single combinatorial strand identification oligonucleotide 16 can be bridged across linker complements and cut to yield two separate modules on the core structure 13.

[00080] The separate modular oligonucleotide approach can be paired with a coupling or docking strand 90. Rather than providing a patch that extends from a surface of the core structure, the different modules are indirectly linked to the core structure (and the binding site 8) via the intervening docking strand. FIG. 8 shows an example supramolecular structure 10 including the docking strand 90, which is a single-stranded universal oligonucleotide that is a linker to modules of a unique identification sequence. The docking strand 90 can be coupled to the core structure 13 via direct covalent linkage or via complementary binding of a base linker portion 94 of the docking strand to a base linker complement 92 that is coupled to the core structure 13. In an embodiment, the docking strand 90 is a common or universal strand between all different supramolecular structures. To assemble the identification sequence, different identification oligonucleotide modules are hybridized to complementary regions on the docking strand 90, as shown in FIG. 9. In the illustrated example, the identification oligonucleotides 16 include four oligonucleotides 100, 102, 104, 106. The identification oligonucleotides 16 include a module region 110 and a linker region 112. Thus, when assembled on the docking strand 90, the oligonucleotides 100, 102, 104, 106 are partially double-stranded and partially single-stranded. [00081] The single-stranded portion, the module region 110, can be hybridized to complementary tagged probes, as shown in FIG. 10. FIG. 10 shows the supramolecular structure of FIG. 9 having a hybridized tagged probe 120a complementary to the particular linked module 1 sequence. The hybridized tagged probe 120a is from a set of probes 120 that, in total, can hybridize to all possible module 1 sequences. In the depicted embodiment, there are four total tagged probes 120a, 120b, 120c, 120d. However, more or fewer are possible. Further, FIGS. 8-10 show a single binding site 8. At other binding sites 8, the other probes 120b, 120c, 120d may find complements. In an embodiment, across the substrate 9, several different binding sites are positive for binding of each of the different tagged probes 120a, 120b, 120c, 120d. While the module 1 probe binding is shown, it should be understood that modules 2, 3, and 4 can be detected using other sets of tagged probes and at different stages as generally disclosed herein. Further, characterization of the unique identification sequence can be based on the unique combination of detected probes at each binding site. In the depicted example, the binding site 8 is positive for binding of the tagged probe 120a, which is associated with (complementary to) a known modular sequence. Thus, positive binding detected for the tagged probe 12a determines a portion of the unique identification sequence, and the detected probes that bind to the modules 2, 3, and 4 are used to resolve the rest of the unique identification sequence.

[00082] FIG. 11 A shows an example supramolecular structure including a rolling circle amplification template that generates an amplification product including multiple copies of the unique identification sequence and that can be associated with the substrate of FIG. 1 according to embodiments of the disclosure. In FIG. 11A, the core structure 13, associated with an individual binding site 8, includes a leash 130 that retains a rolling circle amplification template 132 in association with the core structure. The rolling circle amplification template 132 is a singlestranded circular nucleic acid structure. In embodiments, the template 132v can be a knicked double-stranded template. The leash 130 may be formed from a single or double-stranded oligonucleotide or polymer. In an embodiment, the leash 130 is directly coupled to the core structure 13. In an embodiment, the leash 130 is indirectly coupled via one or more intervening linker molecules, e.g., via hybridization. For example, Fig. 1 IB shows a leash 130 that is a single or double-stranded circular nucleic acid coupled to a linker 137. The leash 130 retains the rolling circle amplification template 132, but permits free movement or rotation of the rolling circle amplification template 132 relative to the leash 130. Thus, amplification around the circular template is not impeded by the presence of the leash 130.

[00083] The rolling circle amplification template 132 at an individual binding site includes the unique identification sequences, shown here as formed by unique combination of complements to individual members of the sets of module 1, module 2, module 3, and module 4 by way of example. However, more or few module complements may be used, and each set for the each module may include two or more members. The number of modules and the size of each set determines the potential plexity, as provided herein. Each rolling circle amplification template 132 is unique, but may carry a universal primer binding region 136, such that amplification of all different templates 132 across the substrate can be achieved by a common primer 138. Using a rolling circle amplification polymerase with strand displacing activity, an amplification product 140 is created with multiple copies of each module sequence. Thus, the potential detectable signal is amplified based on the length of the amplification product 140. The amplification may create 10s, 100s, or 1000s of copies of each module sequence in the amplification product. Contacting the substrate including the amplification product 140 with tagged probes 142 as generally disclosed herein permits resolution of the different module sequences (e.g., which member of the set for each module is present) at each binding site 8.

[00084] While certain techniques disclosed herein are based on hybridization, the techniques in FIGS. 11 A-l IB also includes an amplification reaction to generate the amplification product. To prevent undesired amplification of other nucleic acid species present, such as nucleic acid core molecules in the core structure 13 that function as staples to provide the structural support, the 3’ ends of these nucleic acids may be modified (e.g., via 3'ddC, 3' Inverted dT, 3' C3 spacer, 3' Amino, and 3' phosphorylation) to prevent undesired 5’ to 3’ extension initiated off of the core structure molecules via the polymerase.

[00085] In certain embodiments discussed herein, one or more modules are detected by hybridization of a tagged probe. After a detection step, the tagged probe is removed, either to move on to another stage of module detection or to prepare the substrate for sample analysis by an end user. The removal of the tagged probes can be according to suitable known techniques, including exposure to high temperatures or to basic conditions. However, such conditions may cause degradation of the oligonucleotides and/or the entity. Further, for identification oligonucleotides 16 that are themselves linked by hybridization to the core structure 13, denaturing temperatures may cause undesired release of the identification oligonucleotides 16. Also provided herein are toehold-based displacement techniques for removing hybridized tagged probes that can be conducted in isothermal conditions. FIG. 12 shows an example workflow for toehold-based removal of tagged probes from a unique identification oligonucleotide 16 according to embodiments of the disclosure. The identification oligonucleotide 16 is contacted with a set of tagged probes 150, and the tagged probe 150a having a complementary sequence hybridizes to the complementary portion of the identification oligonucleotide 16. The unbound tagged probes are washed from the substrate 9, and the remaining bound tagged probe 150a is detected at the binding site 8. To remove the bound tagged probe 150a, a set of untagged stripping oligonucleotides 160 is provided. The bound tagged probe 150a includes a complementary region 152 and a toehold region 154 that is noncomplementary. Thus, when the tagged probe 150a is bound to the identification oligonucleotide 16, the toehold region 154 remains unbound and single-stranded. [00086] Contacting the bound tagged probe 150a with a particular stripping oligonucleotide 160a will displace the tagged probe 150a from the identification oligonucleotide 16. The stripping oligonucleotide 160a has higher binding specificity for the tagged probe 150a, because the stripping oligonucleotide 160a, like the identification oligonucleotide 16, includes the complement 162 to the complementary portion 152. Further, the stripping oligonucleotide 160a also includes a toehold complement 164 that is not present on the identification oligonucleotide 16. Thus, contacting the bound tagged probe 150a with the stripping oligonucleotide 160a displaces the tagged probe 150a from the identification oligonucleotide 16. The tagged probe 150a and the stripping oligonucleotide 160a can be released as a double-stranded structure in solution, and can be washed from the substrate 9, leaving the module on the identification oligonucleotide 16 in a single-stranded configuration. In an embodiment, the set of untagged stripping oligonucleotides 160 include a quencher that quenches a tag of the tagged probes 150 to decrease effects of an incomplete wash.

[00087] The set of untagged stripping oligonucleotides 160 can include additional oligonucleotides specific for other modular sequences at different binding sites 8. [00088] In embodiments, the tag is at one end of a tagged probe 150 and the toehold region 154 is at another end of the tagged probe 150. The tag or the toehold region can be at a 5’ end or a 3’ end of the tagged probe or, in embodiments, within the tagged probe 150. In an embodiment, the toehold region is 5-12 bases in length. In an embodiment, the complementary region 152 is at least twice as long as the toehold region 154.

FIG. 13 shows an example workflow for toehold-based displacement of a tagged probe 200 hybridized to a module 210 that is in turn hybridized to a docking oligonucleotide 90 according to embodiments of the disclosure. The tagged probe 200 is displaced by the stripping oligonucleotide 220 that includes a region complementary to a toehold on the tagged probe 200.

[00089] FIG. 14 shows an example of polymerase-based strand displacement. The displaced strand is a fluorescently labeled imager strand. The polymerase can be any strand displacing polymerase, such as phi29, Bst, Bst 2.0, etc. Unlike toehold-based strand displacement, polymerase-based strand displacement does not require the imager strand to have a toehold. That is, the displacement can occur without the presence of a toehold. In the illustrated image, a unique identification oligonucleotide 16 is bound to a tagged probe 250 based on complementarity, permitting identification via imaging of the tagged probe 250.

[00090] The identification oligonucleotide 16 is contacted with a set of tagged probes 250, and the tagged probe 250a having a complementary sequence hybridizes to the complementary portion of the identification oligonucleotide 16. The unbound tagged probes 250 are washed from the substrate, and the remaining bound tagged probe 250a is detected at the binding site 8, shown as being coupled to a supramolecular structure 10 in embodiments. To remove the tagged probe 250, a strand-displacing polymerase 260 is permitted to extend from a template 270 while bound to a different portion of the unique identification oligonucleotide 16. In an embodiment, the workflow may also include providing a set of different template extension oligos 270 designed for and to hybridize to the set of available unique identification oligonucleotide 16 and completing the extension, regardless of whether bound tagged probes 250 are present. Only bound probes 250a, or other bound probes 250 are detected and displaced. Extension via the polymerase 260 displaces the tagged probe 250 starting at the probe 5’ end. The displaced tagged probe can be washed away or otherwise separated from the unique identification oligonucleotide 16. The polymerase extension leaves a double-stranded region of the unique identification oligonucleotide 16 that protects that region from non-specific binding after the detection event. While the illustrated embodiment is discussed with respect to linear unique identification oligonucleotides 16, the polymerase-based displacement may be used in conjunction with or instead of toehold-based displacement and for more complex unique identification oligonucleotide structures (e.g., modular structures).

[00091] In embodiments, the tag is at a 3’ end of the tagged probe 250 to avoid removal during displacement.

[00092] FIG. 15 shows an example of a photocleavable tagged probe 300 that may, in embodiments, be removed via displacement or denaturation-based techniques. The tagged probe 300 is bound to the unique identification oligonucleotide 16, and the fluorophore 310 is attached to the tagged probe 300 via a photo labile group 320. Exposure of the system to the appropriate wavelength of light, e.g., in the UV range, would cleave the fluorophore 310 off of the tagged probe 300 to yield a cleaved fluorophore 310 and a bound but unlabeled probe 330. The unlabeled probe 330 can be washed away or otherwise separated from the unique identification oligonucleotide 16. The photolabile group 320 may include nitrobenzyl, nitrophenethyl compounds, and their dimethoxy derivatives (nitroveratryl). In an embodiment, the photo labile group is a benion group.

[00093] FIG. 16 shows an example of a chemically labile tagged probe 350 that may, in embodiments, be removed via displacement or denaturation-based techniques. The tagged probe 350 is bound to the unique identification oligonucleotide 16, and the fluorophore 310 is attached to the tagged probe 300 via a chemically labile group 360. Exposure of the system to a reducing agent, such as TCEP, would cleave the fluorophore 310 off of the tagged probe 350 to yield a cleaved fluorophore 310 and a bound but unlabeled probe 370. The unlabeled probe 370 can be washed away or otherwise separated from the unique identification oligonucleotide 16.

[00094] Embodiments of the present disclosure include one or more computer-implemented detection systems configured to perform certain methods of the disclosed embodiments. FIG. 17 shows a detection system 1000 that includes a controller 1001. The controller 1001 includes processor 1002 and a memory 1004 storing instructions configured to be executed by the processor 1002. The controller 1001 includes a user interface 1006 and communication circuitry 1008, e.g., to facilitate communication over the internet 1010 and/or over a wireless or wired network. The user interface 1006 facilitates user interaction with characterized detection results as provided herein. The detection may be detection of modular sequences in one or more identification oligonucleotides 16 and/or detection of analyte binding to the substrate 9 in embodiments. [00095] The processor 1002 is programmed to receive detection data and characterize the detected modules of the identification oligonucleotides 16. In one embodiment, the processor generates a report of detected modules after incubation of supram olecular structures 10 immobilized on a substrate 9 with tagged probes. The report may include data of generated optical signals at various binding sites that corresponds to a detected module at the binding site. The report may include processed data, such as a list of detected modules or positive/negative binding results.

[00096] The system 1000 also includes a detector 1020 that operates to detect analyte and/or tagged probe binding. The detector 1020 includes a detection system having one or more sensors 1022. The detector 1020 may also include a reaction controller 1024 that controls sample incubation and appropriate release of reaction reagents and detector molecule assemblies at appropriate time points. The sensor 1022 may be one or more of an optical sensor (e.g., a fluorescent sensor, an infrared sensor), an image sensor, an electrical sensor, or a magnetic sensor. In an embodiment, the sensor 1022 is a metal-oxide semiconductor image sensor device.

[00097] In an embodiment, the sensor 1022 is a sensor that can detect tags of tagged probes as provided herein. The tags may be detectable moieties such as quantum dots, fluorescent labels, fluorescent peptides, and the like. Suitable fluorescent labels include, but are not limited to, Pacific Blue, Pacific Orange, Alexa Fluor dyes (eg. Alexa 350, 430, 488, 555, 647, 700, and 750), fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, Cy7, LC Red 705 and Oregon green. Suitable fluorescent labels also include, but are not limited to, green fluorescent protein, enhanced yellow fluorescent protein, or luciferase. In certain embodiments, labels may include Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750), Pacific Blue, Pacific Orange, Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes) (Eugene, Oreg ), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). The tags may include metafluorophores, which are constructed as DNA nanostructure-based fluorescent probes with digitally tunable optical properties. Each metafluorophore is composed of multiple organic fluorophores, organized in a spatially controlled fashion in a compact sub-100-nm architecture using a DNA nanostructure scaffold. The tags may include nanostructures, such as DNA or RNA origamis as provided herein. In an embodiment, the tags may include self-assembled nanoantennas created by nanoparticles with overlapping plasmonic fields. The selected fluorescent labels may be based on the detection capabilities of the sensor 1022 and detection system 1020. In an embodiment, the fluorescent labels are coupled to associated structures, such as supramolecular structures. The tags may be coupled to probes, which can be single- stranded oligonucleotides that are complementary to sequences of interest. In an embodiment, an individual probe is complementary to only one module sequence. The tags may be coupled to probes via photocleavable or chemically labile groups.

[00098] As described herein, in some embodiments, one or more supramolecular structures 10 immobilized on a mapped substrate 9 enable the detection of one or more analyte molecules in a sample. FIG. 18 is a flow diagram of an analyte detection method 1100. Certain steps of the method 1100 refer to features discussed with respect to FIGS. 1-16. In addition, certain steps of the method 110 can be performed by the system 1000. The method 1100 initiates with providing a mapped substrate 9 (block 1102), prepared as generally discussed herein. The mapped substrate 9 can be a randomly ordered array having immobilized thereon different entities 2 at respective different binding sites 8. The substrate 9, after the immobilization of the entities 2, can be mapped by detecting hybridization of tagged probes to different modular sequences of identification oligonucleotides 16 to assign a particular entity identity with a particular binding site location. The assignments of the entities 2 to binding sites 8 can be provided as map information, that can be accessed by an end user of the substrate (block 1104) performing a sample detection assay.

[00099] The assay can be performed by contacting the substrate 9, including the immobilized supramolecular structures 10, with a sample (block 1106) that includes a plurality of different analyte molecules and/or that is suspected to include certain analytes. The analytes in the sample can be uncharacterized or unknown analytes. In embodiments, the sample may include one or more control analytes.

[000100] When the individual analyte molecule and individual entity 2 have binding specificity for one another, the analyte molecule associates with the entity 2 to form an individual analyte molecule-supramolecular structure complex that can be detected. The reaction conditions permit specific binding of the analyte molecules to specific entities 2. As provided herein, binding specificity may refer to an interaction between the analyte molecule and the entity 2 that remains intact under the reaction conditions and after washing or removal steps for unbound reagents. Binding specificity may include formation of a covalent or non-covalent bonds, ionic bonds, dipole interactions, hydrophilic or hydrophobic interactions, complementary nucleic acid binding, etc. Specific binding may refer to binding to an analyte molecule that binds only to a particular entity 2 and not to other entities 2 under the reaction conditions. Thus, certain entities 2 of the pool of supramolecular structures 10 bind to certain analyte molecules (e.g., binding between a first analyte molecule and a first entity 2 or binding between a second analyte molecule and a first entity 2). Certain entities 2 may have no available binding partners in a given sample and, therefore, do not bind to any analyte molecule with specificity.

[000101] Any unbound analytes can be removed after formation of the complexes before the complexes are provided to a detection system 1020, as provided herein. However, in other embodiments, no wash step is performed.

[000102] In some embodiments, the disclosed techniques provide single molecule capture of analyte molecules in a complex sample. Use of the supramolecular structure as the capture entity permits specific identification and, in embodiments, detection at binding sites. Thus, as provided herein, detectable analyte binding to an individual entity on the substrate generates assay results in which binding characteristics of an analyte pool of multiple different analytes are characterized. In an embodiment, the assay generates detection information indicative of positive binding events at locations on the mapped substrate 9 (block 1108). Using the map information, entities associated with the positive binding event locations can be identified (block 1110). Further, the identified entities representing positive binding events (e.g., that represent detected analyte binding) as well as negative binding events (e.g., entities at binding sites 8 with no detected binding) can be provided as a report or in a user interface to the end user.

[000103] The method 1100 permits a sample having an uncharacterized composition of analytes to be analyzed for the presence and/or concentration of particular analytes of interest. For example, a human sample can be characterized to determine a presence and/or concentration of antibodies with binding specificity to particular antigens in a panel of antigen entities, such that the entities represent a known infectious disease antigen panel. The assay results may show positive binding results associated with a particular antigen, which is indicative of the presence of antibodies in the subject providing the sample. In another embodiment, the identity of analytes in the sample may be at least partially known, but their binding affinity may not be characterized for a particular pool of entities. For example, the entities can be a set of candidate drugs, and the analytes can be molecules in human blood. Binding of a drug candidate to such a protein can be used to assess bioavailability or potential off-target binding. The assay results may show positive binding results associated with a particular drug candidate that can in turn be mapped to a particular analyte, which is based on a detectable binding event

[000104] Di sclosed embodiments relate to analyte detection in which the analytes are present in a sample, such as a biological sample. In some embodiments, the sample comprises an aqueous solution comprising protein, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combinations thereof. In some embodiments, the analyte molecules in the sample comprise protein, peptides, peptide fragments, lipids, DNA, RNA, organic molecules, inorganic molecules, complexes thereof, or any combinations thereof. In some embodiments, the analyte molecules comprise comprises intact proteins, denatured proteins, partially or fully degraded proteins, peptide fragments, denatured nucleic acids, degraded nucleic acid fragments, complexes thereof, or combinations thereof. In some embodiments, the sample is obtained from tissue, cells, the environment of tissues and/or cells, or combinations thereof. In some embodiments, the sample comprises tissue biopsy, blood, blood plasma, urine, saliva, a tear, cerebrospinal fluid, extracellular fluid, cultures cells, culture media, discarded tissue, plant matter, synthetic proteins, bacterial, viral samples, fungal tissue, or combinations thereof. In some embodiments, the sample is isolated from a primary source such as cells, tissue, bodily fluids (e.g., blood), environmental samples, or combinations thereof, with or without purification. In some embodiments, the cells are lysed using a mechanical process or other cell lysis methods (e.g., lysis buffer). In some embodiments, the sample is filtered using a mechanical process (e.g., centrifugation), micron filtration, chromatography columns, other filtration methods, or combinations thereof. In some embodiments, the sample is treated with one or more enzymes to remove one or more nucleic acids or one or more proteins. In some embodiments, the sample comprises intact proteins, denatured proteins, partially or fully degraded proteins, peptide fragments, denatured nucleic acids or degraded nucleic acid fragments. In some embodiments, the sample is collected from one or more individual persons, one or more animals, one or more plants, or combinations thereof. In some embodiments, the sample is collected from an individual person, animal and/or plant having a disease or disorder that comprises an infectious disease, an immune disorder, a cancer, a genetic disease, a degenerative disease, a lifestyle disease, an injury, a rare disease, an age-related disease, or combinations thereof. [000105] The supramolecular structures 10 at individual binding sites 8 can be detected via interaction with analytes that generate a detectable signal upon binding to the entities 2. For example, the analytes may include a signaling element that is optically active and can be measured using a microscope or integrated optical sensor within the substrate 9. In some embodiments, the signaling element is electrically active and may be measured using an integrated electrical sensor. In some embodiments, the signaling element is magnetically active and may be measured using an integrated magnetic sensor. In some embodiments, each signal event is associated with the capture of the same type of analyte molecule (a single copy of the same type of analyte molecule), determined by the corresponding detector and entity, thus counting the number of locations where the signaling element is present gives the quantification of the analyte molecule in the sample. In one embodiment, binding of the analyte to the supramolecular structure 10 causes a detectable configuration change. In one embodiment, binding of the analyte to the supramolecular structure 10 permits binding of additional structures that are detectable by the system 1000. When the analyte is not bound, the additional structures do not bind and, thus, are not detected.

[000106] In some embodiments, the supramolecular structure 10 converts information about the presence of a given analyte molecule in a sample to a DNA signal. In some embodiments, detecting the presence of an analyte molecule, as described herein, comprises controllably releasing a single, or multiple, unique nucleic acid molecules into the solution to be used to identify as well as quantify properties of the analyte molecule from the sample.

[000107] As provided herein, the identification oligonucleotide 16 can be used to uniquely identify individual supramolecular structures 10 and the entity 2. The locations of each entity 2 can stored in a lookup table of the system 1000.

[000108] While certain embodiments of the disclosure have been discussed with respect to supramolecular structures 10, the disclosed embodiments may also be implemented in other contexts to map a randomly ordered substrate or array. For example, other types of linking structures may be provided that physically link a particular entity 2 to particular one or more identification oligonucleotides 16 such that the entity 2 and one or more identification oligonucleotides 16 are co-located at a binding site 8 when randomly immobilized on a substrate 9. The disclosed embodiments may be used to detect identification oligonucleotides 16 having modular sequences and that may or may not include or be associated with supramolecular structures 10. [000109] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS What is claimed is:

1. A method for characterizing entities on a substrate, the method comprising: providing a substrate comprising a plurality of binding sites; contacting the substrate with a pool of supramolecular structures to immobilize the supramolecular structures on respective binding sites of the plurality of binding sites, an individual supramolecular structure of the pool comprising: a core structure comprising a plurality of core molecules; an entity linked to the core structure, wherein different supramolecular structures of the pool comprise different entities having respective different binding specificities; and one or more identification oligonucleotides linked to the core structure, wherein the one or more identification oligonucleotides comprise a unique identification sequence, the unique identification sequence being formed from a plurality of modular sequences selected from a set of modular sequences, wherein the unique identification sequence comprises a unique combination of the modular sequences, and wherein the one or more identification oligonucleotides are unique to each supramolecular structure; contacting the substrate with tagged probes complementary to the plurality of modular sequences; detecting complementary binding of the tagged probes to the one or more identification oligonucleotides of the immobilized supramolecular structures; and associating a location of a binding site on the substrate with an individual entity based on the detected complementary binding.

2. The method of claim 1, wherein the one or more identification oligonucleotides comprise a single-stranded DNA or RNA.

3. The method of claim 1, wherein the one or more identification oligonucleotides comprise a plurality of single-stranded oligonucleotides, each single-stranded oligonucleotide comprising one of the plurality of modular sequences.

4. The method of claim 3, wherein the plurality of single-stranded oligonucleotides are distributed in different regions of the individual supramolecular structure.

5. The method of claim 1, wherein the one or more identification oligonucleotides are coupled to the individual supramolecular structure via one or more nucleic acid linkers.

6. The method of claim 5, wherein the one or more nucleic acid linkers comprises a linker oligonucleotide having a plurality of linker sequences, wherein each linker sequence is hybridized to respective different identification oligonucleotides of the one or more identification oligonucleotides, the one or more identification oligonucleotides comprising a linker complement portion and a single-stranded portion.

7. The method of claim 1, wherein the individual supramolecular structure is planar.

8. The method of claim 1, wherein the one or more identification oligonucleotides comprise an oligonucleotide between 50-150 bases in length.

9. The method of claim 1, wherein the one or more identification oligonucleotides comprise four or more modular sequences that form the unique identification sequence.

10. The method of claim 9, wherein each modular sequence is at least 15 bases long.

11. The method of claim 1, wherein the entity is an antibody.

12. The method of claim 1, wherein the pool of supramolecular structures are randomly immobilized at the plurality of binding sites.

13. The method of claim 1, wherein no more than one supramolecular structure of the pool is immobilized at a single binding site of the plurality of binding sites.

14. The method of claim 1, wherein the plurality of modular sequences comprises a subset of the set of modular sequences.

15. The method of claim 1, wherein the modular sequences of the set of modular sequences can generate at least 250 unique combinations.

16. The method of claim 1, wherein each modular sequence of the set of modular sequences is present in at least one identification oligonucleotide immobilized on the substrate.

17. The method of claim 1, comprising degrading or removing the core structure before the contacting.

18. The method of claim 1, wherein the core structure comprises a nucleic acid origami.

19. The method of claim 1, wherein the core structure comprises a protein origami.

20. A method for characterizing entities on a substrate, the method comprising: providing a substrate comprising a plurality of binding sites having randomly immobilized therein respective supramolecular structures, an individual supramolecular structure comprising: a core structure comprising a plurality of core molecules; an entity linked to the core structure, wherein different supramolecular structures of the supramolecular structures comprise different entities having respective different binding specificities; and one or more identification oligonucleotides linked to the core structure, wherein the one or more identification oligonucleotides comprise a unique identification sequence, the unique identification sequence being formed from a plurality of modular sequences, wherein an individual modular sequence of the plurality of modular sequences is selected from a set of modular sequences, wherein the unique identification sequence comprises a unique combination of the modular sequences, and wherein the one or more identification oligonucleotides are unique to each supramolecular structure; contacting the substrate with a first set of tagged probes complementary to a first subset of the plurality of modular sequences; detecting complementary binding of the first set of tagged probes to the one or more identification oligonucleotides of the immobilized supramolecular structures; removing the bound first set of tagged tagged probes from the substrate; contacting the substrate with a second set of tagged probes complementary to a second subset of the plurality of modular sequences; detecting complementary binding of the second set of tagged probes to the one or more identification oligonucleotides of the immobilized supramolecular structures; and mapping the plurality of binding sites to respective entities based on the detected complementary binding of the first set and the second set, wherein the entities of the supramolecular structures have different binding specificity relative to one another and wherein the mapping comprises associating a location of an individual binding site with an identity of an entity of the entities.

21. The method of claim 20, wherein the mapping comprises determining that the location comprises a first modular sequence and a second modular sequence of the unique identification sequence based on the complementary binding and accessing the identity of the entity associated with the unique identification sequence comprising the first modular sequence and the second modular sequence.

22. The method of claim 20, comprising contacting the substrate with a third set of tagged probes complementary to a third subset of the plurality of modular sequences and a fourth set of tagged probes complementary to a fourth subset of the plurality of modular sequences.

23. The method of claim 22, wherein contacting the substrate with the first set, second set, third set, and fourth set of tagged probes occurs at different times.

24. The method of claim 20, wherein the first set of modular sequences is different from and nonoverlapping with the second set of modular sequences.

25. The method of claim 20, wherein the first set of modular sequences comprises at least four different sequences that are at least 15 bases long.

26. The method of claim 25, wherein the second set of modular sequences comprises at least four different sequences that are at least 15 bases long.

27. The method of claim 20, wherein removing the first set of tagged probes comprises contacting the substrate with stripping oligonucleotides complementary to the first set of tagged probes or displacing the first set of tagged probes using a strand-displacing polymerase.

28. The method of claim 27, wherein each of the stripping oligonucleotides comprises a quencher.

29. The method of claim 27, wherein each of the stripping oligonucleotides does not comprise a tag.

30. The method of claim 27, wherein a tagged probe of the first set of tagged probes comprises a first region complementary to only one modular sequence of the first subset of modular sequences, and a second region that is noncomplementary to the only one modular sequence such that, when the tagged probe is bound to the only one modular sequence, the second region forms a terminal toehold.

31. The method of claim 20, wherein a stripping oligonucleotide of the stripping oligonucleotides is complementary to both the first region and the second region.

32. The method of claim 20, comprising providing mapping information based on the mapping to a purchaser of the substrate.

33. The method of claim 20, comprising storing mapping information based on the mapping.

34. A randomly ordered array, comprising: a substrate comprising a plurality of binding sites; a plurality of supramol ecul ar structures immobilized on the substrate such that a binding site of the plurality of binding sites comprises no more than one supram olecular structure of the plurality of supram olecular structures, and wherein an individual supramolecular structure of the plurality of supramolecular structures comprises: a core structure comprising a plurality of core molecules; an entity linked to the core structure, wherein different supramolecular structures comprise different entities having respective different binding specificities; and one or more identification oligonucleotides linked to the core structure, wherein the one or more identification oligonucleotides comprise a unique identification sequence, the unique identification sequence being formed from a plurality of modular sequences selected from a set of modular sequences, wherein the unique identification sequence comprises a unique combination of the modular sequences, and wherein the one or more identification oligonucleotides are unique to the one supramolecular structure; and wherein unique identification sequences of identification oligonucleotides of the plurality of supramolecular structures present on the substrate represent a majority of possible combinations of the set of modular sequences.

35. The randomly ordered array of claim 34, wherein entities of the plurality of supramolecular structures present on the substrate have different binding specificities relative to one another.

36. The randomly ordered array of claim 34, wherein the one or more identification oligonucleotides comprise a single-stranded DNA or RNA.

37. The randomly ordered array of claim 34, wherein the one or more identification oligonucleotides comprise a plurality of single-stranded oligonucleotides, each single-stranded oligonucleotide comprising one of the plurality of modular sequences.

38. The randomly ordered array of claim 34, wherein the plurality of single-stranded oligonucleotides are distributed in different regions of the individual supramolecular structure.

39. The randomly ordered array of claim 34, wherein the one or more identification oligonucleotides are coupled to the individual supramolecular structure via one or more nucleic acid linkers.

40. The randomly ordered array of claim 39, wherein the one or more nucleic acid linkers comprises a linker oligonucleotide having a plurality of linker sequences, wherein each linker sequence is hybridized to respective different identification oligonucleotides of the one or more identification oligonucleotides, the one or more one or more identification oligonucleotides comprising a linker complement portion and a single-stranded portion.

41. The randomly ordered array of claim 40, wherein the linker oligonucleotide extends away from the core structure.

42. The randomly ordered array of claim 40, wherein the linker oligonucleotide is coupled to the core structure via complementary binding to a core structure oligonucleotide.

43. The randomly ordered array of claim 34, wherein the one or more identification oligonucleotides comprise an oligonucleotide between 50-150 bases in length.

44. The randomly ordered array of claim 34, wherein the one or more identification oligonucleotides comprise four or more modular sequences that form the unique identification sequence.

45. The randomly ordered array of claim 34, further comprising a set of tagged probes complementary to only a subset of the modular sequences.

46. The randomly ordered array of claim 45, wherein each supramolecular structure comprises only one tagged probe of the set of tagged probes, wherein the only one tagged probe is hybridized to a modular sequence of the subset.

47. The randomly ordered array of claim 43, wherein the only one tagged probe comprises a noncomplementary terminal region that is not hybridized to the modular sequence of the subset.

48. The randomly ordered array of claim 47, wherein the subset comprises at least four different modular sequences, and wherein the set of tagged probes comprises different probes complementary to only one of each of the four different modular sequences.

49. The randomly ordered array of claim 34, wherein the set of the modular sequences comprises a plurality of nonoverlapping subsets, and wherein each individual unique identification sequence of the substrate comprises only one modular sequence selected from each of the plurality of nonoverlapping subsets to form the unique combination.

50. A detection system, comprising: a substrate comprising a plurality of binding sites; a plurality of supramol ecul ar structures immobilized on the substrate such that a binding site of the plurality of binding sites comprises no more than one supram olecular structure of the plurality of supramolecular structures, and wherein an individual supramolecular structure of the plurality of supramolecular structures comprises: a core structure comprising a plurality of core molecules; an entity linked to the core structure, wherein different supramolecular structures comprise different entities having respective different binding specificities; and one or more identification oligonucleotides linked to the core structure, wherein the one or more identification oligonucleotides comprise a unique identification sequence, the unique identification sequence being formed from a plurality of modular sequences selected from a set of modular sequences, wherein the unique identification sequence comprises a unique combination of the modular sequences, and wherein the one or more identification oligonucleotides are unique to the one supramolecular structure; and wherein unique identification sequences of identification oligonucleotides of the plurality of supramolecular structures present on the substrate represent a majority of possible combinations of the set of modular sequences; a sensor that detects complementary binding of tagged probes to the modular sequences of the one or more identification oligonucleotides at the plurality of binding sites; and a controller that operates to: receive data from the sensor; determine unique identification sequences of the plurality of supramolecular structures at the plurality of binding sites based on the data; access identities of entities associated with the unique identification sequences; and generate a map of the identified entities at locations of the plurality of binding sites on the substrate.

51. The detection system of claim 50, wherein the controller operates to receive user input of selected entities of the plurality of supramolecular structures and perform a quality check of the unique identification sequences based on the received user input.

52. The detection system of claim 50, wherein the controller operates to communicate the map to a purchase of the substrate.

53. A plurality of supramolecular structures, wherein an individual supram olecul ar structure of the plurality of supramolecular structures comprises: a core structure comprising a plurality of core molecules; an entity linked to the core structure, wherein different supramolecular structures comprise different entities having respective different binding specificities; and one or more identification oligonucleotides linked to the core structure, wherein the one or more identification oligonucleotides comprise a unique identification sequence, the unique identification sequence being formed from a plurality of modular sequences selected from a set of modular sequences, wherein the unique identification sequence comprises a unique combination of the modular sequences, and wherein the one or more identification oligonucleotides are unique to the one supramolecular structure; and wherein unique identification sequences of identification oligonucleotides present in the plurality of supramolecular structures represent a majority of possible combinations of the set of modular sequences.

54. The plurality of supramolecular structures of claim 53, wherein the plurality of supramolecular structures is pooled in solution.

55. An array, comprising: a substrate comprising a plurality of binding sites, wherein each binding site comprises: an entity immobilized thereon, wherein different binding sites of the plurality of binding sites comprise different entities having respective different binding specificities; and a plurality of separate identification oligonucleotides, each separate identification oligonucleotide comprising only a portion of a unique identification sequence, wherein each identification oligonucleotide comprises a module having a modular sequence, wherein the unique identification sequence comprises a unique combination of the modular sequences on the separate identification oligonucleotides, and wherein the unique identification sequence is associated with the entity; and wherein unique identification sequences of identification oligonucleotides of the plurality of supramolecular structures present on the substrate represent a majority of possible combinations of the set of modular sequences.

56. The array of claim 55, wherein each binding site comprises four or more separate identification oligonucleotides.

57. The array of claim 55, wherein each identification oligonucleotide of the plurality is less than 50 bases long.

58. The array of claim 55, wherein the module of each identification oligonucleotide is selected from a fixed set of at least four modules comprising different modular sequences, and wherein the module of each identification oligonucleotide is selected from a different fixed set.

59. The array of claim 55, further comprising a set of tagged probes, wherein a number of different tagged probes is equal to a total number of modules represented on the substrate.

60. The array of claim 59, wherein the tagged probes are tagged with a chemically labile or photocleavable tag.

61. The array of claim 55, wherein the entity and the plurality of separate identification oligonucleotides are physically linked by a core structure comprising a plurality of core molecules at each binding site.

62. The array of claim 55, wherein the entity and the plurality of separate identification oligonucleotides are physically linked by a bead at each binding site.

63. A method for characterizing entities on a substrate, the method comprising: providing a substrate comprising a plurality of binding sites, wherein each binding site comprises: an entity; and a circular single-stranded nucleic acid template comprising a common primer binding region and a complement of a unique identification sequence, the unique identification sequence being formed from a plurality of modular sequences selected from a set of modular sequences, wherein the unique identification sequence comprises a unique combination of the modular sequences, and wherein the one or more identification oligonucleotides are unique to each entity; amplifying the circular single-stranded nucleic acid template at each binding site using a common primer to generate a rolling circle amplification product comprising multiple copies of the unique identification sequence; contacting the substrate with tagged probes complementary to the plurality of modular sequences; detecting complementary binding of the tagged probes to portions of the rolling circle amplification product; and associating a location of a binding site on the substrate with an individual entity based on the detected complementary binding.

64. The method of claim 63, wherein each binding site comprises a core structure, and wherein the circular single-stranded nucleic acid template is coupled to the core structure via a leash.

65. The method of claim 64, wherein the circular single-stranded nucleic acid template and the leash are coupled to permit rotation of the circular single-stranded nucleic acid template relative to the leash.

66. The method of claim 63, wherein each binding site comprises a bead, and wherein the circular single-stranded nucleic acid template is coupled to the bead via a leash.