WO2024261215A1 - High throughput screens of translation and stability of mrna using barcoded xrna display and its variations - Google Patents

Abstract

Suggested is a xRNA-display construct comprising, consisting or essentially consisting of: (a) at least one xRNA molecule comprising an amino-acid polymer encoding sequence; (b) a nucleic acid molecule functioning as a distinct barcode capable of reconstructing the potential molecular composition of said at least one xRNA molecule; and (c) an aminoacylated tRNA mimic; wherein said xRNA molecule, said nucleic acid molecule and said aminoacylated tRNA are connected through a chemical brancher.

Description

High throughput screens of translation and stability of mRNA using barcoded xRNA display and its variations

PRIORITY CLAIM

[0001] The present application claims priority of US provisional application 63522482 dated June 22, 2023.

AREA OF THE INVENTION

[0002] The present invention belongs to the area of mRNA therapeutics and specifically pertains to methods and compounds for evaluating chemical modifications and RNA genetic elements in mRNA molecules. More specifically, the invention encompasses a high-throughput approach, referred to as xRNA display, to decode the structure-activity relationship (SAR) principles governing the translatability and stability of mRNA molecules with improved therapeutic properties.

TECHNOLOGICAL BACKGROUND

[0003] mRNA therapeutics is an emerging therapeutics modality that has gained significant attention in recent years. The therapeutic effect of this modality relies on the ability to translate the mRNA into the desired therapeutic proteins or peptides. To enhance therapeutic outcome, it is crucial to maximize mRNA stability and translatability. Stable mRNA molecules persist longer within cells, thereby providing more opportunities for the ribosomes to translate them into protein. Furthermore, the translatability of mRNA - the efficiency with which mRNA is translated into protein - is another significant factor in the design of mRNA therapeutics. mRNA molecules with high translatability yield larger amounts of therapeutic protein per mRNA molecule, which is essential for maximizing the therapeutic benefit while reducing dosage requirements.

[0004] Therefore, the development of methods for understanding and manipulating the stability and translatability of mRNA could dramatically improve the design and effectiveness of mRNA therapeutics. However, the field has yet to decode the structure-activity relationship (SAR) of various chemical building blocks and their composition into mRNA elements on one hand to the translatability and stability of the molecule on the other hand.

[0005] The problem is exacerbated by the sheer combinatorial space of possible mRNA molecules. There are on average 3.2 codons to encode each amino acid, meaning that an mRNA that encodes a peptide with 30 amino acids can have over 10¹⁵ options just its coding region. Each of these changes can modulate the stability and translatability of the mRNA molecule. In addition, there is a massive number of possible cap structures, 5'UTRs, 3'UTRs, and tail lengths and compositions. Each instance of these elements can have an effect on the ability to produce the therapeutic protein and modulate the half-life of the mRNA. On top of that, therapeutic mRNA can utilize non-standard building blocks beyond the four standard occurring nucleotides. These can include position-dependent modifications to the nucleobase such as m6-adenine, pseudouridine and N1 -methyl-pseudouridine, modifications to the sugar ring, such as 2'-F, 2'-0Me, and 2'-H, modifications to the phosphate backbone, such as phosphorothioate, phosphorothioate, and peptidyl links, introducing various 5'-cap structures, and incorporating non-natural elements such as fluorophores, morpholino based nucleotides, biotinylation, DNA bases, and fatty acid chains. Each one of these changes and their exact position can change the translatability of the mRNA molecule and its stability.

[0006] Efforts to decode the SAR principles governing mRNA molecules have been hampered by the lack of high-throughput methodologies capable of evaluating the effects of chemical modifications, genetic element compositions, and amino acid sequences on translatability and stability. Conventional methods have proven to be time-consuming, labor-intensive, and insufficient to comprehensively analyze the vast array of possible mRNA variants.

RELEVANT STATE OF THE ART

[1] Sample, Paul J., et al. "Human 5' UTR design and variant effect prediction from a massively parallel translation assay." Nature biotechnology 37.7 (2019): 803-809.

[2] R. Lorenz et al. (2011), "ViennaRNA Package 2.0", Algorithms for Molecular Biology, 6:26.

[3] I.L. Hofacker (1994), "Fast folding and comparison of RNA secondary structures’ , Monatshefte fur Chemie, Volume 125, Issue 2, pp 167-188

[4] P. Oikonomou et. Al. (2020), "In vivo mRNA display enables large-scale proteomics by next generation sequencing" https://doi.org/10.1073/pnas.200265Q117

[5] EP 4114943 A1 (COLUMBIA UNIV) describes a method for producing in vivo mRNA displayed proteins by linking of in vivo expressed proteins to in vivo expressed RNA sequences that enable specific downstream identification of the individual proteins through nucleic acid-based sequencing. RNA-protein linkage is enabled by fusing an RNA-binding domain (e.g., MS2 coat protein) to a protein of interest that is expressed in a cell or compartment in which is also expressed the RNA sequence harboring both the recognition element for the RNA-binding domain (e.g., MS2 stem-loop) and an identifying sequence that uniquely maps to the protein of interest. The identifying sequence can be the coding sequence corresponding to the protein of interest or any RNA barcode that by design uniquely corresponds to the protein of interest. As such, libraries of such in vivo mRNA displayed proteins can be assayed in parallel for a variety of protein behaviors and functions.

PROBLEM TO BE SOLVED

[0007] mRNA display is a molecular strategy to screen large peptide libraries. This technique relies on in-vitro translation of an mRNA library into a peptide library, with each peptide molecule physically linked to the mRNA that encodes it. This covalent linking is mediated by introducing a puromycin moiety to the 3' end of each mRNA molecule. As the ribosome stalls at the end of the coding sequence, the puromycin can intercalate the ribosome, creating a peptide bond with the last amino acid, and connecting the peptide to the mRNA. Next, the library is incubated with a protein of interest. The library is thoroughly washed to remove non -specific interactions and the RNA of the bound peptides can be amplified or sequenced. As such, this technology is as an excellent tool for identifying high-affinity peptides for proteins. The inventors took a radically different approach. In our approach, the mRNA display is not used to screen peptides. Rather, the aim of the current invention is to screen chemical modifications of mRNA therapeutic molecules, a distinct modality, by meticulously reengineering the basic components of mRNA display.

[0008] However, comparing the translation efficiency of a large set of mRNAs remains still a serious problem. In unconventional mRNA display, the sole purpose of the technology is to evaluate peptide binders, whereas the mRNA itself serves as a construct that is translated in in- vitro cell extracts to produce the peptide. Therefore, the inventors sought to develop a technology that can monitor the translation and stability of mRNA molecules. As artificial cell extracts do not necessarily reflect in cell translation and may lead to results that do not represent in cell translation, the inventors intended developing an in-vivo mRNA display translation, where the peptide is produced inside cells. Therefore, one task of the present invention is resolving this issue and allowing monitoring and quantifying translation efficiency inside the natural cellular environment.

[0009] Another problem refers to the comparison of the translation capacity of a large set of chemically-modified mRNAs. Chemical modifications of mRNA are currently essential for the stability and function of multiple RNA based therapies. However, the polymeric nature of mRNA creates an immense searching space: Each mRNA can be differently modified in x ways, where x = [number of nucleotides] [^{number of wa}y^{s to mod|f}y^{a nuc|eot|de}], mRNA display can be adapted to monitor translation efficiency of large numbers of different mRNAs, however, when it comes to chemical modifications - the nature of the modifications is lost during the process (specifically - when the mRNA is converted to cDNA). To solve this problem, the inventors intended to develop novel mRNA display constructs that retain the information encoding the mRNA modification pattern in a distinct, non-chemically modified DNA barcode. [0010] Finally, the previously described in vivo mRNA display relies on non-covalent linkage between an RNA of a certain structure and an RNA binding domain. This approach has three major disadvantages: any screened RNA should encode these two elements; a non-covalent linkage between the structural RNA element and the RNA binding domain results in high false negatives, where this interaction breaks during processing; and trans-interactions (intramolecular) between a structural RNA element, and an RNA binding domain that was transcribed by a different element leads to false positives.

The covalent linkage approach wherein the peptide and the mRNA are linked covalently overcome these three limitations and therefore resulting in a superior and more reliable assay.

BRIEF DESCRIPTION OF THE INVENTION

[0011] A first object of the present invention refers to a first xRNA-display construct comprising, consisting or essentially consisting of:

(a 1) at least one xRNA molecule comprising an amino-acid polymer encoding sequence;

(bl) a nucleic acid molecule functioning as a distinct barcode capable of reconstructing the potential molecular composition of said at least one xRNA molecule; and

(cl) an aminoacylated tRNA mimic; wherein said xRNA molecule, said nucleic acid molecule and said aminoacylated tRNA are connected through a chemical brancher.

[0012] A second object of the present invention refers to a second xRNA-display construct comprising, consisting or essentially consisting of:

(a2) an xRNA molecule comprising an amino-acid polymer encoding sequence;

(b2) a first nucleic acid molecule and a second nucleic acid molecule;

(c2) an aminoacylated tRNA mimic; and

(d2) a ribosomal stalling sequence; and wherein the 3' of first nucleic acid is covalently linked to the 5' of the xRNA molecule, the 3' of the xRNA molecule is covalently linked to the 5' of the ribosomal stalling sequence and the 3' of the ribosomal stalling sequence is covalently linked to the aminoacylated tRNA mimic; and wherein the first and second nucleic acid molecules are functioning as a distinct untranslated barcode that identifies the profile of the xRNA molecule. [0013] A third object of the present invention refers to a third xRNA-display construct comprising, consisting or essentially consisting of:

(a3) an xRNA molecule comprising an amino-acid polymer encoding sequence;

(c3) an aminoacylated tRNA mimic; and

(d3) a ribosomal stalling sequence; wherein the xRNA molecule comprises a unique watermark signature inside or outside the encoding sequence functioning as a distinct barcode that identifies the profile of the xRNA molecule; and wherein the 3' end of the xRNA molecule is covalently linked to the 5' of the ribosomal stalling sequence and the 3' end of the ribosomal stalling sequence is linked to the aminoacylated tRNA mimic.

[0014] Surprisingly, the present invention addresses the aforementioned by introducing the xRNA display platform. This system enables the high-throughput identification and evaluation of chemical modification, RNA genetic elements, and encoded amino acids sequences in mRNA molecules. Through a comprehensive analysis of these factors, the xRNA display platform facilitates the rational design of mRNA molecules with enhanced therapeutic properties. By encoding for the mRNA chemical modifications signature/identity on a separate (but covalently attached) DNA barcode, the technology described herein enables to retain the chemical signature information in a way that allows for high throughput measurements via deep sequencing.

[0015] According to some embodiments, provided herein are system, platform, reagents, compounds, and method for high throughput identification and evaluation of chemical modifications, usage of RNA genetic elements, and encoded amino-acid sequences to decode the SAR principles of mRNA molecules. According to some embodiments, provided herein a system (also referred to herein as "xRNA display") enabling a massively parallel method of evaluating and predicting the effect of chemical modifications, genetic element composition, and amino-acid sequences on the duration of the effect of mRNA molecules and their translatability. xRNA display lays out a platform to generate, assay, and analyse massive amounts of data in order to rationally design mRNA molecules with improved therapeutic properties.

[0016] The present invention utilizes the xRNA display technique, a molecular strategy for creating large peptide libraries, to evaluate the effect of chemical modifications of oligonucleotide molecules. This method involves a library of mRNA molecules that encompass various chemical modifications, genetic elements, and/or amino acid sequences in specific positions, compositions, and patterns, collectively referred to as variants. [0017] Each molecule in the library encodes the identity of the variants it carries, enabling their recovery using high throughput genomics methods such as nucleic acid sequencing or array hybridization. The encoded information is stored in one or more specific regions collectively called the barcode. Additionally, each molecule contains a special moiety that facilitates covalent linking between the mRNA molecule and its translated peptide.

[0018] To initiate the evaluation process, the initial library, referred to as tO, is subjected to translation for a desired period termed t1. Following translation, the t1 library is separated into at least two fractions. One fraction consists of xRNA molecules that have successfully conjugated to a peptide, while the second fraction comprises xRNA molecules that have not formed a connection with a peptide. Optionally, additional fractions can be isolated based on the size of the created peptides, the size of the xRNA, and/or the presence of a 5' cap.

[0019] Each fraction obtained from the t1 library, along with the tO library, undergoes a distinct reaction to extract the barcode information. In some cases, the t1 library before fractionation is also subjected to barcode extraction. By analyzing the distribution of barcodes in the t1 fractions in comparison to tO, and optionally the t1 fraction prior to fractionation, the inventors can determine the translatability and stability of the molecules.

Definitions

[0020] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0021] As used herein, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "nucleic acid" may also include a plurality of nucleic acids.

[0022] The term "barcode" refers to one or more nucleic acid sequences, typically short and collectively consisting of about 5 to 50 bases, which are utilized to faithfully reconstruct the entire molecular composition of one or more mRNA molecules. These nucleic acid barcode sequences can be attached to target mRNA molecules of interest or their amplification products. Barcodes may exist as single or double-stranded nucleic acids and can be chemically attached to mRNA molecules, engage in electrostatic interactions with the mRNA molecules, or be enclosed within the mRNA molecules. Each barcode sequence enables the identification of a specific mRNA molecule.

[0023] The terms "polynucleotide" and "nucleic acid" are used herein interchangeably. They refer to a polymeric form of nucleotides of any length. Polynucleotides may have any three- dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, synthetic polynucleotides, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified, such as by conjugation with a labeling component. Polynucleotides depicted herein are in 5' to 3' direction unless otherwise stated.

[0024] |f not stated otherwise, the term "nucleic acid" refers to any nucleic acid such as ribonucleic acid, deoxyribonucleic acid, xeno nucleic acid, single stranded or double stranded. [0025] The terms "codon" or "triplet" refers to a sequence of three nucleotides. A codon may encode an amino acid in a defined reading frame of a polynucleotide.

[0026] The terms "polypeptide", "peptide", and "protein" relate to oligo- and polypeptides and refers to substances which comprise two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more consecutive amino acids linked to one another via peptide bonds. The term "protein" refers to large peptides, preferably peptides having at least 151 amino acids, but the terms "(poly)peptide" and "protein" are used herein usually as synonyms. Polypeptides may further comprise according to the invention substances which contain not only amino acid components but also non-amino acid components such as sugars and phosphate structures, as well as substances containing bonds such as ester, thioether or disulfide bonds. According to the present invention, a nucleic acid such as RNA may encode a polypeptide or protein. Accordingly, a transcribable polynucleotide or a transcript thereof such as mRNAs may contain an open reading frame (ORF) encoding a polypeptide. Said polynucleotide may express the encoded polypeptide or protein. For example, said polynucleotide may be a nucleic acid encoding a therapeutical polypeptide.

[0027] The tables, text, and figures provided below may refer to numerous polynucleotide modification structures that comprise a natural or unnatural polynucleotide. These structures are shown in 5’ to 3’ orientation and may be prepared by phosphoramidite synthesis, and are commercially available from custom oligonucleotide vendors such as Integrated DNA Technologies (Coral ville, Iowa, USA), or GeneLink (Orlando, Florida, USA). There are numerous of non-standard phosphoramidite monomer unit "building blocks" published and commercially available from custom polynucleotide vendors that can be easily incorporated into custom synthesized polynucleotides. Many of these non-standard monomer units are classified as spacers (e.g., "iSp"), and affinity tags (e.g., "[Bio-dT]"). All polynucleotide modification structures in the description below are described using well-known polynucleotide synthesis nomenclature to indicate the non-standard monomer units. (See e.g., the web-site of Integrated DNA Technologies (IDT) at https://eu.idtdna.com/site/catalog/Modifications/GetAIIMods, or GenLink at http://www.genelink.com/newsite/products/OligoModifications.asp for further details of commonly used oligonucleotide nomenclature.) For example, non-standard monomer units are enclosed in forward slashes ("/") or in brackets ("["and "]"), and an asterisk "*" between units indicates a phosphorothioate linkage. xRNA molecules

[0028] in a first preferred embodiment of the present invention the xRNA-display construct contains an xRNA molecule (components a) which is a nucleic acid polymer that is made of any combination of the following monomers: RNA or chemically modified RNA nucleotides (for example sugar and/or backbone modifications), DNA or chemically modified DNA nucleotides, chemical spacers, linkers, chemical doublers, branchers, conjugates (for example - puromycin), XNA, or combination thereof. Said display may further comprise polypeptides which are encoded by the xRNA molecule, wherein the polypeptide is preferably conjugated via the aminoacylated tRNA mimic. The xRNA-display construct may also contain a unique watermark signature, said signature representing preferably a combination of 1 to 10,000 codons within the sequenced region.

Nucleic acids

[0029] in another preferred embodiment of the present invention said first and optionally said second nucleic acids (components b) respectively, can be independent from each other singlestranded, double-stranded, or a combination thereof. More preferably the acids are selected from the group comprising RNA, DNA, RNA-DNA hybrid, chemically modified RNA, chemically modified DNA, XNA, linkers, chemical doublers, branchers, conjugates (for example - puromycin), or any combination thereof.

Aminoacylated tRNA mimics

[0030] in another preferred embodiment, said aminoacylated tRNA mimic (component (c) is puromycin or a puromycin-like molecule that has the capacity to stall the progress of the ribosome.

Ribosomal stalling sequence

[0031] in another preferred embodiment the display may also encompass a ribosomal stalling sequence (component d) located between the end of the amino-acid polymer encoding sequence and the brancher. This element can be DNA, or chemically modified RNA/DNA nucleotide(s) or XNA or a codon for which the cognate tRNA is rare within the target cells of interest or the brancher itself or a chemical doubler/trebler. Chemical brancher

[0032] in another preferred embodiment said chemical brancher is selected from symmetric doubler, asymmetric doubler (Lev), asymmetric doubler (FMoc), symmetric brancher, asymmetric brancher (Lev), asymmetric brancher (FMoc), symmetric Trebler, asymmetric Trebler (Lev), asymmetric Trebler (FMoc).

Separator

[0033] In another preferred embodiment the xRNA-display according to the present invention may further comprise a separator, wherein the xRNA molecule, the nucleic acid molecule, and the separator are connected through a chemical brancher and the aminoacylated tRNA is linked to the 3' end of the separator or to the 3' or 5' end of the nucleic acid barcode. Suitable separators can be for example selected from the group consisting of natural or non-natural nucleic acids, chemical polymers, spacers, conjugates or any combination thereof.

High-affinity ligands

[0034] in another preferred embodiment the xRNA-display according to the present invention may also comprise at least one high affinity ligand covalently bound to the chemical brancher, the nucleic acid molecule, the separator, the xRNA, or combination thereof. Suitable high affinity ligands may be selected from the group consisting of biotin, PC Biotin, Desthio-Biotin, Dual-biotin, biotin-TEG. Particularly preferred high affinity ligands are represented by the polypeptide encoded by the xRNA molecule.

Method for xRNA display

[0035] A fourth object of the present invention refers to a method for xRNA display in live mammalian cells, comprising:

(i) transfecting multiplexed xRNA display libraries prior to peptide conjugation into living cells;

(ii) extracting xRNA libraries under conditions that preserve xRNA-ribosome interactions post lysis;

(iii) inducing peptide conjugation post lysis by subjecting the cell lysates to suitable conditions, such as high-salt incubation; or conjugating the xRNA to the peptides without lysing the cells, for example - by freezing and thawing the cells under conditions that preserve cell viability.

(iv) purifying the xRNA libraries from the lysate using affinity-based purification methods; (v) separating the xRNA library into at least two fractions, corresponding to peptide- conjugated or unconjugated xRNA fractions;

(vi) isolating and purifying the various fractions;

(vii) assessing the relative abundance of each xRNA sequence in the peptide-conjugated, and optionally also in the unconjugated fractions to determine a Translatability Score, indicating the relative translatability of a given xRNA sequence; and

(viii) assessing the stability of xRNA sequences by transfecting libraries into cells, isolating them at one or multiple time points post transfection, and determining a Stability Score based on the abundance of xRNA sequences over time; wherein each of steps (iv), (v) and (viii) are optionally.

[0036] In a first preferred embodiment of step (i) said xRNA display libraries are transfected into mammalian cells using transfection reagents suitable for efficient delivery. In the alternative, the xRNA display libraries are taken up into mammalian cells via delivery mediated conjugate (for example GalNAc or Cholesterol) or by passive diffusion.

[0037] in another preferred embodiment, cell lysis (step ii) is performed using a lysis buffer optimized to maintain the integrity of xRNA-ribosome interactions.

[0038] In another preferred embodiment the conditions for inducing peptide conjugation (step iii) include varying factors such as salt concentration, temperature, or other suitable methods known in the art.

[0039] in another preferred embodiment purification of xRNA libraries (step iv) is performed using affinity-based techniques, including but not limited to biotin-Streptavidin affinity purification, antibody pulldown against the translated peptide or other suitable methods. In the alternative, purification of xRNA is performed using an affinity method directed specifically to the translated polypeptide, such as antibodies, the fractionation step can be skipped.

[0040] in another preferred embodiment separation of the xRNA library into peptide- conjugated and unconjugated fractions (step v) is accomplished by techniques such as polyacrylamide gel electrophoresis (PAGE), chromatography, HPLC, or other suitable methods. [0041] in another preferred embodiment the sequencing libraries are prepared using methods including but not limited to cDNA synthesis, amplification, and adapter ligation.

[0042] Preferably said Translatability Score is determined by quantitatively assessing the relative abundance of each xRNA sequence in the peptide-conjugated and unconjugated fractions, while the Stability Score is determined by assessing the abundance of xRNA sequences at multiple time points post transfection, relative to the initial library, to infer the relative stability of the xRNA sequences over time. [0043] Translatability Score can be defined as TPM(high)/((TPM(high)+TPM(low)), where TPM(high) is transcripts per million from the upper peptide-conjugated band post separation by polyacrylamide gel electrophoresis (PAGE), and TPM(low) is transcripts per million from the lower unconjugated band.

[0044] Stability Score can be defined as TPM(extract)/ ((TPM(extract)+TPM(input)), where TPM(extract) is transcripts per million from the library following incubation in cells and extracted, and TPM(input) is transcripts per million from the input sample.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: xRNA display construct design. In (a) and (b) the barcode is separated from the mRNA coding region via a chemical doubler. In (c) the barcode is positioned both upstream and downstream to the mRNA coding region. In (d) the coding region serves as the barcode.

Figure 2: xRNA display compatibility with peptide conjugation, a. The xRNA display design in use. b. xRNA-peptide conjugation following 1 hour translation in HeLa cell extract, affinity purification from the extract and high-salt treatment. Up to 32% xRNA-peptide conjugation efficiency is observed. Arrow represents an uncharacterized xRNA cleavage product.

Figure 3: xRNA display in cells, a. The xRNA display design in use. Exemplary sequence of the depicted xRNA is:

[m7GPPP][mG]GGAGAGCCACCAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUUAGCAAAAA AAAAAA[BiodT]AAAAAAAAA (SEQ ID NO 1) [Sp9] [Sp9] [Sp9] ACQPuro], wherein nucleotides 1 - 48 are RNA, nucleotides 49 onwards are DNA. [mG] is the 1st nucleotide and [m7GPPP] is not counted and can be converted to an mRNA cap structure before transfection, b. xRNA-peptide conjugation following 6 hours translation in cultured A549 cell, extraction of RNA-ribosome complexes and high-salt treatment in various temperatures. Up to 39% xRNA-peptide conjugation efficiency is observed. Right panel: A control xRNA without Puromycin, that underwent the same experimental procedures described above.

Figure 4: Identification of translatable mRNAs by xRNA display, a. The xRNA display design in use. Exemplary sequence of the depicted xRNA is [m7GPPP][mG]GGAGAGCCACCAUGUCAGGUGGAGGAAGUUCUGGUGUGAGCGGCUGGCGGCU GUUCAAGAAGAUUAGCGGUUCCAGUGGUGGUGGAUCUGCGAAAAAAAAAAA[BiodT]AAAAAA AAA (SEQ ID NO 2) [Sp9] [Sp9] [Sp9] ACC[Puro], wherein nucleotides 1 -90 are RNA, nucleotides 91 onwards are DNA. [mG] is the 1st nucleotide and [m7GPPP] is not counted and can be converted to an mRNA cap structure before transfection, b. xRNA-peptide conjugation following 10 minutes (left) and 1 hour (right) translation in HeLa cell extract and high-salt treatment. Up to 22% and 41% xRNA-peptide conjugation efficiency is observed after 10 minutes or 1 hour of in vitro translation, respectively.

Figure 5: Illustrates the designed library of 2,000 sequences encoding the HiBit peptide flanked by Glycine/Serine linkers. Upper polypeptide sequence is MSGGGSSGVSGWRLFKKISGSSGGGS (SEQ ID NO 3), amino acids 2-8 (SGGGSSG, SEQ ID NO 4) and 20-26 (GSSGGGS, SEQ ID NO 5), also shown in the design illustrations, are Serine/Glycine (S/G)-linkers.

Figure 6A-C: Correlations between translation scores across biological replicates. Shown are scatter-plots of the translation score of each sequence (among n=2,000) sequences across each of three pairs of biological replicates, as well as a trend line (fitted via linear regression). The Spearman correlation coefficient and its p-value are shown on the top left of each panel.

Figure 7A-B: Measures of the assay capability to distinguish between positive and negative controls. 7a. depicts ROC curves at the 10 minutes (orange) and 1 hour (cyan) time points. The ROC-AUC values are shown in the legend. 7b. presents a histogram showing the distribution of translation scores of negative controls (cyan) and positive controls (orange) at the 10 minutes time point.

Figure 8A-B: Depicts the average translation scores across five different groups of sequences differing in the presence/absence of start and stop codons, at the 10 minutes time point (left) and the 1 hour time point (right). Error bars represent 95% confidence intervals, computed via 1,000 statistical bootstrap samples.

Figure 9A-C: The correlation between stability score, translation score, and minimum free energy. Shown are scatter plots displaying the association between translation score, stability score, and minimum free energy (each panel shows the association between two of these three values), as well as a trend line fitted via linear regression. The Spearman correlation coefficient and its p-value are shown on the top left of each panel.

Figure 10: Schematic of xRNA display in cells protocol.

Figure 11: HiBiT activity (RLU) from A549 cells transfected with an HiBiT-encoding xRNA and an unmodified mRNA control.

Figure 12: mRNA display library post cell incubation and peptide conjugation, size separated on a polyacrylamide-Urea sequencing gel

Figure 13A-B: Correlation of the translation score between different replicates

Figure 14A-B: ROC and Precision-recall curves to estimate separation between positive and negative controls.

Figure 15A-B: Comparison of translation score distributions in positives vs. negative controls.

Figure 16: Translation score distributions

Figure 17A-D: Design of five Barcode Display constructs with variable length RNA and variable 3'-ends. The Barcode Display constructs were generated via ligation of in vitro transcribed RNA (red sequences) to chemically synthesized DNA constructs containing an asymmetric doubler and 3'-Puromycin.

17A/B: HiBiT_66 & Oligo_1 : upper strand sequence 5' to asymmetric doubler is: [m7GPPP][mG]GGAGAGCCACCAUGUCAGGUGGAGGAAGUUCUGGUGUGAGCGGCUGGCGGCU GUUCAAGAAGAUUAGCGGUUCCAGUGGUGGUGGAUCUCCGCTTACTAGCGCGTACCAATGGT (SEQ ID NO 6), wherein nucleotides 1 -48 are RNA, nucleotides 49 onwards are DNA and [mG] is the 1st nucleotide and [m7GPPP] is not counted. Lower strand sequence 5' to asymmetric doubler is TCAGGCTATGAATCTTCCTGCTCAGTTATGTTAA (SEQ ID NO 7)

[PCL][BiodT][Sp18][Sp18],

HiBiT_66 & Oligo_2: upper strand sequence 5' to asymmetric doubler is:

[m7GPPP][mG]GGAGAGCCACCAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUUAGCCCGCT TACTAGCGCGTACCAATGGT (SEQ ID NO 8), wherein nucleotides 1 -48 are RNA, nucleotides 49 onwards are DNA and [mG] is the 1st nucleotide and [m7GPPP] is not counted. Lower strand sequence 5' to asymmetric doubler is TCAGGCTATGAATCTTCCTGCTCAGTTATGTTAA (SEQ ID NO 7) [PCL][BiodT][Sp18][Sp18],

17C/D: HiBiT_69 & Oligo_1 : upper strand sequence 5' to asymmetric doubler is: [m7GPPP][mG]GGAGAGCCACCAUGUCAGGUGGAGGAAGUUCUGGUGUGAGCGGCUGGCGGCU GUUCAAGAAGAUUAGCGGUUCCAGUGGUGGUGGAUCUCCGCTTACTAGCGCGTACCAATGGT (SEQ ID NO 23), wherein nucleotides 1 -90 are RNA, nucleotides 91 onwards are DNA, and [mG] is the 1st nucleotide and [m7GPPP] is not counted. Lower strand sequence 5' to asymmetric doubler is TCAGGCTATGAATCTTCCTGCTCAGTTATGTTAA (SEQ ID NO 7)

[PCL][BiodT][Sp18][Sp18],

HiBiT_69 & Oligo_2: upper strand sequence 5' to asymmetric doubler is:

[m7GPPP][mG]GGAGAGCCACCAUGUCAGGUGGAGGAAGUUCUGGUGUGAGCGGCUGGCGGCU GUUCAAGAAGAUUAGCGGUUCCAGUGGUGGUGGAUCUCCGCTTACTAGCCAAAAA (SEQ ID NO 9), wherein nucleotides 1 -90 are RNA, nucleotides 91 onwards are DNA, and [mG] is the 1st nucleotide and [m7GPPP] is not counted. Lower strand sequence 5' to asymmetric doubler is TCAGGCTATGAATCTTCCTGCTCAGTTATGTTAA (SEQ ID NO 7) [PCL] [BiodT] [Sp 18] [Sp 18] .

17E: HiBiT_69 & Oligo_3: upper strand sequence 5' to asymmetric doubler is: [m7GPPP][mG]GGAGAGCCACCAUGUCAGGUGGAGGAAGUUCUGGUGUGAGCGGCUGGCGGCU GUUCAAGAAGAUUAGCGGUUCCAGUGGUGGUGGAUCUCCGCTTACTAGCCAAAAAAAA (SEQ ID NO 10), wherein nucleotides 1 -90 are RNA, nucleotides 91 onwards are DNA, and [mG] is the 1st nucleotide and [m7GPPP] is not counted. Lower strand sequence 5' to asymmetric doubler is TCAGGCTATGAATCTTCCTGCTCAGTTATGTTAA (SEQ ID NO 7) [PCL][BiodT][Sp18][Sp18],

Figure 18: Barcode Display constructs are efficiently translated in transfected A549 cells. HiBiT activity (RLU) measured 6 hours post transfection from A549 cells transfected with Barcode Display constructs of various designs (Figure 3) and a linear mRNA display positive control (Twist library).

Figure 19: HiBiT activity (RLU) following transfection of Barcode Display constructs into A549 cells, post-lysis conjugation (- / + salt) and purification using Streptavidin beads.

Figure 20A-B: Representative construct used for 1 K barcode display synthesis. A Part4- RNA ligation is [Sp9]GsGGAGAGCCACCAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUUAGC (SEQ ID NO 13), Part 2A is pCCGCTTACTAGC (SEQ ID NO 14), Part 1 B is GTAGGTAGGCTTTTG (SEQ ID NO 15), Part3 is NNNNNNNNNNGTATGCAG (SEQ ID NO 16), Part 2B CAGTCAGA (SEQ ID NO 17), Part 1A is TTGCACTCT (SEQ ID NO 21), Part 0 is [PCL] [BiodT] [Spl 8] [Spl 8] connected to [asymmetric doubler] connected to AAAAAAAA[Sp9] [Sp9] [Sp9]ACC[Puro] (SEQ ID NO 18, [Sp9] and [Puro] is omitted in the sequence listing). Figure 21 : Results of single barcode Sanger sequencing, results show the presence of the target barcode (highlighted). Peaks show raw data from the Sanger sequencing, each color corresponds to a different base assignment (Red = T, green = A, Black = G and blue = C, light red = Unassigned at by auto processing). Depicted nucleic acid sequences are TTTCCTACACGACGCTCTTCCGATCCTTCAGGCTATGAATCTTCCTGCTCAGTTATGTTA (SEQ ID NO 19), depicted amino acid sequence is FPTRRSSDPSGYESSCSVML (SEQ ID NO 20).

Figure 22: Distribution of positions of each barcode sequence across the entire library.

Figure 23: Joint distribution of the number of occurrences of each of the 1,024 barcodes.

EXAMPLES

[0045] The following examples illustrate the present invention, and are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the statements of the invention which follow thereafter. The Examples described below are provided to illustrate aspects of the present invention and are not included for the purpose of limiting the invention.

Example 1

Assessing Translatability and Stability ofxRNA Display Libraries in Live Mammalian Cells

[0046] The present invention uses a new method to evaluate the translatability and stability of libraries of xRNA display molecules in live mammalian cells. This method involves the following steps:

1. Transfection of multiplexed xRNA display libraries into living mammalian cells.

2. Cell lysis at different time points post-transfection (tp) using a modified polysome lysis buffer that preserves xRNA-ribosome interactions.

3. Incubation of cell lysates in high-salt conditions at room temperature for 1 -2 hours to induce peptide conjugation after lysis.

4. Purification of biotinylated xRNA libraries from the lysate using Streptavidin beads.

5. Separation of the xRNA library into at least two fractions through PAGE, representing peptide-conjugated and unconjugated xRNA fractions.

6. Isolation and purification of the various fractions.

7. Preparation of sequencing libraries by generating first-strand cDNA, second-strand cDNA, and performing PCR amplification.

8. Assessment of the relative abundance of each xRNA sequence in the peptide-conjugated and unconjugated fractions to determine a Translatability Score, providing insights into the relative translatability of a given xRNA sequence.

9. Additionally, xRNA libraries can be transfected into cells and isolated at multiple time points (tp) post-transfection without inducing peptide conjugation.

10. Purification of these libraries as a pool using Streptavidin beads, followed by sequencing to determine the abundance of xRNA sequences at each tp relative to the tO library. 11. Calculation of a Stability Score by comparing the abundance of xRNA sequences over time, enabling the assessment of the relative stability of a given xRNA sequence in transfected cells.

[0047] The method described herein provides a comprehensive approach for assessing the translatability and stability of xRNA sequences in live mammalian cells. By combining transfection, cell lysis, purification, PAGE separation, and sequencing techniques, the method allows for the determination of relative translatability and stability scores of xRNA display libraries.

[0048] The process of generating an xRNA display molecule with an asymmetric doubler, as described in Figure 2, involved ligating an in vitro transcribed mRNA molecule (encoding Hi BiT), which was enzymatically capped (cap-1), to a chemically synthesized DNA oligo containing the asymmetric doubler and a 3'-Puromycin (using a ssDNA splint with partial complementary to both sequences). Subsequently, the xRNA molecule with the asymmetric doubler underwent a 1 -hour in vitro translation reaction in HeLa extract, followed by 1 hour incubation at room temperature with increasing salt concentrations. After purification of the xRNA molecules, using Streptavidin beads and separation through PAGE, the inventors observed up to 32% peptide conjugation for xRNA molecules containing an asymmetric doubler in high salt conditions.

[0049] An xRNA display molecule was generated by ligating (a) an in vitro transcribed mRNA molecule encoding a HiBiT tag, which was enzymatically capped (cap-1), to (b) a chemically synthesized DNA oligo containing a 3'-Puromycin. Additionally, a negative control lacking the 3'-Puromycin was generated using the same method. A549 cells were seeded into a 24-well plate, and the following day, 300ng/well of the xRNA display constructs were transfected into cells using Lipofectamine MessengerMax. Following transfection and 6 hours of incubation, the cells were lysed in an ice-cold modified Polysome lysis buffer at different salt concentrations and frozen overnight at -20C. The cell lysates were then incubated at 4C or at room temperature for 2 hours to induce peptide conjugation post lysis. Purification of the xRNA libraries using Streptavidin beads, followed by PAGE separation (peptide- conjugated vs. unconjugated xRNA factions), revealed up to 39% peptide conjugation for xRNA display molecules treated with high salt conditions at room temperature. Importantly, no peptide conjugation was detected in the negative control (Figure 3). Example 2

Library Design and Codon Composition Sensitivity Testing for Comprehensive Validation

[0050] The inventors performed a large-scale experiment to validate that the invention works as intended. To this end, they designed a library of 2,000 sequences encoding the HiBiT peptide flanked by Glycine/Serine linkers. The design of each sequence in the library is shown in Figure 5. The library design included positive controls (sequences expected to generate a strong peptide-conjugation), negative controls (sequences expected not to generate peptide- conjugation, and test sequences with different codon compositions, to test for the sensitivity of the experiment to different translation rates. Codon frequencies were obtained from the codon usage database [Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nakamura, Y., Gojobori, T. and Ikemura, T. (2000) Nucl. Acids Res. 28, 292] (https://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=9606).

[0051] in cases where the inventors modified sequence elements were outside the sequenced region, they created a unique signature, referred to as "watermark signature", using a specific combination of several synonymous substitutions that do not affect the produced peptide within the sequenced region. The watermark signature was designed such that it did not overlap with any of the other sequenced sequences. The library composition is described in Table 1. Unless mentioned otherwise, all the sequences had the same 5' UTR, as described in Figure 5. Table 2 shows the codons frequency ranges used to design the "HiBiT frequent codons" sequences; Table 3 the codons frequency ranges used to design the "HiBiT rare codons" sequences and finally Table 4 watermark signatures for distinguishing changes in non-sequenced regions

Table 1

Description of the 2000 test library sequences

*AII the sequences with different 5' UTR had a G at the 5' end. Their coding region was comprised of randomly selected codons encoding HiBiT. Since the 5' UTR is not sequenced, a specific combination for amino acids S,R,L was chosen to encode this group, as defined in Table 4.

Table 2

Codons frequency ranges used to design the "HiBiT frequent codons" sequences

Table 3

Codons frequency ranges used to design the "Hi BiT rare codons" sequences

Table 4

Watermark signatures for distinguishing changes in unsequenced regions

[0052] To perform the experiment, the library was ordered from Twist Biosciences, PCR amplified, and served as a template for in-vitro transcription. The resulting mRNA pool was purified, enzymatically capped (Cap-1), and ligated to a DNA oligo containing a 3'-Puromycin. Enzymatic capping was performed using the One-Step Capping and 2'-O-Methylation protocol and reagents (Vaccinia Capping Enzyme and mRNA Cap 2'-0-Methyltransferase) from NEB. The ligation product was isolated and purified through PAGE to generate the library of 2000 xRNA sequences. Additionally, the xRNA library underwent either a 10-minute or 1 -hour in vitro translation reaction in HeLa extracts, followed by incubation under high salt conditions at room temperature. The inventors observed approximately 22% peptide conjugation after a 10- minute translation reaction and approximately 41% peptide conjugation after a 1 -hour translation reaction (Figure 4.).

[0053] The inventors sequenced various libraries to verify that the experiment worked as intended, including:

1. Input: Two aliquots of the input library (a library with the original sequences prior to running the experiment)

2. Unconjugated: Three different aliquots of sequences in the unconjugated band, representing three different biological replicates of the experiment

3. Peptide-conjugated: Three different aliquots of sequences in the peptide-conjugated band, representing three different biological replicates of the experiment

4. Unseparated: A single aliquot of sequences at the 10 minutes time point and a single aliquot of sequences at the 60 minutes time point (a 4th biological replicate), not separated by gel electrophoresis.

[0054] To analyze the data, they were first processed and then defined a translation score and a stability score for each xRNA sequence at each time point, denoting the relative rate at which the sequence gets translated into protein by that time point, and the relative survival rate of the sequence at that time point, respectively (all measures are relative to other sequences in the library). The inventors briefly outline these steps below. To process the sequencing data in each library, the following protocol was exercised:

(i) extracted read counts

(ii) removed duplicate sequences under the assumption that duplicate sequences originate from PCR-induced duplications

(iii) removed sequences that failed library mapping due to not identifying sequences corresponding to the PCR primer annealing sites) and

(iv) removed sequences that could not be perfectly matched to one of the 2,000 sequences designed for the experiment by extracting the watermark barcode from the sequence and comparing it to the designated barcode collection.

[0055] Then, the translation score of a given sequence at a given time point was defined via the per-replicate average of the quantity

+ t^low) (averaged across three biological replicates). Here, j_s the read count of the sequence in the peptide-conjugated library (at the given time point) divided by the total number of read counts in the peptide-conjugated library, and t^lowt^low j_s defined analogously for the unconjugated library. The stability score of a given sequence at a given time point was defined via the quantity neutral ( ^neutral | jinputj neutral ( ^neutral | jinputj

[0056] Here, t'^ieutroIt^ziei‘^traI j_s the _read _COunt of the sequence in the unseparated library (at the given time point) divided by the total number of read counts in the unseparated library, and _tmp t_tmput j_{s avera}g_e read count of the sequence in the input libraries divided by the total number of read counts in the input library (averaging across multiple aliquots of the input library). Several analyses using these quantities as detailed below were performed.

Example 3

Verifying per-replicate consistency of translation scores

[0057] To verify that the experiment is robust and replicable, the inventors computed the translation score of each sequence in each biological replicate separately (without averaging across biological replicates) and computed the Spearman correlation between the translation scores across pairs of biological replicates. The inventors obtained extremely high and statistically significant correlations (r=0.99, p< 10“¹⁶) across all pairs of biological replicates (Figure 6.). These indicated that the experiment was robust and highly replicable.

Example 4

Testing for differences between translation scores of positive and negative controls

[0058] To verify that the assay is able to distinguish between sequences that get translated to protein and sequences that do not get translated to protein, the inventors selected 35 high- confidence positive controls (defined as Test HiBiT and HiBiT frequency codons, as defined in Table 1) and 182 high-confidence negative controls (defined as sequences without a start codon or with a premature stop codon). The inventors tested for substantial differences between these control sequences in several ways:

1. Computing the area under the receiver-operating curve (ROC-AUC): The ROC-AUC was given by 0.99 (after 10 minutes) and by 0.92 (after 60 minutes), indicating strong separation between the two groups. The ROC curve is shown in Figure 7a.

2. Testing for a statistically significant difference between positive and negative controls: The inventors performed a Manny-Whitney U test and obtained p<3.87E-15, indicating strong separation between the two groups.

3. Plotting the histograms of translation scores across positive and negative controls: The histogram provides a visual representation of the strong separation observed between positive and negative controls. The histogram at the 10 minutes time-point is shown in Figure 7b. [0059] These results all demonstrate that the assay accurately distinguishes between sequences that get translated to protein and sequences that do not get translated to protein.

Example 5

Testing the effects of modifying the start and stop codons

[0060] To further verify that the assay works as intended, the inventors verified that the assay only reports high translation scores to sequences that (a) have a start codon; and (b) lack a stop codon. Specifically, the inventors compared translation scores across five different groups of sequences:

1. No start: Sequences without a start codon

2. Out-of-frame start+stop: sequences with an alternative start codon and a stop codon in the same reading frame (these will not be peptide-conjugated)

3. Out-of-frame start, no stop: Sequences with an alternative start codon and without a stop codon.

4. In-frame start+stop: Sequences with premature stop codons (these will not be peptide- conjugated)

5. In-frame start, no stop: Sequences with a regular start codon and without a stop codon [0061] As expected, sequences without a start codon or with a stop codon had extremely low translation scores, compared to sequences with a start codon and without a stop codon (Figure 8). The results demonstrate that the assay requires the presence of a start codon and the absence of a stop codon in order to generate a high translation score.

Example 6

Investigating the relationship between translation score, stability score, and minimum free energy

[0062] To demonstrate that the assay provides meaningful information about the dynamics of xRNA translation and survival rates, the inventors investigated the relationship between translation score, stability score, and minimum free energy (MFE). Specifically, the inventors considered all 1,474 sequences with an in-frame start codon, without a stop codon, and with codon frequencies within the range defined in Table 2 and computed their minimum free energy via the RNA fold tool in the ViennaRNA package, using default parameters (R. Lorenz et al. (2011), and I.L. Hofacker et al. (1994)). The results demonstrate a strong and statistically significant relationship between these quantities (Figure 9). Translation scores have a strong positive correlation with MFE, stability scores have a strong negative correlation with MFE, and translation and stability scores are negatively correlated. These results demonstrate that the assay provides meaningful information about the dynamics of xRNA translation and survival rates.

Example 7

Synthesis of DNA Oligonucleotides Using CPG Beads and Doubler

[0063] DNA oligonucleotide synthesis was carried out using CPG beads that were loaded with puromycin. The synthesis followed the established solid phase DNA synthesis protocol and employed DMTr chemistry to construct the desired DNA sequence. Asymmetric doubler consisting of a DMTr and a Levulinyl (Lev) group was introduced at the required position. The DMTr group from the first arm of the doubler was removed, and Biotin was coupled. Subsequently, the Lev group on the second arm of the doubler was removed, allowing for the synthesis of the remaining DNA strand. Once the chemical synthesis was completed, the DNA was liberated from the solid support and ligated to the HiBiT RNA coding region using a DNA splint as a ligation scaffold (Figure 2a).

[0064] An exemplary nucleic acid sequence from the 5' end to the asymmetric doubler comprising the HiBit Coding sequence and DNA (dashed line) of Fig. 2 a. is: [m7GPPP][mG]GGAGAGCCACCAUGUCAGGUGGAGGAAGUUCUGGUGUGAGCGGCUGGCGGCU GUUCAAGAAGAUUAGCGGUUCCAGUGGUGGUGGAUCUGCGCTACATGGC (SEQ ID NO 24), wherein nucleotides 1 -90 are RNA, nucleotides 91 onwards are DNA, and [mG] is the 1st nucleotide and [m7GPPP] is not counted. The DNA downstream of the asymmetric doubler may be AAAAAAAAAA (SEQ ID NO 25) [Sp9][Sp9][Sp9]ACC[Puro],

Example 8

Barcode display screen in cells

[0065] Supra, the synthesis of an mRNA display library (ordered from TWIST biosciences) and its utilization to monitor RNA stability and translatability in cell extracts was described. It was also demonstrated that surprisingly, the mRNA technique can be utilized to monitor translation inside living cells (but not in a screen setup yet). Now, Example 8 provides the results of a full mRNA display screen done in mammalian cells, which is probably the first time that an mRNA display technique that is based on covalent linkage between the mRNA and its transcribed product is performed inside live cells. The library composition is described in Tables 1 -4 and Figure 5.

[0066] In this protocol, the inventors further developed a method for xRNA display in transfected human cells as shown in Figure 10. Cell lysis and peptide conjugation conditions were optimized to facilitate peptide conjugation post-lysis by incubating the lysate in high salt conditions. The method utilizes a modified Polysome buffer for cell lysis, which maintains ribosome-mRNA association post-lvsis, followed by a high salt incubation to maximize peptide conjugation. In addition to developing xRNA display in cells to screen for translatability, an extended xRNA stability time course was also performed, based on the observation that HiBiT activity is significantly prolonged when expressed from a modified xRNA compared to an unmodified mRNA control (Figure 11).

Protocol - xRNA display in cells

[0067] A549 cells were transfected with the mRNA display library described in Figure 5 using Lipofectamine MessengerMax, according to the manufacturer protocol. Cells were lysed at 1 ,5h and 6h post-transfection using a modified polysome lysis buffer that preserves xRNA-ribosome interactions. mRNA - peptide conjugation was performed post lysis by incubation at 22C for 2 hours in the presence of salts. The mRNA display library was purified from the lysates using Dynabeads™ MyOne™ Streptavidin C1 (Invitrogen) according to the manufacturer recommendations, and was loaded on a polyacrylamide-Urea sequencing gel for separation of peptide conjugated from unconjugated mRNA display constructs based on their size (Figure 12). The peptide conjugated and unconjugated mRNA display constructs were excised from the gel, eluted via passive diffusion, and dialysed using 10KDa filter columns. The mRNA display constructs were reverse transcribed to cDNA and sequencing libraries prepared. The libraries were sequenced on a NovaSeq machine.

Analysis

[0068] The input library, and the two bands (low and high) at two different time points (after 1.5 hours and after 6 hours) were sequenced. An extensive analysis of the sequencing data to demonstrate the feasibility and potential utility of our platform was performed. The analysis demonstrates reproducibility across biological replicates, clear separation between positive and negative controls, and the ability to infer some of the factors that drive mRNA translation rate.

Reproducibility across biological replicates

[0069] A translation score for each sequence at each time point was defined. The translation score of a sequence is defined as its TPM (transcripts per million) in the high band, divided by the sum of its TPM in the high and low bands. The inventors next computed the correlation between the translation scores at both replicates at both time points. The results demonstrate a strong correlation (Spearman r~0.7) at both time points (Figure13), demonstrating strong reproducibility.

Separating positive from negative controls

[0070] Two sets of positive and negative controls were defined. Negative controls included sequences with no start codon (90 sequences) or with a premature stop codon (108 sequences), whereas positive controls include the 35 sequences with the highest codon adaptation index (i.e., sequences using codons with some of the most frequent tRNA frequencies). To estimate separation between position and negative controls, the inventors computed a receiver- operating-characteristic (ROC) curve, and a Precision-Recall curve, based on the translation scores of the positive I negative controls at both time points, and then computed the areas under the curves.

At both time points, the area under the curves is near-perfect, indicating an extremely strong separation between positive and negative control (Figure 14). Also, the distribution of translation scores among negative and positive controls were plotted, which also shows the strong separation between these two groups (Figure 15).

Inferring the factors driving mRNA translation rate

[0071] To infer the factors driving mRNA translation rates, the sequences were portioned into groups, as specified in previous Table 1. Different trends in the translation scores distribution in the different groups were observed (Figure 16). As expected, negative controls have a significantly lower translation score than non-translated ones. However, one can see that some groups clearly have different translation dynamics than others. This demonstrates that the platform helps infer the factors driving mRNA translation rate.

Example 9

Barcode display synthesis and function

Barcode Display constructs are efficiently translated in transfected A549 cells

[0072] As explained supra Figure 1 shows the design of barcode display contracts. In the following it is shown: their successful synthesis; their ability to be translated in mammalian cells; and their compatibility with the mRNA display method (i.e., covalent conjugation of the mRNA to its protein product).

[0073] To test the translatability of Barcode Display constructs in transfected human cells, 5 Barcode Display constructs of various designs were created (Figure 17) by ligating an in vitro transcribed RNA (red sequences) to various chemically synthesized DNA constructs containing an asymmetric doubler and 3'-Puromycin. Following PAGE-purification, the Barcode Display constructs were transfected into A549 cells, and Hi BiT activity (RLU) was measured at 6 hours post transfection (Figure 18). Alongside the various Barcode Display constructs, the inventors also transfected a linear mRNA display positive control (Twist library, described in the provisional patent and above). The inventors detected a robust luminescence signal from all Barcode Display constructs tested, with RLU values comparable to the linear positive control. This experiment demonstrates that the Barcode Display constructs are efficiently translated when transfected into human cells. This is surprising as these contracts are significantly different from the canonical structure of mRNA.

Efficient post-lysis conjugation of Barcode Display constructs to the translated HiBiT peptide

[0074] To confirm efficient peptide conjugation from the Barcode Display constructs, the constructs were transfected into A549 cells, followed by lysis in a modified Polysome buffer 5 hours post transfection, to allow time for translation to occur. The lysates were then incubated in the presence (+) or absence (-) of salt to facilitate peptide conjugation, followed by Streptavidin pulldown to recover the Barcode Display constructs and wash away any nonconjugated HiBiT. DNasel treatment was applied to release the constructs from beads, and the presence of conjugated HiBiT was assessed via luminescence detection. The results show efficient peptide conjugation in the presence of salt (RLU is greater than the linear positive control Twist library), but not in the absence of salt (Figure 19). This experiment demonstrates efficient conjugation (post-lysis) of Barcode Display constructs conjugated to the HiBiT peptide after being translated inside human cells and further proves the feasibility of the barcodedisplay method.

Example 10

Synthesis and sequencing of a IK barcode display library

[0075] A sequencing analysis was performed to demonstrate our ability to successfully synthesize a library with over 1,000 different sequences. The library construct is provided in Figure 20. Barcode display library synthesis consists of orthogonal solid phase chemical synthesis of paralleled oligonucleotides with split and pool approach at various stages of the synthesis. The overall synthesis efforts consist of various steps as depicted in the above schematic.

[0076] Synthesis of Part 0: It's started on puromycin nucleotide attached to the of 2000 A solid support i.e. (CPG) beads. Along with the deoxy adenosine, this part of the synthesis consists of an incorporation of unnatural phosphoramidite monomers including Spacer-9, Spacer-18, biotinylated dT, and ends with the addition of the photocleavable linker. A coupling time 50s for unmodified and 10 mins for modified monomers were implemented to ensure maximum coupling at each addition of the monomer. This part is synthesized in multiple columns (24) to obtain a large stock of the solid support for subsequent library synthesis. Part of the solid support from representative columns was removed to cleave and deprotect oligo for further LCMS analysis to confirm the synthesis quality. After which, all the columns at this stage are mixed and the solid support is divided in 16 columns each containing equal quantity of the solid support.

[0077] Synthesis of Part 1: Part 1A synthesis consist of 16 distinct barcodes which were synthesized in 16 separate columns obtained from step-0 utilizing standard DMTr chemistry and 10 min coupling. Fmoc-dT was added as a last nucleotide monomer in this section before starting the synthesis of Part 1 B which consists of different 3' UTR's. Along with positive and negative controls, these UTR contains different chemical modifications. Synthesis of the Part 1 B continued on the top strand of the doubler after Olev deprotection, utilizing standard DMTr chemistry and 10 min coupling. Upon completion of the par 1 B quality of the synthesis were assessed by LC-MS by taking the Aliquot from representative columns. The solid support from all 16 columns were pulled together, mixed well to produce a homogenous library. At this stage the solid support is redistributed into 8 columns prior to the synthesis of the next section.

[0078] Synthesis of Part 2 : Part 2A synthesis consist of the remaining nucleotides of 3' UTR and the ligation section which was completed utilizing standard DMTr chemistry and 10 min coupling. Last monomer used in this section is CPR 2 which was added using double 10 min coupling and the DMTr group was removed resulting in the hydroxyl group being capped so that it will not interfere with the rest of synthesis. At the end upon cleavage and deprotection of the library the 5’ phosphate group will be revealed on the top strand which will be used for the subsequent ligation of the RNA. Next the part 2B was synthesized first by deprotecting the Fmoc group followed by the addition of the second barcode corresponding to the second half of the 3' UTR. The solid support from all 8 columns were pulled together, mixed well to produce a homogenous sample. Solid support is then redistributed into 8 columns prior to the synthesis of the next section of the library.

[0079] Synthesis of Part 3: Part 3 of the library consists of barcode 3 which is corresponding to the RNAs that will be ligated to the construct and the UMI which is synthesized using standard DMTr chemistry with 10 min coupling time. After completion of the synthesis as before solid support from all 8 columns were combined together. Finally, this library is cleaved from the CPG beads and deprotected, revealing a 5' phosphate group on the top strand and a 5' free hydroxyl group on the bottom strand. The cleaved and deprotected library is purified using denaturing PAGE and processed for the sequencing library preparation.

[0080] Barcode sequencing: To confirm our ability to retrieve the barcode information from the barcode display library, the library was prepared for sequencing using IDT low DNA prep kit, according to the manufacturer recommendations.

[0081] The library was submitted for Sanger sequencing. From the Sanger sequencing the inventors found the presence of the full barcode highlighted in red below (Figure 21).

[0082] The ability to retrieve the barcode information on a high throughput scale was demonstrated by sequencing the library on Illumina NGS iSeqWO. In total the inventors synthesized the 1024 sequence library on a doubler, UV cleaved the barcodes and prepared them for sequencing. After PhiX removal, adapter trimming and merging of paired end reads, the inventors obtained 231,000 reads covering 89.8% of the 1,024 barcode combinations (with 79.1% of the combinations appearing at least 10 times). The number of occurrences of each barcode was evaluated (allowing up to one indel vs. the sequence), finding a roughly uniform distribution for each of the three barcodes as shown in the following Table 5:

Table 5

Barcode occurrences (max #indels: 1)

[0083] It was further verified that the barcode positions were consistent with the expected order, with the barcodel position mostly distributed across the 3' end of the reads, barcode 3 positions mostly distributed across the 5' end of the reads, and barcode 2 mostly distributed in the middle (Figure 22). [0084] Finally, the joint distribution of the three barcodes was investigated (Figure 23). Here, rows correspond to barcode 1, columns correspond to barcode 2, and each row/column combination is divided into eight cells corresponding to the three barcode 3 sequences. Figure 23 shows that the joint barcode distribution is roughly uniform, though exceptions exist, such as the absence of barcode2 sequences 4, 5, 6, 7, 8 when barcodel =4.

Claims

WHAT CLAIMED IS

1. A first xRNA-display construct comprising, consisting or essentially consisting of:

(a1) at least one xRNA molecule comprising an amino-acid polymer encoding sequence (b1) a nucleic acid molecule functioning as a distinct barcode capable of reconstructing the potential molecular composition of said at least one xRNA molecule; and

(cl) an aminoacylated tRNA mimic; wherein said xRNA molecule, said nucleic acid molecule and said aminoacylated tRNA are connected through a chemical brancher.

2. The xRNA-display construct according to Claim 1, wherein said xRNA molecule is an RNA or chemically modified RNA molecule, DNA building blocks, chemical spacers, RNA with conjugates, XNA, modified DNA, or combination thereof.

3. The xRNA-display construct according to Claim 1 and/or 2, further comprising polypeptide encoded by the xRNA molecule, wherein the polypeptide is conjugated via the aminoacylated tRNA mimic.

4. The xRNA-display construct according to any of the preceding Claims 1 to 3, further comprising a ribosomal stalling sequence located between the end of the amino-acid polymer encoding sequence and the brancher.

5. The xRNA-display construct according to any of the preceding Claims 1 to 4, wherein said nucleic acid is single-stranded, double-stranded, or a combination thereof.

6. The xRNA-display construct according to any of the preceding Claims 1 to5 , wherein said nucleic acid is selected from the group comprising RNA, DNA, RNA-DNA hybrid, chemically modified RNA, chemically modified DNA, XNA, or any combination thereof.

7. The xRNA-display construct according to any of the preceding Claims 1 to 6, wherein said aminoacylated tRNA mimic is puromycin.

8. The xRNA-display construct according to any of the preceding Claims 1 to 7, wherein said chemical brancher is selected from symmetric doubler, asymmetric doubler (Lev), asymmetric doubler (FMoc), symmetric brancher, asymmetric brancher (Lev), asymmetric brancher (FMoc), symmetric Trebler, asymmetric Trebler (Lev), asymmetric Trebler (FMoc).

9. The xRNA-display construct according to any of the preceding Claims 1 to 8, further comprising a separator, wherein the xRNA molecule, the nucleic acid molecule, and the separator are connected through a chemical brancher and the aminoacylated tRNA is linked to the 3' end of the separator or to the 3' or 5' end of the nucleic acid barcode.

10. The xRNA-display construct according to Claim 9, wherein the separator is selected from the group consisting of natural or non-natural nucleic acids, chemical polymers, spacers, conjugates or any combination thereof.

11. The xRNA-display construct according to any of the preceding Claims 1 to 10, further comprising at least one high affinity ligand covalently bound to the chemical brancher, the nucleic acid molecule, the separator, the xRNA, or combination thereof.

12. The xRNA-display construct according to Claim 11, wherein said high affinity ligand is selected from the group consisting of biotin, PC Biotin, DesthioBiotin, Dual-biotin, biotin- TEG.

13. The xRNA-display construct according to Claim 11 or 12, wherein said high affinity ligand is the polypeptide encoded by the xRNA molecule.

14. A second xRNA-display construct comprising:

(a2) an xRNA molecule comprising an amino-acid polymer encoding sequence;

(b2) a first nucleic acid molecule and a second nucleic acid molecule;

(c2) an aminoacylated tRNA mimic;

(d2) a ribosomal stalling sequence; and wherein the 3' of first nucleic acid is covalently linked to the 5' of the xRNA molecule, the 3' of the xRNA molecule is covalently linked to the 5' of the ribosomal stalling sequence and the 3' of the ribosomal stalling sequence is covalently linked to the aminoacylated tRNA mimic; and wherein the first and second nucleic acid molecules are functioning as a distinct untranslated barcode that identifies the profile of the xRNA molecule.

15. The xRNA-display construct According to Claim 14, wherein said xRNA molecule is an RNA or chemically modified RNA molecule, RNA with DNA building blocks, RNA with chemical spacers, RNA with conjugates, XNA or combination thereof.

16. The xRNA-display construct according to Claim 14 and/or 15, further comprising polypeptide encoded by the xRNA molecule, wherein the polypeptide is conjugated via the aminoacylated tRNA mimic.

17. The xRNA-display construct according to any of the preceding Claims 14 to 16, wherein said first and second nucleic acid each independently selected from single-stranded, double-stranded, or combination thereof.

18. The xRNA-display according to any of the preceding Claims 14 to 17, wherein said first and second nucleic acid are each independently selected from RNA, DNA, RNA-DNA hybrid, chemically modified RNA, chemically modified DNA, XNA, or any combination thereof.

19. The xRNA-display construct according to any of the preceding Claim 14 to 18, wherein said aminoacylated tRNA mimic is puromycin.

20. The xRNA-display construct according to any of the preceding Claims 14 to 19, further comprising at least one high affinity ligand covalently bound to the first nucleic acid, second nucleic acid or combination thereof.

21. The xRNA-display construct according to Claim 20, wherein said high affinity ligand is selected from biotin, PC Biotin, DesthioBiotin, Dual-biotin, and biotin-TEG.

22. The xRNA-display construct according to Claim 20 and/or 21, wherein said high affinity ligand is the polypeptide encoded by the xRNA molecule.

23. A third xRNA-display construct comprising, consisting or essentially consisting of:

(a3) an xRNA molecule comprising an amino-acid polymer encoding sequence;

(c3) an aminoacylated tRNA mimic; and

(d3) a ribosomal stalling sequence; wherein the xRNA molecule comprises a unique watermark signature inside or outside the encoding sequence functioning as a distinct barcode that identifies the profile of the xRNA molecule; and wherein the 3' end of the xRNA molecule is covalently linked to the 5' of the ribosomal stalling sequence and the 3' end of the ribosomal stalling sequence is linked to the aminoacylated tRNA mimic.

24. The xRNA-display construct according to Claim 23, wherein said xRNA molecule is an RNA or chemically modified RNA molecule, RNA with DNA building blocks, RNA with chemical spacers, RNA with conjugates, XNA or combination thereof.

25. The xRNA-display construct according to Claim 23 and/or 24, wherein said aminoacylated tRNA mimic is puromycin.

26. The xRNA-display construct according to any preceding claim 23 to 25, wherein said ribosome stalling is selected from triple nucleotide RNA codon for which there is no tRNA available in the reaction, or alternatively, a modified RNA, a DNA, a modified DNA, XNA, spacers, conjugates and combination thereof combinations.

27. The xRNA-display construct according to any of the preceding Claims 23 to 26, further comprising at least one high affinity ligand.

28. The xRNA-display construct according to Claim 27, wherein said high affinity ligand is selected from biotin, PC Biotin, DesthioBiotin, Dual-biotin, biotin-TEG.

29. The xRNA-display construct according to any of the preceding Claims 23 to 28, further comprising polypeptide encoded by the xRNA molecule, wherein the polypeptide is conjugated via the aminoacylated tRNA mimic.

30. The xRNA-display construct according to any of the preceding Claims 23 to 29, wherein said watermark signature is a combination of 1 to 10,000 codons within the sequenced region.

31. A method for xRNA display in live mammalian cells, comprising:

(i) transfecting multiplexed xRNA display libraries prior to peptide conjugation into living cells;

(ii) extracting xRNA libraries under conditions that preserve xRNA-ribosome interactions post lysis;

(iii) inducing peptide conjugation post lysis by subjecting the cell lysates to suitable conditions, such as high-salt incubation, or conjugating the xRNA to the peptides without lysing the cells;

(iv) purifying the xRNA libraries from the lysate using affinity-based purification methods;

(v) separating the xRNA library into at least two fractions, corresponding to peptide- conjugated or unconjugated xRNA fractions;

(vi) isolating and purifying the various fractions;

(vii) assessing the relative abundance of each xRNA sequence in the peptide- conjugated, and optionally also in the unconjugated fractions to determine a Translatability Score, indicating the relative translatability of a given xRNA sequence; and

(viii) assessing the stability of xRNA sequences by transfecting libraries into cells, isolating them at one or multiple time points post transfection, and determining a Stability Score based on the abundance of xRNA sequences over time; wherein each of steps (iv), (v) and (viii) are optionally.

32. The method of claim 31, wherein the xRNA display libraries are transfected into mammalian cells using transfection reagents suitable for efficient delivery.

33. The method of claim 31, wherein the xRNA display libraries are taken up into mammalian cells via a delivery ligand/conjugate (for example GalNAc or Cholesterol) or via passive diffusion.

34. The method according to any of the preceding Claims 31 to 33, wherein the cell lysis is performed using a lysis buffer optimized to maintain the integrity of xRNA-ribosome interactions.

35. The method according to any of the preceding Claims 31 to 34, wherein the conditions for inducing peptide conjugation include varying factors such as salt concentration, temperature, or other suitable methods known in the art.

36. The method according to any of the preceding Claims 31 to 35, wherein the purification of xRNA libraries is performed using affinity- based techniques, including but not limited to biotin-Streptavidin affinity purification, antibody pulldown against the translated peptide or other suitable methods.

37. The method according to any of the preceding Claims 31 to 35, wherein the purification of xRNA is performed using affinity method directed specifically to the translated polypeptide, such as antibodies.

38. The method according to any of the preceding Claims 31 to 37, wherein the separation of the xRNA library into peptide-conjugated and unconjugated fractions is accomplished by techniques such as polyacrylamide gel electrophoresis (PAGE), chromatography, HPLC, or other suitable methods.

39. The method according to any of the preceding Claims 31 to 38, wherein the sequencing libraries are prepared using methods including but not limited to cDNA synthesis, amplification, and adapter ligation.

40. The method according to any of the preceding Claims 31 to 39, wherein the Translatability Score is determined by quantitatively assessing the relative abundance of each xRNA sequence in the peptide-conjugated and unconjugated fractions.

41. The method according to any of the preceding Claims 31 to 40, wherein the Stability Score is determined by assessing the abundance of xRNA sequences at multiple time points post transfection, relative to the initial library, to infer the relative stability of the xRNA sequences over time.