Click display compositions and methods

Abstract

The present disclosure relates to click display compositions and methods of use thereof.

Description

[0001] Docket No.11164-018WO1 CLICK DISPLAY COMPOSITIONS AND METHODS CROSS-REFERENCE TO RELATED APPLICATION This PCT application claims priority to, and the benefit of, U.S. Provisional Patent Application No.63 / 509,349, filed June 21, 2023, which is incorporated by reference herein in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with Government Support under Grant No. DP2AI136600 awarded by the National Institutes of Health. The Government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING The sequence listing submitted on June 21, 2024, as an .XML file entitled “11164- 018WO1_ST26” created on June 17, 2024, and having a file size of 57,223 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). FIELD The present disclosure relates to click display compositions and methods of use thereof. BACKGROUND Rational design of proteins with desirable properties is challenging due to the complexity of a protein’s structure-function relationship. Directed evolution, which mimics natural evolution and entails the screening of large libraries of variants to identify those with desired functions, offers a powerful alternative to rational protein design as it does not require a priori knowledge of a protein’s structure-function relationship. A prerequisite for directed evolution is a link between the protein and its coding DNA / RNA and the choice of display method dictates the size / diversity of the protein library accessible for discovery. Over the years, many cell-based (e.g., yeast / bacterial / phage-display) and cell-free (in vitro) protein display methods have been developed. Since cell-free protein display methods are typically able to access a library size orders of magnitude larger than cell-based methods, in vitro protein display methods are often preferred for drug development. Docket No.11164-018WO1 Given the complexities of rational protein design, there is need to develop improved protein display methods to further improve drug development efforts. The compositions and methods disclosed herein address these and other needs. SUMMARY The present disclosure provides compositions and methods of preparing a nucleic acid- polypeptide complex for the use of screening proteins and rational protein design. In one aspect, disclosed herein is a modified linker comprising a first nucleic acid linked to a second nucleic acid, wherein the first nucleic acid comprises a ribosome binding molecule and a first click chemistry molecule, and wherein the second nucleic acid comprises a second click chemistry molecule and a crosslinking nucleotide. In some embodiments, the second nucleic acid comprises a sequence complementary to a target nucleic acid sequence. In some embodiments, the ribosome binding molecule comprises puromycin. In some embodiments, the first click chemistry molecule comprises an alkyne compound. In some embodiments, the alkyne comprises a dibenzocyclooctyne (DBCO). In some embodiments, the second click chemistry molecule comprises an azide (N3). In some embodiments, the crosslinking nucleotide comprises a 3-cyanovinylcarbazole nucleotide (CnvK). In one aspect, disclosed herein is a method of preparing a modified linker, the method comprising providing a first nucleic acid comprising a first click chemistry molecule and a ribosome binding molecule, providing a second nucleic acid comprising a second click chemistry molecule and a crosslinking nucleotide, and reacting the first and second click chemistry molecules to link the first nucleic acid to the second nucleic acid forming the modified linker. In some embodiments, the first click chemistry molecule and the ribosome binding molecule are at opposite ends of the first nucleic acid. In some embodiments, the second nucleic acid comprises a nucleic acid sequence complementary to a DNA template sequence. In some embodiments, the ribosome binding molecule comprises puromycin. In some embodiments, the first click chemistry molecule reacts with the second click chemistry molecule. In some embodiments, the first click chemistry molecule comprises an alkyne. In some embodiments, the alkyne comprises a dibenzocyclooctyne (DBCO). In some embodiments, the second click chemistry molecule comprises an azide (N3). In some embodiments, the crosslinking nucleotide comprises a 3-cyanovinylcarbazole nucleotide (CnvK). Docket No.11164-018WO1 In one aspect, disclosed herein is a method of preparing a complementary DNA (cDNA)- polypeptide complex, the method comprising preparing the modified linker of any preceding aspect, providing a DNA template, transcribing the DNA template into an mRNA, crosslinking the modified linker to the mRNA, wherein a crosslinking nucleotide binds to the mRNA, degrading the DNA template, translating the mRNA into a polypeptide, wherein the polypeptide is transferred to the ribosome binding molecule, and reverse transcribing the mRNA into a complementary DNA (cDNA), wherein the cDNA and polypeptide are bound by the modified linker forming the cDNA-polypeptide complex. In one aspect, disclosed herein is a method of sequentially inducing transcription, translation, and reverse transcription in one vessel, the method comprising preparing the modified linker of any preceding aspect, providing a DNA template, transcribing the DNA template into an mRNA, crosslinking the modified linker to the mRNA, wherein the crosslinking nucleotide binds to the mRNA, degrading the DNA template, translating the mRNA into a polypeptide, wherein the polypeptide is transferred to the protein translation inhibitor, and reverse transcribing the mRNA into a complementary DNA (cDNA), wherein the cDNA and polypeptide are bound by the modified linker forming the cDNA-polypeptide complex. In one aspect, disclosed herein is a method of screening a panel of proteins, the method comprising preparing the modified linker of any preceding aspect, providing a DNA template, transcribing the DNA template into an mRNA, crosslinking the modified linker to the mRNA, wherein the crosslinking nucleotide binds to the mRNA, degrading the DNA template, translating the mRNA into a polypeptide, wherein the polypeptide is transferred to the ribosome binding molecule, reverse transcribing the mRNA into a complementary DNA (cDNA), wherein the cDNA and polypeptide are bound by the modified linker forming the cDNA-polypeptide complex, and combining and incubating the cDNA-polypeptide complex with a panel of proteins to capture one or more proteins that bind to the polypeptide. In some embodiments, the second nucleic acid comprises a sequence complementary to the DNA template. In some embodiments, a light source enhances crosslinking of the modified linker to the mRNA. In some embodiments, the light source comprises a wavelength of about 360nm or greater. In some embodiments, the light source comprises an ultraviolet light source. In some embodiments, the DNA template is transcribed into the mRNA by one or more enzymes selected from RNA polymerases and transcription factors. In some embodiments, the mRNA is translated into the polypeptide by one or more biomolecules selected from a ribosome, a peptidyl transferase, and a transfer RNA synthetase. In some embodiments, the mRNA is reverse Docket No.11164-018WO1 transcribed into the cDNA by one or more enzymes selected from a reverse transcriptase and a DNA polymerase. In some embodiments, the method is performed within 3 hours. In some embodiments, the vessel comprises a tube, a cuvette, a dish, a microplate, or a partially enclosed or fully enclosed apparatus. In some embodiments, the one or more proteins are identified using one or more protein detection techniques comprising enzyme-linked immunosorbent assay (ELISA), western blot, mass spectrometry, high performance liquid chromatography, protein immunoprecipitation, and protein immunostaining. BRIEF DESCRIPTION OF FIGURES The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below. FIGS. 1A, 1B, and 1C show the click display of proteins on their encoding cDNA molecules. FIG. 1A show the design of the modified linker ML. FIGS. 1B and 1C show the overview of click display steps. Purified DNA template (1 µg / 25 µL reaction) is added directly to the in vitro protein synthesis reaction together with ML (final 2 µM). In vitro transcription, ML- mRNA crosslinking, in vitro translation, and reverse transcription reactions are carried out sequentially in a single tube to yield click-displayed protein-cDNA complexes. FIGS. 2A, 2B, and 2C show the ML synthesis by copper-free click chemistry. FIG. 2A shows the design of oligo #1 and oligo #2. FIG. 2B shows that the ML can be efficiently synthesized under ambient conditions. Oligo #1 and oligo #2 were mixed in at an equimolar ratio (250 µM of each oligo) and incubated at room temperature for 10 minutes.100 pmol of each oligo, either alone or following the 10-minute click reaction, was analyzed on a 15% Urea-PAGE gel that was first visualized under UV, and then stained with SYBR Green and visualized again. Only oligo #1 and ML are visible under UV without dye staining. FIG. 2C shows that the ML can be efficiently crosslinked to mRNA. After hybridization, the mixtures of mRNA and ML were irradiated under UV for 30, 60 or 90s before being analyzed on a 6% Urea-PAGE gel. Only ML- crosslinked mRNA is visible on unstained gels. Due to its small size, ML is not visible on the 6% Urea-PAGE gel but can be seen on a 15% Urea-PAGE gel (FIG.9). FIGS.3A, 3B, and 3C show the protein display. FIG.3A shows the comparison of protein display efficiency of mRNA-ML at different concentrations. The product was analyzed on 12% SDS-PAGE gels and visualized under UV without staining. Only ML (containing the fluorescent oligo #1) and protein covalently linked to ML are visible on the gel. p.mRNA-ML: partially Docket No.11164-018WO1 digested mRNA-ML. FIG.3B shows the comparison of the protein display efficiency of reactions containing 1 µg of DNA template and 2 or 3 µM of ML to that with 0.5 µM of mRNA-ML. FIG. 3C shows the schematic for terminating the transcription reaction. Red triangle represents the BsaI recognition site. FIG. 4 shows the confirmation of one-pot reverse transcription reaction. RT: Reverse Transcriptase. SS-II: SuperScript II; SS-IV: SuperScript IV. Ct: cycle threshold. The qPCR product was further analyzed on a 1% agarose gel. FIGS. 5A and 5B show the characterization of click displayed D3G86. FIG. 5A shows the scheme of the pull-down experiment. FIG.5B shows the qPCR quantification of the D3G86-cDNA complex pulled down by streptavidin beads and the agarose gel electrophoresis of the PCR product. One representative experiment with the mean of two technical replicates ± standard error is shown. FIG.6 shows the frequency of D3G86sequence in each round during the mock enrichment. FIGS. 7A, 7B, 7C, and 7D show the characterization of ClfA-binding DARPin clones. ClfA-binding affinity measurement by ELISA (FIG. 7A) and SDS-PAGE (FIG. 7B) of IMAC- purified DARPins selected from six rounds of panning on ClfA. FIG.7C shows the illustration of the sequences in the variable residues of the different DARPins. FIG. 7D shows the AlphaFold- predicted structure of D1F8. Variable residues comprising the binding interface (randomized during library creation) are shown in red / stick form. FIGS. 8A and 8B show the gel analysis of ML synthesis reaction. Oligo #1 and Oligo #2 were mixed in at equal molar ratio (250 µM of each oligo) and reacted at room temperature for 10 minutes or 1, 2 or 3 days. The equivalent amount of each oligo (100 pmol) was analyzed on a 15% Urea-PAGE gel that was first visualized under UV, and then stained with SYBR Green and visualized again. Only Oligo #1 and ML are visible under UV without dye staining. FIG. 9 shows that the ML can be efficiently crosslinked to mRNA. After hybridization, the mixtures of mRNA and ML were irradiated under UV for 30, 60 or 90s before being analyzed on 15% or 6% Urea-PAGE gels. Only Oligo #1, ML and ML-crosslinked mRNA are visible on unstained gels. The band intensity for unreacted ML at 0s appears to be reduced with the appearance of a smear above the original band. This is likely due to hybridization of ML with mRNA which is partially denatured during electrophoresis. FIGS. 10A and 10B shows qRT-PCR and ELISA datasets. FIG. 10A shows the Ct differences (ΔCt) between samples from representative rounds incubated in the absence and presence ClfA. FIG.10B shows the ELISA-based screen to identify individual DARPin molecules able to bind ClfA. Docket No.11164-018WO1 FIG.11 shows the sequences of the selected ClfA-binding DARPins from ELISA screen. FIG.12 shows the counts of different DARPin sequences from the R6 library. DETAILED DESCRIPTION The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof. Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Terminology Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise. The following definitions are provided for the full understanding of terms used in this specification. The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within Docket No.11164-018WO1 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%. As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient. “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. A “protein”, "polypeptide", or “peptide” each refer to a polymer of amino acids and does not imply a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, antibody, and enzyme are included within the definition of polypeptide. This term also includes polypeptides with post-expression modification, such as glycosylation (e.g., the addition of a saccharide), acetylation, phosphorylation, and the like. The term “incubate”, or other forms of “incubate”, such as “incubating” or “incubated”, refers to a process of establishing or maintaining an environment under conditions favorable to induce an event, such as an interaction, a reaction, or a development. The term “screening” refers to a method especially used in drug discovery in which data processing / control software, liquid handling devices, and sensitive detectors can allow for quick conductions of chemical, genetic, or pharmacological tests. This process allows one to quickly recognize active compounds, antibodies, or genes that modulate a particular biomolecular pathway. The results of these processes provide starting points for drug design. As used herein, a “linker” refers to a DNA molecule comprising at least 2 DNA oligonucleotides fused or linked together. In some embodiments, the linkers can be at least 10 or Docket No.11164-018WO1 more base pairs (bps). In some embodiments, the linkers can be modified to incorporate one or more modifications, including but not limited to a chemical moiety (i.e.; azides, alkynes, methyl groups, poly adenylate tails, or untranslated regions (UTRs)), an antibiotic, an inhibitor, a modified nucleotide, or any other molecules or compounds. A “nucleotide” is a compound consisting of a nucleoside, which consists of a nitrogenous base and a 5-carbon sugar, linked to a phosphate group forming the basic structural unit of nucleic acids, such as DNA or RNA. The four types of nucleotides are adenine (A), cytosine (C), guanine (G), and thymine (T), each of which are bound together by a phosphodiester bond to form a nucleic acid molecule. A “crosslinking nucleotide” refers to a nucleotide derivative that recognizes and binds at least one nucleic acid molecule. The crosslinking nucleotide binds said nucleic acid molecules by covalently connecting the nucleic acids, wherein the crosslink links nucleotide residues within the same nucleic acid (intrastrand crosslinking) or from opposite nucleotide residues of two separate nucleic acid (interstrand crosslinking). A “nucleic acid” is a chemical compound that serves as the primary information-carrying molecules in cells and make up the cellular genetic material. Nucleic acids comprise nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). A chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material. The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No.7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol.215:403410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. Docket No.11164-018WO1 “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above). Percent identity may be measured over the length of an entire defined polynucleotide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured. A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett.174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide. As used herein, the term “complementary” refers to the property of nucleic acids, including DNA or RNA, wherein a single strand comprises base pairs that are attracted to the base pairs of another single strand. The base pairs or nucleotides that are attracted to each other are as follows: adenine (A) to uracil (U) or thymine (T), guanine (G) to cytosine (C) , and vice versa. An “enzyme” is a biological molecule, usually a protein or peptide, that acts under certain conditions, such as pH, temperature, and / or salt concentration, to accelerate biochemical reactions, either inside or outside of a tissue or cell. The molecules upon which enzymes initiate a reaction are called “substrates”, and the enzyme converts the substrates into different molecules called “products”. Enzyme functions are usually measured based on the enzyme “activity” which refers to the amount of substrate converted into a product or products by the enzyme within a given amount of time. As used herein, unless otherwise indicated, the term "alkynyl" alone or in combination refers to a monovalent aliphatic hydrocarbon radical having the indicated number of carbon atoms and at least one carbon-carbon triple bond. The radical may be a linear or branched chain and, where specified, optionally substituted with one to three suitable substituents as defined above. Illustrative examples of alkynyl groups include, but are not limited to, ethynyl, 1-propynyl, 2- Docket No.11164-018WO1 propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-pentynyl, 3-pentynyl, 1- hexynyl, 2-hexynyl, 3-hexynyl and the like. Preferred alkynyl groups include ethynyl, 1-propynyl and 2-propynyl. Preferred optional suitable substituents include halo, methoxy, ethoxy, cyano, nitro and amino. An “azide” refers to a linear, polyatomic anion comprising two double bonds binding three nitrogen atoms. Modified Linker Compositions The present disclosure provides a modified linker for use of preparing a complementary DNA (cDNA)-polypeptide complex for an in vitro protein display. Protein therapeutics, such as antibodies, have the potential to treat a plethora of human diseases. However, the number of available protein therapeutics remain quite limited due to difficulty associated with the engineering of protein binding molecules (referred to herein as “protein binders”). One of the most efficient methods for engineering protein binders is directed evolution in which a library of protein variants are screened to identify those with desired functions. A prerequisite for directed evolution is the display (linkage) of protein on (to) its coding nucleic acid sequence (DNA or RNA). Thus, there is a need to develop modified linker compositions that can effectively and efficiently bind and display a protein and its coding nucleic acid sequence. In one aspect, disclosed herein is a modified linker comprising a first nucleic acid linked to a second nucleic acid, wherein the first nucleic acid comprises a ribosome binding molecule and a first click chemistry molecule, and wherein the second nucleic acid comprises a second click chemistry molecule and a crosslinking nucleotide. In some embodiments, the first nucleic acid comprises two or more nucleotides selected from adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). In some embodiments, the first nucleic acid comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more nucleotides selected from adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). In some embodiments, the second nucleic acid comprises two or more nucleotides selected from adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). In some embodiments, the second nucleic acid comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, Docket No.11164-018WO1 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more nucleotides selected from adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). In some embodiments, the first nucleic acid and the second nucleic acid comprises DNA. In some embodiments, the first nucleic acid and the second nucleic acid comprises RNA. In some embodiments, the first nucleic acid and the second nucleic acid are covalently bound together. In some embodiments, the second nucleic acid comprises a sequence complementary to a target nucleic acid sequence. In some embodiments, the ribosome binding molecule comprises puromycin. As used herein, a “ribosome binding molecule” refers to a molecule, including but not limited to an antibiotic, an inhibitor, a peptide, a protein, or nucleic acid that prevents protein synthesis by targeting and binding a ribosomal site. In some embodiments, the ribosome binding molecule binds at ribosome site A to prevent the addition of incoming aminoacyl-transfer RNA (tRNA). In some embodiments, the modified linker is a component of a click chemistry reaction, click chemistry assay, or a composition thereof. “Click chemistry” refers to a chemical philosophy introduced by K. Barry Sharpless of The Scripps Research Institute, describing chemistry tailored to generate covalent bonds quickly and reliably by joining biomolecules comprising reactive groups, including but not limited to alkyne, azides, nitrones, and other chemical groups. Click chemistry does not represent a specific reaction, but is a class of reactions that mimic reactions found in nature. Click chemistry is used for joining two molecular entities of choice in a one-pot synthesis reaction. As used herein, a “one-pot” synthesis reaction refers to a strategy to improve the efficiency of one or more reactions, wherein the reactant (or starting material) is subjected to one or more reactions within one reaction vessel. The click chemistry approach was originally conceived as a method to rapidly generate complex substances by joining small subunits together in a modular fashion (Kolb et al. 2004, Angew Chem Int Ed 40: 3004-3031; Evans, 2007, Aust J Chem 60: 384-395). Various forms of click chemistry reactions are known in the art, such as the Huisgen 1,3-dipolar cycloaddition copper catalyzed reaction (Tornoe et al., 2002, J Organic Chem 67: 3057-3064), which is often referred to as the “click reaction”. Other alternatives include, but are not limited to cycloaddition reactions, such as, for example Diels-Alder, nucleophilic substitution reaction (especially to small, strained rings like epoxy and aziridine compounds), carbonyl chemistry formation of urea Docket No.11164-018WO1 compounds, and reactions involving carbon-carbon double bonds, such as, for example alkynes in thiol-yne reactions. The azide alkyne Huisgen cycloaddition reaction uses a copper catalyst in the presence of a reducing agent to catalyze the reaction of a terminal alkyne group attached to a first molecule. In the presence of a second molecule comprising an azide moiety, the azide reacts with the activated alkyne to for a 1,4-disubstituted 1,2,3-triazole. The copper catalyzed reaction occurs at room temperature and is sufficiently specific that purification of the reaction product is often not required (Rostovstev et al., 2002, J Org Chem 67: 3057). The azide and alkyne functional groups are largely inert towards biomolecules in an aqueous medium, allowing the reaction to occur in complex mediums. The triazole formed is chemically stable and is not subject to enzymatic cleavage, making the click chemistry product highly stable in biological systems. However, it should be noted that the copper catalysts are toxic to living cells, thus in some embodiments the modified linker is a component of a copper-free click chemistry reaction, click chemistry assay, or a composition thereof. A copper-free click reaction comprises covalent modifications of biomolecules to be compatible with living systems (Agard et al., 2004, J Am Chem Soc 126: 15046-15047). The copper-free reaction uses ring strain in place of the copper catalyst to promote a [3+2] azide- alkyne cycloaddition reaction. For example, cyclooctyne is a 8-carbon ring structure comprising an internal alkyne bond. The closed ring structure induces a substantial bond angle deformation of the acetylene, which is highly reactive with azide groups to form a triazole. This, cyclooctyne derivatives, such as, for example dibenzocyclooctyne (DBCO), may be used for copper-free click reaction without the toxic effects of copper catalysts. In some embodiments, the click reactions are modular, wide in scope, give high chemical yields, generate inoffensive byproducts, are stereospecific, exhibit a large thermodynamic driving force (>84 kJ / mol) to favor a reaction generating a single reaction product, and / or can be carried out under physiological conditions. In some embodiments, the click chemistry reaction exhibits high atom economy, can be carried out under simple reaction conditions, and use readily available starting materials and reagents. In some embodiments, the first click chemistry molecule comprises an alkyne compound, or any derivatives or variants thereof. In some embodiments, the first click chemistry molecule comprises a cyclooctyne compound, or any derivatives or variants thereof. In some embodiments, the alkyne comprises a dibenzocyclooctyne (DBCO) , or any derivatives or variants thereof. In some embodiments, the first click chemistry molecule comprises a nitrone compound, or any Docket No.11164-018WO1 derivatives or variants thereof. In some embodiments, the first click chemistry molecule comprises an azide compound, or any derivatives or variants thereof. In some embodiments, the second click chemistry molecule comprises an azide (N3) , or any derivatives or variants thereof. In some embodiments, the second click chemistry molecule comprises a sodium azide compound. In some embodiments, the second click chemistry molecule comprises a nitrone compound. In some embodiments, the second click chemistry molecule comprises an alkyne compound. In some embodiments, the second click chemistry molecule comprises a cyclooctyne compound. Examples of click chemistry reactants and reagents can be found in U.S. Patent No. 8,632,752 B2, herein incorporated by reference in its entirety for teaching alkyne, nitrone, azide, or other reactive compounds. In some embodiments, the crosslinking nucleotide comprises a photo sensitive or a light sensitive crosslinking nucleotide. In some embodiments, the crosslinking nucleotide is a UV- sensitive crosslinking nucleotide. In some embodiments, the crosslinking nucleotide comprises a 3-cyanovinylcarbazole nucleotide (CnvK). Methods of preparing an in vitro protein display Click chemistry encompasses a group of diverse linking reactions that are simple to perform, provide high yields, require no or minimal purification, and are versatile in joining diverse structures without the prerequisite of protection steps. Examples of click chemistry reactions can be found in “Click Chemistry, a Powerful Tool for Pharmaceutical Sciences, Pharm. Res. 2008 Oct; 25(10): 2216-2230”, herein incorporated by reference in its entirety for teaching the different types of click chemistry reactions. In one aspect, disclosed herein is a method of preparing an in vitro protein display using a click chemistry reaction. In some embodiments, the click chemistry reaction comprises a copper- free azide-alkyne cycloaddition reaction, a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, a strain- promoted alkyne-nitrone cycloaddition (SPANC) reaction, a strained alkene and azide [3+2] cycloaddition, a strained alkene and tetrazine inverse demand Diels-Alder reaction, a strained alkene and tetrazole photoclick reaction, or other variable reactions. In one aspect, disclosed herein is a method of preparing the modified linker of any preceding aspect, the method comprising providing a first nucleic acid comprising a first click chemistry molecule and a ribosome binding molecule, providing a second nucleic acid comprising a second click chemistry molecule and a crosslinking nucleotide, and reacting the first and second Docket No.11164-018WO1 click chemistry molecules to link the first nucleic acid to the second nucleic acid forming the modified linker. In some embodiments, the first click chemistry molecule comprises an alkyne compound, or any derivatives or variants thereof. In some embodiments, the first click chemistry molecule comprises a cyclooctyne compound, or any derivatives or variants thereof. In some embodiments, the alkyne comprises a dibenzocyclooctyne (DBCO) , or any derivatives or variants thereof. In some embodiments, the first click chemistry molecule comprises a nitrone compound, or any derivatives or variants thereof. In some embodiments, the first click chemistry molecule comprises an azide compound, or any derivatives or variants thereof. In some embodiments, the second click chemistry molecule comprises an azide (N3) , or any derivatives or variants thereof. In some embodiments, the second click chemistry molecule comprises a sodium azide compound. In some embodiments, the second click chemistry molecule comprises a nitrone compound. In some embodiments, the second click chemistry molecule comprises an alkyne compound. In some embodiments, the second click chemistry molecule comprises a cyclooctyne compound. In some embodiments, the first click chemistry molecule and the ribosome binding molecule are at opposite ends of the first nucleic acid. In some embodiments, the first click chemistry molecule is located at the five prime (5’) end of the first nucleic acid, and the ribosome binding molecule is located at the three prime (3’) end of the first nucleic acid. In some embodiments, the first click chemistry is located at the 3’ end of the first nucleic acid, and the ribosome binding molecule is located at the 5’ end of the first nucleic acid. In some embodiments, the second click chemistry molecule is located at the 5’ end of the second nucleic acid. In some embodiments, the second click chemistry molecule is located at the 3’ end of the second nucleic acid. In some embodiments, the second nucleic acid comprises a nucleic acid sequence complementary to a DNA template sequence. In some embodiments, the ribosome binding molecule comprises puromycin. In some embodiments, the ribosome binding molecule binds at ribosome site A to prevent the addition of incoming aminoacyl-transfer RNA (tRNA). In some embodiments, the first click chemistry molecule reacts with the second click chemistry molecule. In some embodiments, the crosslinking nucleotide comprises a 3-cyanovinylcarbazole nucleotide (CnvK). In some embodiments, the crosslinking nucleotide is located near the 5’ end of the second nucleic acid. In some embodiments, the crosslinking nucleotide is located at or near Docket No.11164-018WO1 the 3’ end of the second nucleic acid. In some embodiments, the crosslinking nucleotide is located anywhere between the 5’ end and the 3’ end of the second nucleic acid. The present disclosure teaches of the central dogma of molecular biology, which relates to the flow genetic information from DNA, to RNA, usually in the form of messenger RNA (mRNA), to polypeptides, or from RNA directly to polypeptides. One exception to this dogma relates to the reverse central dogma, which teaches that genetic information can flow in the reverse direction from RNA, usually mRNA, to DNA, also called reverse transcription. It should be noted that the reverse central dogma describes producing a copy of DNA that is complementary to the RNA sequence. Further teachings of the central dogma and reverse central dogma can be found at “Central Dogma of Molecular Biology, Nature. 08 August 1970; 227, 561-563”, “What history tells us XIII. Fifty years of the Central Dogma, Journal of Biosciences. June 2008, 171-175”, or “Reverse Transcription and the Central Dogma, Progress in Molecular and Subcellular Biology. 1973; 3, 1-14”, herein incorporated by reference for the teachings of transcription (flow of genetic information from DNA to mRNA), translation (flow of genetic information from mRNA to polypeptides), and / or reverse transcription (flow of genetic information from mRNA to DNA) processes. Thus in one aspect, disclosed herein is a method of preparing a complementary DNA (cDNA)-polypeptide complex, the method comprising preparing the modified linker of any preceding aspect, providing a DNA template, transcribing the DNA template into an mRNA, crosslinking the modified linker to the mRNA, wherein a crosslinking nucleotide binds to the mRNA, degrading the DNA template, translating the mRNA into a polypeptide, wherein the polypeptide is transferred to the ribosome binding molecule, and reverse transcribing the mRNA into a complementary DNA (cDNA), wherein the cDNA and polypeptide are bound by the modified linker forming the cDNA-polypeptide complex. In one aspect, disclosed herein is a method of sequentially inducing transcription, translation, and reverse transcription in one vessel, the method comprising preparing the modified linker of any preceding aspect, providing a DNA template, transcribing the DNA template into an mRNA, crosslinking the modified linker to the mRNA, wherein the crosslinking nucleotide binds to the mRNA, degrading the DNA template, translating the mRNA into a polypeptide, wherein the polypeptide is transferred to the protein translation inhibitor, and reverse transcribing the mRNA into a complementary DNA (cDNA), wherein the cDNA and polypeptide are bound by the modified linker forming the cDNA-polypeptide complex. In some embodiments, a light source enhances crosslinking of the modified linker to the mRNA. In some embodiments, the light source comprises a wavelength of about 100 nanometers Docket No.11164-018WO1 (nm) or greater. In some embodiments, the light source comprises a wavelength of about 100nm to about 400nm. In some embodiments, the light source comprises a wavelength of about 100nm, 101nm, 102nm, 103nm, 104nm, 105nm, 106nm, 107nm, 108nm, 109nm, 110nm, 111nm, 112nm, 113nm, 114nm, 115nm, 116nm, 117nm, 118nm, 119nm, 120nm, 121nm, 122nm, 123nm, 124nm, 125nm, 126nm, 127nm, 128nm, 129nm, 130nm, 131nm, 132nm, 133nm, 134nm, 135nm, 136nm, 137nm, 138nm, 139nm, 140nm, 141nm, 142nm, 143nm, 144nm, 145nm, 146nm, 147nm, 148nm, 149nm, 150nm, 151nm, 152nm, 153nm, 154nm, 155nm, 156nm, 157nm, 158nm, 159nm, 160nm, 161nm, 162nm, 163nm, 164nm, 165nm, 166nm, 167nm, 168nm, 169nm, 170nm, 171nm, 172nm, 173nm, 174nm, 175nm, 176nm, 177nm, 178nm, 179nm, 180nm, 181nm, 182nm, 183nm, 184nm, 185nm, 186nm, 187nm, 188nm, 189nm, 190nm, 191nm, 192nm, 193nm, 194nm, 195nm, 196nm, 197nm, 198nm, 199nm, 200nm, 201nm, 202nm, 203nm, 204nm, 205nm, 206nm, 207nm, 208nm, 209nm, 210nm, 211nm, 212nm, 213nm, 214nm, 215nm, 216nm, 217nm, 218nm, 219nm, 220nm, 221nm, 222nm, 223nm, 224nm, 225nm, 226nm, 227nm, 228nm, 229nm, 230nm, 231nm, 232nm, 233nm, 234nm, 235nm, 236nm, 237nm, 238nm, 239nm, 240nm, 241nm, 242nm, 243nm, 244nm, 245nm, 246nm, 247nm, 248nm, 249nm, 250nm, 251nm, 252nm, 253nm 254nm, 255nm, 256nm, 257nm, 258nm, 259nm, 260nm, 261nm, 262nm, 263nm, 264m, 265nm, 266nm, 267nm, 268nm, 269nm, 270nm, 271nm, 272nm, 273nm, 274nm, 275nm, 276nm, 277nm, 278nm, 279nm, 280nm, 281nm, 282nm, 283nm, 284nm, 285nm, 286nm, 287nm, 288nm, 289nm, 290nm, 291nm, 292nm, 293nm, 294nm, 295nm, 296nm, 297nm, 298nm, 299nm, 300nm, 301nm, 302nm, 303nm, 304nm, 305nm, 306nm, 307nm, 308nm, 309nm, 310nm, 311nm, 312nm, 313nm, 314nm, 315nm, 316nm, 317nm, 318nm, 319nm, 320nm, 321nm, 322nm, 323nm, 324nm, 325nm, 326nm, 327nm, 328nm, 329nm, 330nm, 331nm, 332nm, 333nm, 334nm, 335nm, 336nm, 337nm, 338nm, 339nm, 340nm, 341nm, 342nm, 343nm, 344nm, 345nm, 346nm, 347nm, 348nm, 349nm, 350nm, 351nm, 352nm, 353nm, 354nm, 355nm, 356nm, 357nm, 358nm, 359nm, 360nm, 361nm, 362nm, 363nm, 364nm, 365nm, 366nm, 367nm, 368nm, 369nm, 370nm, 371nm, 372nm, 373nm, 374nm, 375nm, 376nm, 377nm, 378nm, 379nm, 380nm, 381nm, 382nm, 383nm, 384nm, 385nm, 386nm, 387nm, 388nm, 389nm, 390nm, 391nm, 392nm, 393nm, 394nm, 395nm, 396nm, 397nm, 398nm, 399nm, 400nm, or more. In some embodiments, the light source comprises an ultraviolet light source. In some embodiments, the method is performed within 3 hours. In some embodiments, the method is performed in about 30 minutes. In some embodiments, the method is performed in about 1 hour. In some embodiments, the method is performed in about 2 hours. In some embodiments, the method is performed in about 3 hours. In some embodiments, the vessel comprises a tube, a cuvette, a dish, a microplate, or a partially enclosed or fully enclosed apparatus. Docket No.11164-018WO1 Methods of screening, analyzing, measuring, and / or detecting proteins Acceleration of drug discovery research and development requires identification of protein targets and protein-protein interactions using target-based screening techniques. However, existing approaches require substantial investments of resources and time, and are often unsuccessful. Techniques of protein display (linkage), including mRNA display, ribosome display, and TRAP display are cell-free target-based screening techniques that provide protein library sizes up to 1014, however such techniques are labor intensive, unstable, and have low yields. Therefore, there is a need to develop a quick, high-yielding, and efficient technique to improve drug discovery screening practices. In one aspect, disclosed herein is a method of screening a panel of proteins, the method comprising preparing the modified linker of any preceding aspect, providing a DNA template, transcribing the DNA template into an mRNA, crosslinking the modified linker to the mRNA, wherein the crosslinking nucleotide binds to the mRNA, degrading the DNA template, translating the mRNA into a polypeptide, wherein the polypeptide is transferred to the ribosome binding molecule, reverse transcribing the mRNA into a complementary DNA (cDNA), wherein the cDNA and polypeptide are bound by the modified linker forming the cDNA-polypeptide complex, and combining and incubating the cDNA-polypeptide complex with a panel of proteins to capture one or more proteins that bind to the polypeptide. In one aspect, disclosed herein is a method for analyzing, measuring, and / or detecting a panel of proteins, the method comprising preparing the modified linker of any preceding aspect, providing a DNA template, transcribing the DNA template into an mRNA, crosslinking the modified linker to the mRNA, wherein the crosslinking nucleotide binds to the mRNA, degrading the DNA template, translating the mRNA into a polypeptide, wherein the polypeptide is transferred to the ribosome binding molecule, reverse transcribing the mRNA into a complementary DNA (cDNA), wherein the cDNA and polypeptide are bound by the modified linker forming the cDNA-polypeptide complex, combining and incubating the cDNA-polypeptide complex with a panel of proteins to capture one or more proteins that bind to the polypeptide, and detecting and / or measuring the interaction between the cDNA-polypeptide complex and the one or more proteins using one or more biochemical protein detection methods including but not limited to western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, antibody detection, gas or liquid chromatography (LC or GC), and enzyme linked immunospot assay (ELISpot). Docket No.11164-018WO1 In some embodiments, the method analyzes, measures, and / or detects protein-protein interactions between the protein display and the one or more proteins. In some embodiments, the one or more proteins are identified using one or more protein detection techniques including, but is not limited to enzyme-linked immunosorbent assay (ELISA), western blot, mass spectrometry, high performance liquid chromatography, protein immunoprecipitation, and protein immunostaining. In some embodiments, the method of screening, analyzing, measuring, and / or detecting a panel of proteins comprises a DNA template that is transcribed into the mRNA by one or more enzymes selected from RNA polymerases and transcription factors. In some embodiments, the method of screening, analyzing, measuring, and / or detecting a panel of proteins comprises the mRNA being translated into the polypeptide by one or more biomolecules selected from a ribosome, a peptidyl transferase, and a transfer RNA synthetase. In some embodiments, the method of screening, analyzing, measuring, and / or detecting a panel of proteins comprises the mRNA being reverse transcribed into the cDNA by one or more enzymes selected from a reverse transcriptase and a DNA polymerase. In some embodiments, the method of screening, analyzing, measuring, and / or detecting a panel of proteins comprises a light source for enhancing the crosslinking of the modified linker to the mRNA. In some embodiments, the light source comprises a wavelength of about 100 nanometers (nm) or greater. In some embodiments, the light source comprises a wavelength of about 100nm to about 400nm. In some embodiments, the light source comprises a wavelength of about 100nm, 101nm, 102nm, 103nm, 104nm, 105nm, 106nm, 107nm, 108nm, 109nm, 110nm, 111nm, 112nm, 113nm, 114nm, 115nm, 116nm, 117nm, 118nm, 119nm, 120nm, 121nm, 122nm, 123nm, 124nm, 125nm, 126nm, 127nm, 128nm, 129nm, 130nm, 131nm, 132nm, 133nm, 134nm, 135nm, 136nm, 137nm, 138nm, 139nm, 140nm, 141nm, 142nm, 143nm, 144nm, 145nm, 146nm, 147nm, 148nm, 149nm, 150nm, 151nm, 152nm, 153nm, 154nm, 155nm, 156nm, 157nm, 158nm, 159nm, 160nm, 161nm, 162nm, 163nm, 164nm, 165nm, 166nm, 167nm, 168nm, 169nm, 170nm, 171nm, 172nm, 173nm, 174nm, 175nm, 176nm, 177nm, 178nm, 179nm, 180nm, 181nm, 182nm, 183nm, 184nm, 185nm, 186nm, 187nm, 188nm, 189nm, 190nm, 191nm, 192nm, 193nm, 194nm, 195nm, 196nm, 197nm, 198nm, 199nm, 200nm, 201nm, 202nm, 203nm, 204nm, 205nm, 206nm, 207nm, 208nm, 209nm, 210nm, 211nm, 212nm, 213nm, 214nm, 215nm, 216nm, 217nm, 218nm, 219nm, 220nm, 221nm, 222nm, 223nm, 224nm, 225nm, 226nm, 227nm, 228nm, 229nm, 230nm, 231nm, 232nm, 233nm, 234nm, 235nm, 236nm, 237nm, 238nm, 239nm, 240nm, 241nm, 242nm, 243nm, 244nm, 245nm, 246nm, 247nm, 248nm, 249nm, 250nm, 251nm, 252nm, 253nm 254nm, 255nm, 256nm, 257nm, 258nm, 259nm, 260nm, 261nm, 262nm, 263nm, 264m, 265nm, 266nm, Docket No.11164-018WO1 267nm, 268nm, 269nm, 270nm, 271nm, 272nm, 273nm, 274nm, 275nm, 276nm, 277nm, 278nm, 279nm, 280nm, 281nm, 282nm, 283nm, 284nm, 285nm, 286nm, 287nm, 288nm, 289nm, 290nm, 291nm, 292nm, 293nm, 294nm, 295nm, 296nm, 297nm, 298nm, 299nm, 300nm, 301nm, 302nm, 303nm, 304nm, 305nm, 306nm, 307nm, 308nm, 309nm, 310nm, 311nm, 312nm, 313nm, 314nm, 315nm, 316nm, 317nm, 318nm, 319nm, 320nm, 321nm, 322nm, 323nm, 324nm, 325nm, 326nm, 327nm, 328nm, 329nm, 330nm, 331nm, 332nm, 333nm, 334nm, 335nm, 336nm, 337nm, 338nm, 339nm, 340nm, 341nm, 342nm, 343nm, 344nm, 345nm, 346nm, 347nm, 348nm, 349nm, 350nm, 351nm, 352nm, 353nm, 354nm, 355nm, 356nm, 357nm, 358nm, 359nm, 360nm, 361nm, 362nm, 363nm, 364nm, 365nm, 366nm, 367nm, 368nm, 369nm, 370nm, 371nm, 372nm, 373nm, 374nm, 375nm, 376nm, 377nm, 378nm, 379nm, 380nm, 381nm, 382nm, 383nm, 384nm, 385nm, 386nm, 387nm, 388nm, 389nm, 390nm, 391nm, 392nm, 393nm, 394nm, 395nm, 396nm, 397nm, 398nm, 399nm, 400nm, or more. In some embodiments, the light source comprises an ultraviolet light source. In some embodiments, the method of screening, analyzing, measuring, and / or detecting a panel of proteins is performed within 3 hours. In some embodiments, the method of screening, analyzing, measuring, and / or detecting a panel of proteins is performed in about 30 minutes. In some embodiments, the method of screening, analyzing, measuring, and / or detecting a panel of proteins is performed in about 1 hour. In some embodiments, the method of screening, analyzing, measuring, and / or detecting a panel of proteins is performed in about 2 hours. In some embodiments, the method of screening, analyzing, measuring, and / or detecting a panel of proteins is performed in about 3 hours. In some embodiments, the method of screening, analyzing, measuring, and / or detecting a panel of proteins is performed within a vessel including, but not limited to a tube, a cuvette, a dish, a microplate, or a partially enclosed or fully enclosed apparatus. In some embodiments, the method of screening, analyzing, measuring, and / or detecting of a panel of proteins is coupled with a sequencing technique, wherein the mRNA or cDNA are sequenced. In some embodiments, the sequencing technique comprises next generation sequencing (NGS), RNA-seq, high-throughput sequencing, and polymerase chain reactions (including, but not limited to PCR and quantitative PCR (qPCR)). A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. Docket No.11164-018WO1 By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below. EXAMPLES The following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art. Example 1: Click display - A rapid and efficient in vitro protein display method for directed evolution A novel method for in vitro protein display– click display – was developed that does not depend on maintaining RNA integrity during biopanning and yields covalently linked protein- cDNA complexes from double-stranded input DNA within 2 hours. The display is achieved in a one-pot format encompassing transcription, translation, and reverse transcription reactions in series. Stable linkage between proteins and the encoding cDNA is mediated by a modified DNA linker – ML – generated via a click reaction between a puromycin-containing oligo and a cDNA synthesis primer. Biopanning of a click-displayed mock library coupled with next-generation sequencing analysis revealed >600-fold enrichment of target binders within a single round of panning. A synthetic library of designed ankyrin repeat proteins (DARPins) with ~1012individual members was generated using click display in a 25-µL reaction and six rounds of library panning against a model protein yielded a panel of nanomolar binders. This example establishes click display as a powerful tool for protein binder discovery / engineering and provides a convenient platform for in vitro biopanning selection even in RNase-rich environments such as on whole cells. One of the earliest cell-free display technologies, mRNA display, takes advantage of the ability of puromycin to enter the ribosomal A site and attach to the nascent peptide. Specifically, a single-stranded DNA (~30 nt) harboring a puromycin molecule at its 3’ end is ligated to the coding mRNA to enable the formation of protein-mRNA conjugates upon translation. This method, for the first time, enabled cell-free protein engineering and greatly expanded the library size accessible to directed evolution. However, major drawbacks of this system are the poor ligation efficiency, making the method highly labor-intensive, and dependence of RNase-free conditions to preserve RNA integrity. Docket No.11164-018WO1 Ribosome display, another in vitro protein display technology, utilizes a spacer sequence and a stem loop structure at the 3’ end of the mRNA to ‘trap’ the ribosome, thus bypassing the need for mRNA-DNA ligation. Upon translation, this spacer sequence remains attached to the peptidyl tRNA and occupies the ribosomal tunnel, allowing the protein of interest to protrude out of the ribosome. Although ribosome display is procedurally simpler than mRNA-display, its dependence on mRNA and ribosomes for display confers significant limitations: i) since the ribosome is a large protein-RNA complex, unpredictable interactions between the ribosome and the displayed protein can bias the selection of binder proteins; ii) the peptidyl- tRNA / mRNA / ribosome complex is intrinsically unstable, requiring the selection steps to be performed before reverse transcription (RT), under a high magnesium concentration and at low temperatures (e.g.4˚C), fostering the selection of variants with restricted biological relevance; iii) a strictly RNase-free environment is necessary to preserve the integrity of the mRNA during panning, making the success of ribosome display experiments highly sensitive to even small differences in experimental practices and reagents / consumables. TRAP technology is another in vitro display approach that couples mRNA transcription, protein translation and reverse transcription to dramatically reduce the time needed for protein display. In TRAP display, a single-stranded DNA linker functionalized with a puromycin molecule at its 3’ end is hybridized to the mRNA in the transcription / translation reaction mixture that lacks Release Factor. The mRNA contains a STOP codon immediately preceding the mRNA / DNA hybridization region which stalls the ribosome and enables the puromycin to be incorporated into the nascent protein. The display efficiency was later increased by using 2’-Ome- modified DNA linker to suppress promoter-independent transcription and unwanted DNA / RNA duplexes. Most recently, the same group created a branched DNA to covalently link the nascent protein with the reverse transcribed 1ststrand cDNA to obviate the need for RNase-free conditions during biopanning. Although conceptually elegant and simple, it is a non-trivial task to synthesize the branched DNA linker which is not currently commercially available and suffers from very low synthesis yield (<0.5% to 4%). In this example, a method for in vitro protein display – termed click display – is described that employs reagents that are readily available from commercial sources and produces covalently linked protein-cDNA product from coding DNA in a one-pot reaction within 2 hours. Covalent linkage of the protein variants and their encoding cDNA molecules is achieved using a modified DNA linker – ML – generated by copper-free click reaction between two oligos (FIG.1A): oligo #1 contains a puromycin molecule at the 3’ end and a click chemistry moiety (i.e., dibenzocyclooctyne (DBCO)) at the 5’ end; oligo #2 is complementary to the 3’ end of the mRNA Docket No.11164-018WO1 and harbors a matching click chemistry moiety (i.e. azide (N3)) at its 5’ end. The highly efficient and specific click chemistry reaction between DBCO and N3 enables the modified oligo ML to be produced with very high efficiency under physiological conditions, obviating the need for purification. Oligo #2 also contains a 3-cyanovinylcarbzaole nucleotide (CnvK, red line in FIG. 1A), enabling ML to be covalently crosslinked to the mRNA with nearly 100% efficiency upon brief exposure to long wavelength UV (>360 nm). Both oligos #1 and #2 are commercially available and the crosslinked mRNA-ML can be efficiently produced using a nail spa UV lamp, allowing click display to be readily performed in any molecular biology lab. The click display reaction was optimized for the in vitro protein synthesis mixture based on the PURExpress In Vitro Protein Synthesis Kit (NEB) which contains all the components needed for in vitro transcription and translation. Template DNA harboring a T7 promoter and ribosome binding site (RBS) is added directly to the in vitro protein synthesis reaction mixture (1 µg DNA per 25 µL reaction) alongside ML (final 2 µM, FIG. 1B). Transcription, ML-mRNA crosslinking, translation, and reverse transcription are carried out sequentially in a one-pot reaction to yield nascent protein-cDNA complexes that can be used directly for biopanning (FIG. 1C). Synthesis of protein-cDNA complex from the input DNA template is complete within 2 hours (Table 1). The ability of click-displayed libraries to support the selection of rare binders to a desired target protein was demonstrated using two complementary approaches. First, a click-displayed mock DARPin library containing 0.01% of a known eGFP-binding DARPin was panned against biotinylated eGFP. This approach revealed that 625-fold enrichment of the eGFP-binding DARPin was achieved in a single-round of selection, representing a 6-12-fold enhancement in enrichment efficiency over conventional mRNA-display. Next, a naïve DARPin library with ~1012variants was displayed in a 25-µL click-display reaction and used to identify a panel of binders to a model target protein – clumping factor A (ClfA) – following 6 rounds of panning. Several of the selected ClfA binders showed nanomolar target binding affinity as determined by ELISA. Materials and Methods DNA / mRNA Synthesis and Purification. The template DNA sequence used for the click display of D3G86is shown in SEQ ID NO: 1. For optimizing the reaction condition using mRNA- ML, this DNA template was PCR-amplified using primers 1938 and 2643 (Table 2) and use as the template for mRNA synthesis (1 µg per 20 µL reaction, HiScribe™ T7 High-Yield RNA Synthesis Kit (New England Biolabs, E2040S)). At the end of the reaction, 70 µL nuclease-free water, 10 µL 10x DNase I Buffer, and 2 µL DNase I (2 U / µL, New England Biolabs, M0303S) Docket No.11164-018WO1 were added and the mixture was incubated at 37 ºC for another 15 minutes to degrade the template DNA. The synthesized mRNA was purified using EZNA MicroElute RNA Cleanup Kit (VWR, 101319-190), eluted in nuclease-free water and stored at -80ºC in aliquots until use. To construct a naïve DARPin library for click display, synthetic oligos AR-F2 and AR-R2 (Table 2, final 1.6 µM), encoding all the variable regions, were incubated in 1x Isothermal Amplification Buffer (New England Biolabs, B0537S) in the presence of MgSO4 (final 6 mM) and dNTP (final 1.4 mM each) at 94 ºC for 2 minutes followed by slow cooling (-0.1 ºC / second) to 65 ºC before the addition of Bst2.0 (final 320 U / mL). The mixture was incubated at 65ºC for ~30 minutes to generate double-stranded DARPin variable region (DVR). The synthesized DNA product was purified from agarose gel. The N-cap and C-cap region of D3G86 were amplified by PCR using primers 2617 / 2701 and 2702 / 2581, respectively, digested with BsaI and ligated to BsaI-digested DVR. The final ligation product (700 bp) was loaded on an agarose gel, and, after gel purification, 650 ng DNA product was recovered. This amount of DNA corresponds to 1.4 pmol or 8.5 x 1011different DNA molecules. The entire amount of DNA was used as template for PCR amplification using primers 1938 / 2643 and Taq polymerase in a total of 3.2 mL of PCR reaction to preserve the library diversity. The final DNA product was purified again on agarose gel and used for click display (1 µg per 25 µL reaction). Synthesis of ML and mRNA-ML. Oligo #1 and #2 (Table 2) were custom-synthesized by IDT (Integrated DNA Technologies, Inc., IA) and resuspended in DNA duplex buffer (Integrated DNA Technologies, Inc., 11-01-03-01) to achieve a final concentration of 0.5 mM. An equal volume of oligo #1 and oligo #2 were mixed in a thin-wall PCR tube (Fisher Scientific, 14230225) and incubated at room temperature in the dark. The reaction product – ML – was diluted 10-fold in DNA duplex buffer and stored at -80 °C until use. To estimate the reaction efficiency, an aliquot of the reaction mix was removed at 10 minutes, 1 day or 2 days, diluted 10- fold immediately in water (final 25 µM of each oligo) and stored at -20ºC until analysis on a 15% Urea-PAGE gel (Novex, EC6885BOX) and visualized under UV exposure both before and after staining with SYBR Green dye. mRNA-ML was prepared by incubating purified mRNA (final 1-8 µM) with ML (1:1.2 molar ratio) in DNA duplex buffer in the presence of RNasin Plus RNase inhibitor (final 1 U / µL, Promega, PRN2611) in a thin-wall PCR tube. To maximize the hybridization efficiency between mRNA and ML, the mixture was heated at 94 ºC for 1 minute, slowly cooled to 70 ºC at -0.1 ºC / second, incubated at 70 ºC for 5 minutes and finally slowly cooled to 25 ºC at the same rate and held at 25°C for an additional 15 minutes. After hybridization, the mixture was irradiated Docket No.11164-018WO1 under a nail spa UV lamp (SUNUV Gel Nail Light, Model SUNONEWT, Amazon) for 30-90s. The samples were resuspended in 2xRNA buffer (TBE-Urea Sample Buffer 2x, G-Biosciences, 786-474) and incubated at 70 ºC for 10 minutes to denature the mRNA. The crosslinking efficiency was analyzed using 15% and 6% Urea-PAGE gels (2 pmol mRNA per lane, Novex™ TBE-Urea Gels, EC6885BOX, EC6865BOX) in warm 1xTBE buffer (55 ºC) under a constant voltage of 180V and 100 V, respectively. Click Display Translation Optimization. To compare the amount of click displayed product (FIG. 3A), different amount of input mRNA-ML was added to the translation reaction mixture (New England Biolabs, PURExpress in vitro protein synthesis, E6800). The mixture was incubated at 37 ºC for 30 minutes with shaking followed by 20 minutes incubation at room temperature without shaking. After translation, RNase A (final 1 µg / µL, Omega Bio-Tek, AC118) was added to degrade the mRNA, and the mixture was incubated at 37 ºC for another 30 minutes. For gel analysis, the samples were first diluted 2-fold in 2x gel shift buffer (50%(v / v) formamide, 125 mM Tris-HCl pH 6.8, 10mM DTT, 20 mM EDTA and 2% (w / v) SDS), heat inactivated at 95 ºC for 5 minutes, and then analyzed on 12% SDS-PAGE gels (Bio-Rad, Cat# 4561043). After electrophoresis, the gel was visualized under UV exposure without staining. To evaluate the click display efficiency from DNA template (FIG. 3B), 1 µg (600 nM) template DNA encoding D3G86, T7 promoter and terminator (SEQ ID NO: 1) was added directly to 1 reaction volume of PURExpress in vitro protein synthesis reaction together with 2 or 3 µM of ML. The reaction mixture was incubated at 37 ºC for 20 minutes to allow for mRNA transcription before the addition of BsaI-HF (final 1.6 U / µL, New England Biolabs, R3535) to cleave the template DNA and terminate the transcription. The mixture was then incubated at 37 ºC for 10 minutes without shaking, for DNA digestion, and 30 minutes with vigorous shaking for protein translation, and was finally incubated for another 20-30 minutes at room temperature to allow for protein transfer. RNase A (Omega Bio-Tek, AC118, final 1µg / µL) was then added to degrade the mRNA and the mixture was diluted 2-fold in 2x gel shift buffer before being analyzed on 12% SDS-PAGE gels (Bio-Rad, 4561043). After electrophoresis, the gels were visualized under UV directly without staining. Click Display Reverse Transcription Condition Optimization. Click display was carried out as described above using mRNA-ML (final 0.5 µM, 25 µL reaction) as the template. After translation, EDTA (final 30 mM, ThermoFisher Scientific, AM9260G) was added to the reaction to inactivate the ribosome, followed by MgCl2(300 mM x 3 µL) to neutralize the excess EDTA. The reaction volume was then expanded 3-5 fold before the addition of dNTP (final 0.5mM), SuperScript II (SS-II, final 2.7 U / µL) or SuperScript IV (SS-IV, final 2.7 U / µL) reverse Docket No.11164-018WO1 transcriptase and the appropriate reaction buffer. The mixture was incubated at 42 ºC (for SS-II) or 50 ºC (for SS-IV) for 30 minutes, diluted 100-fold in ddH2O and the amount of the synthesized cDNA was quantified using Forget-Me-Not™ EvaGreen® qPCR Master Mix (Biotium, 31045) with primers 1892 and 2488 (Table 2) in a qPCR instrument (Bio-Rad, C1000 Touch™ Thermal Cycler and CFX 96 Real-Time PCR Detection Systems). After the reaction, the PCR product was also analyzed on 1% agarose gels to ensure the amplification of the correct target DNA. Protein Expression and Purification. ClfA (SEQ ID NO: 3), eGFP (SEQ ID NO: 2) and all the DARPin molecules were expressed in E. coli Bl21 (DE3) cells in LB medium and purified using gravity Ni-NTA agarose beads following the standard procedure. Protein purity was determined using 12% SDS-PAGE gels. Streptavidin Pull-Down Assay. eGFP and ClfA were biotinylated using EZ-Link Sulfo- NHS-LC-Biotin (1:4 molar ratio, Fisher Scientific, PI21335). Click-displayed D3G86 was diluted 10-fold in blocking buffer SBTD0.3 (StartingBlock™ Blocking Buffer (Thermo Fisher Scientific, 37578) supplemented with 0.05% Tween-20, and 0.3 mg / mL ssDNA (Deoxyribonucleic acid sodium salt from salmon testes, Sigma, D1626)) in the absence or presence of 200 nM biotinylated eGFP. The mixture was incubated at room temperature for 1 hour before the addition of streptavidin-coated magnetic beads (Dynabeads™ MyOne™ Streptavidin T1, Thermo Fisher Scientific, 65-601) to pull-down the biotinylated eGFP and the associated cDNA. The beads were washed 8 times with SBTD0.3. After the final wash, the beads were resuspended 1x Taq ThermoPol®Reaction Buffer (New England Bio Labs, B9004S) and the amount of cDNA associated with the beads was quantified using Forget-Me-Not qPCR kit with primers 1892 and 2488. To purify click-displayed product via IMAC, the reaction mixture after reverse transcription was either buffer-exchanged into PBS (50 mM NaH2PO4, 300 mM NaCl,) via ultrafiltration using 30 kDa MWCO Amicon columns (Amicon™ Ultra-0.5 Centrifugal 30kDa MWCO Filter Units, MilliporeSigma™, UFC503024) or diluted 10-fold in PBS+10 mM imidazole to reduce the DTT concentration before the addition of Ni-NTA agarose bead slurry (company Pierce™ Ni-NTA Magnetic Agarose Beads, 78605). His-tagged proteins were purified following the standard protocol. Library Enrichment. To estimate the enrichment efficiency, 1 µg of DNA encoding a naïve N3C DARPin library was mixed with 0.1 ng of DNA encoding D3G86 and used as template for click display (Table 1). After reverse transcription, the mixture was purified by one-step IMAC and incubated with 200 nM biotinylated eGFP at room temperature for 1 hour. Streptavidin T1 or neutravidin-coated magnetic beads (Cytiva SpeedBeads Magnetic Neutravidin Coated Particles, Docket No.11164-018WO1 Fisher Scientific, 09981154) were added to the mixture to pull down biotinylated eGFP and the associated click-displayed product. The beads were washed 3 to 8 times for rounds 1 to 3. After the final wash, the beads were resuspended in 1x Standard Taq buffer and the cDNA was amplified by PCR using Taq polymerase (NEB, M0267S) and primers 1938 and 2643, which contain the T7 promoter and terminator region, respectively. The PCR product was used as the template for the subsequent round of panning after gel purification. To enrich binders to ClfA, click-displayed naïve DARPin library made from 1 µg of DNA was used. This library was purified by IMAC and incubated with biotinylated ClfA (final 200 nM). ClfA-binders were pulled down using streptavidin- or neutravidin-coated beads in alternative rounds. Next-Generation Sequencing (NGS). NGS samples were prepared by amplifying the PCR product from different rounds of panning (R0-R3) using NGS primers containing the Illumina adapter sequences (Table 2). The different rounds were distinguished using a two- nucleotide barcode inserted between the Illumina adapter and the complementary region of the primers. The selected cDNA from each round was amplified with its respective primer pair using Q5 High-Fidelity Polymerase (New England Biolabs, M0491). Each round was then purified using the ZymoClean Gel DNA Recovery Kit (Zymo Research), after which 150 ng of the DNA from each round was combined. Samples were sequenced using a Mi-Seq Next-Generation Sequencing system from Amplicon-EZ (Azenta Life Sciences). Paired-end reads were merged using FLASH. Sequences were converted to FASTA format, translated, and analyzed using SeqKit. ELISA. The ELISA experiment was carried out essentially. Briefly, the MaxiSorp immune plates (Nunc MaxiSorp plates, Fisher Scientific, 50-712-278) was coated with ClfA (4 µg / mL) over night. All DARPins contain Myc- and His-tag at the N-terminus. The next day, after blocking the plate with DPBSTB (DPBS with 0.1% Tween-20 and 2% BSA), serially diluted IMAC-purified DARPin was added to the plate. After incubation and washing, well-bound DARPin proteins were detected using mouse anti-c-Myc antibody (Invitrogen, 13-2500) and horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Jackson Immuno Research, 115-035-146) followed by color development using TMB (3,3', 5,5”-tetramethylbenzidine) substrate. Model Generation. The 3D model of D1F8 was generated using AlphaFold at Texas A&M High Performance Research Computing (HPRC) facility. The top 5 predicted models were nearly identical, and the lowest ranked model was used in this study. Docket No.11164-018WO1 Results Synthesis of ML and mRNA-ML The puromycin-containing linker ML was produced by reacting to oligo #1 and oligo #2 (FIG. 2A). Oligo #1 contains a fluorescently labeled nucleotide (TAMRA) to facilitate the subsequent gel analysis. Oligo #2 is complementary to the mRNA and can be crosslinked to the mRNA via the cnvK (K) nucleotide upon UV irradiation. These oligos were mixed at an equimolar ratio (250 µM each oligo) in DNA duplex buffer (IDT) and incubated at room temperature. After only 10 minutes of incubation, nearly all the input oligos have reacted to yield ML (FIG. 2B and FIG.8). The reaction efficiency is estimated to be >90% by densitometry analysis. To simplify the calculation of the ML input amounts, for subsequent steps, 100% ML synthesis efficiency was assumed. Next, the ability of ML to be specifically crosslinked to mRNA was confirmed. Purified mRNA (final 1 µM), whose 3’ end is complementary to oligo #2 (FIG.2A), was mixed with ML at 1:1.2 molar ratio in DNA duplex buffer in a thin-walled PCR tube (20 µL total). The mixture was heated to 94 ºC for 1 minute to denature the mRNA and then slowly cooled to 25ºC for hybridization. Finally, the mixture was irradiated under a nail spa UV lamp for 30, 60 and, 90 seconds and analyzed on Urea-PAGE gels. Over 70% of the input mRNA was crosslinked to ML after 90 seconds UV irradiation as determined by densitometric analysis, with free ML being nearly completely depleted (FIG. 2C and FIG. 9). Although the crosslinking efficiency was marginally higher with 90 seconds UV irradiation, to maximally preserve the mRNA integrity, 30 seconds irradiation time was selected for all subsequent experiments. To simplify the calculation of mRNA-ML input amounts for subsequent steps, it was assumed that all the input mRNA is converted to mRNA-ML. Optimization of Conditions for Protein Display To demonstrate the ability for protein display, mRNA-ML was used as template for protein translation using the PURExpress In Vitro Protein Synthesis Kit (NEB). Considering that each PURExpress in vitro protein synthesis reaction contains 2.4 µM of ribosome (60 pmol per 25 µL reaction), and that ribosomes in association with the mRNA are not actively released due to mRNA-ML crosslinking at the cnvK site, the yield of protein display with 0.5, 0.75 and 1 µM mRNA-ML was compared. The protein translation reaction was carried out at 37 ºC for 30 minutes followed by another 20 minutes incubation at room temperature to facilitate the transfer of the nascent protein to puromycin. Due to the large size of mRNA, it is difficult to distinguish free mRNA and mRNA crosslinked to ML and protein. To simplify the gel analysis, RNase A was added to the reaction mixture to degrade the single-stranded mRNA. Similar amounts of protein- Docket No.11164-018WO1 ML were observed in all reactions, suggesting that the display efficiency is likely saturated at 0.5 µM mRNA-ML (FIG.3A), corresponding to 7.5x1012mRNA molecules per 25 µL reaction. Since mRNA in duplex with ML is partially protected from RNase A digestion, both ML and partially digested mRNA-ML (p.mRNA-ML) are visible on the gel. In the reaction containing 1 µM mRNA-ML, a large amount of p.mRNA-ML is also visible on the gel, indicating that excess of mRNA-ML is present at this condition. In order to avoid handling purified mRNA, which is highly sensitive to RNase contamination, the reaction conditions were optimized to enable DNA to be used directly as the template for click display. DNA template (2 pmol per 25 µL reaction) and ML (final 2 and 3 µM) were added directly to the in vitro protein synthesis reaction and the transcription reaction was carried out at 37 ºC for 20 minutes before being UV irradiated for 30s. Excess ML was used in this reaction to compensate for the anticipated reduced hybridization efficiency between ML and nascent mRNA due to the lack of the heat denaturation and slow cooling steps. Since mRNA synthesized after UV irradiation will not be crosslinked to ML but can potentially compete with the crosslinked mRNA-ML for ribosomes in the reaction, the restriction enzyme BsaI, which cleaves the DNA template immediately after the T7 promoter, was added to the mixture to terminate the transcription reaction (FIG. 3C). The mixture was then incubated at 37ºC for 40 minutes to allow both BsaI digestion and protein translation, and then treated with RNase A before electrophoresis. The amount of click-displayed protein appeared to be similar regardless of the amount of the ML (FIG.3B) and is comparable to the control reaction using 0.5 µM mRNA-ML as the template, indicating that DNA template can be used directly for click display without compromising the protein display efficiency. A large ML band was visible on the gel, especially in the reaction containing 3 µM ML, showing that the concentration of ML is likely in excess at this condition. Optimization of Conditions for Reverse Transcription The final component of the click display reaction series – reverse transcription was then optimized. To avoid mistaking the input DNA template for the synthesized cDNA, click display was carried out using mRNA-ML (final 0.5 µM) as the template in these studies. At the conclusion of translation, EDTA (final 30 mM) was added to inactivate the ribosomes followed by an equimolar amount of MgCl2to neutralize the EDTA. The mixture was then diluted 3-fold before the reverse transcription reaction. The cDNA synthesis efficiency of SuperScript II (SS-II) was compared to SuperScript IV (SS-IV) whose optimum reaction temperatures are 42 ºC and 50 ºC, respectively. The amount of the synthesized cDNA was quantified using qPCR. To ensure that the measured cycle threshold (Ct) values correspond to the amplification of the target DNA, the Docket No.11164-018WO1 reaction mixture after qPCR was also analyzed via electrophoresis. The amplified PCR product was present only in samples incubated in the presence of reverse transcriptase (RT) (FIG. 4), confirming the synthesis of cDNA under these conditions. Similar amounts of cDNA were present in both reactions, with SS-IV offering a slight advantage in the synthesis efficiency (slightly lower cycle threshold (Ct) value). Evaluation of Target Binding Ability of a Click-Displayed Protein The final one-pot transcription, translation, and reverse transcription reaction protocol is summarized in Table 1. To evaluate the efficiency of click display, DARPin 3G86 (D3G86) was displayed, which strongly binds eGFP (KD~9 nM), and determined its ability to be pulled down by biotinylated eGFP in a streptavidin pull-down assay (FIG.5A). Briefly, click-displayed D3G86 was diluted 10-fold in blocking buffer with or without biotinylated eGFP (final 200 nM). After 30 minutes incubation at room temperature, streptavidin-coated magnetic beads were added to the mixture to pull-down biotinylated eGFP and the associated click-displayed D3G86. A much larger amount of cDNA was pulled-down in samples incubated in the presence of eGFP than that in buffer alone, as manifested in the significantly lower cycle threshold (Ct) values in quantitative PCR (qPCR, FIG.5B) analysis, confirming that click displayed D3G86is active and retains the ability to associate with eGFP. To ensure that the amplification is specific, the qPCR product was analyzed on agarose gels. A faint band of the amplified cDNA was visible in the sample incubated in the absence of eGFP, likely resulting from non-specific association of the parental template DNA with the beads. To further reduce the background signal, the 6xHis tag was exploited at the N-terminus of D3G86and purified the click-displayed D3G86product via one-step immobilized metal affinity chromatography (IMAC) prior to incubation with eGFP or buffer. IMAC purification of the input click-displayed protein significantly reduced the background signal resulting in further elevated Ct values for samples incubated in buffer alone and the reduction of PCR product on agarose gels (FIGS. 5A-5B). Although the Ct values for samples incubated in the presence of eGFP also increased slightly, the ΔCt between samples incubated with and without eGFP increased significantly from 5.6 (original) to 11.7 (after IMAC purification). This result indicates that IMAC purification can efficiently reduce the background signal from click display. The dsDNA template present in the click reaction is likely responsible for the background signal. Mock Library Enrichment To evaluate the efficiency of click display to support the selection of rare target protein binders from a large library, a mock library enrichment experiment was performed. DNA encoding the eGFP-binding DARPin D3G86(0.1 ng) was mixed with a naïve DARPin library (1 µg) at Docket No.11164-018WO1 1:10,000 molar ratio and used as the template for click display. The click displayed product was purified via IMAC and underwent standard panning / selection against biotinylated eGFP (final 200 nM). Streptavidin and neutravidin beads were used to capture the eGFP-binding DARPin(s) in alternative rounds. To quantify the amount of D3G86 present in the output from different rounds of selection, approximately 150 ng of the final PCR product from each round was sequenced with a Mi-Seq Next-Generation Sequencing system yielding a dataset with 149,768 full-length protein sequences. The frequency of D3G86increased from 0.0024% in R0 to 1.5% in R1 and peaked at 17.5% in R2 (FIG.6). The enrichment efficiency from R0 to R1 is estimated to be 625-fold, which is 6-12-fold higher than conventional mRNA-display. Somewhat unexpectedly, the frequency of D3G86 decreased in R3 to 9.6%. It was contemplated that this phenomenon may be due to the selection of other eGFP-binding DARPins from the library that may out-compete D3G86for eGFP binding. Nevertheless, these results confirm the ability of click display to rapidly enrich binders to a desired target protein. Discovery of DARPin Binders to a Model Protein using Click Display Next, a click-displayed synthetic DARPin library was used to pan for binders to a model target protein – clumping factor A (ClfA), an S. aureus surface virulence protein. A click- displayed DARPin library prepared from 1 µg of DNA template (~1012different molecules) was purified via IMAC and underwent standard selection against biotinylated ClfA (final 200 nM). After 5 rounds of panning, significantly more DARPin-cDNA complexes were pulled down by streptavidin beads incubated in the presence of the target protein ClfA, which is manifested as a higher ΔCt (FIG.10). The PCR product from Round 6 was subcloned into the pET28a expression vector and used to transform BL21(DE3) cells. One hundred and eighty-eight (two 96-well plates minus 4 control wells per plate) clones were picked and grown in LB. The next day, the cell lysates after protein induction were harvested and their ability to bind ClfA was determined via ELISA. Overall, >50% of the clones showed >3 standard deviations above the background binding to ClfA, with >30% showing >10 standard deviations above the background binding (FIG.10). Seven clones that showed strong ClfA-binding when applied as crude cell lysates were purified via IMAC and re-evaluated for binding to ClfA via ELISA (FIGS. 7A-7B). The sequences of these clones were found to be distinct although they cluster into two large families, with DARPin 1E5 (D1E5) adopting a different set of target-binding residues than all the other clones. Clones D1E10, D2D2, D2G3 and D2E7 share the identical target-binding residues but contain different point mutations in the scaffold (FIGS. 7C-7D and FIG. 11). The very different ClfA- binding ability of D1E10 and D2E7 highlights the influence of framework residues on target binding. To evaluate the diversity, the PCR product from R6 was subjected to next-generation sequencing Docket No.11164-018WO1 (NGS). A total of 27,026 full-length DARPin sequences were obtained, among which D2G3was the most abundant, representing 627 reads (2.3%, FIG. 12). This result shows that significant polyclonal diversity remains in the click-display-selected pools even after 6 rounds of panning. Since the diversity of the naïve library (estimated to be ~1012) greatly exceeds the capacity of NGS, this initial library was not sequenced. The successful enrichment of ClfA-binding DARPins, as well as the preservation of high library diversity after 6 rounds of enrichment, underscores the power of click display for protein binder discovery. Discussion Current in vitro protein display methods are complex, labor-intensive, and often require strictly RNase-free conditions for purification and / or library panning, putting them out-of-reach for many researchers. For example, RNA is used to link the displayed protein to its coding sequence in both mRNA-display and ribosome-display, limiting their applications to highly purified and RNase-free targets. Although a recently developed in vitro display method, TRAP display, enables simultaneous mRNA transcription and protein translation, it requires a branched oligo that is not commercially available (Integrated DNA Technologies, Inc. does not offer synthesis of the branched oligos at the time of preparing this disclosure) and whose synthesis is non-trivial and inefficient (<0.5% to 4%,), severely limiting the utility of this approach. In this example, an alternative in vitro protein display technology – click display – was developed that allows the production of covalently linked protein-cDNA complex from double- stranded input DNA in a one-pot reaction within 2 hours. A modified oligo linker (ML) with two 3’ ends – one functionalized with puromycin and the other a primer for cDNA synthesis – was generated using copper-free click chemistry and was used to link the coding cDNA and the displayed protein. The constituents of ML, Oligo #1, and Oligo #2 are commercially available (e.g. IDT) and no purification step is needed for ML synthesis. The estimated cost of ML is ~$4 per 25 µL in vitro protein synthesis reaction. Since the protein is covalently linked to the cDNA, there is no need to maintain RNase-free conditions during biopanning, allowing selections to be potentially carried out in RNase-rich environments such as on whole cells. Using a previously engineered eGFP-binding DARPin – D3G86 – and next-generation sequencing (NGS), the enrichment efficiency of click-displayed libraries was estimated to be 625- fold per round, which is 6-12-fold higher than the conventional mRNA-display techniques, demonstrating a high protein display efficiency of click display. A naïve DARPin library was used to enrich binders to a model protein: ClfA. Click displayed DARPin library was prepared from 1 µg of DNA template in 25 µL translation reaction, Docket No.11164-018WO1 corresponding to a library size of ~1012. A high-throughput ELISA screen of 188 randomly picked DARPin clones from the Round 6-selected library showed a >50% hit rate for ClfA binders (FIG. 10B). All seven of the randomly picked clones showed nanomolar ClfA-binding affinity based on ELISA (FIG. 6) and have different sequences, although some of the sequences differ only by substitutions in DARPin framework region (FIG. 11). NGS confirmed that the enriched library retains high diversity even after 6 rounds of panning (FIG.12), which indirectly corroborates the large library diversity in the initial rounds. In conclusion, disclosed herein is the development of a powerful new tool for in vitro protein display and protein engineering – click display – that does not demand an RNase-free cleanliness standard for panning success and requires only readily available reagents for implementation. The utility of click display was demonstrated through the discovery of a panel of novel binders to a S. aureus surface virulence protein, clumping factor A, from a synthetic DARPin library with ~1012distinct members. Although a DARPin library was used in this disclosure, click display can be used to display / engineer any protein amenable to in vitro translation. It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0002] Docket No.11164-018WO1 TABLES Table 1. Overview of one-pot click display of proteins to their encoding cDNA. Components Vol needed Final Conc. / Note Solution A 10 μL te

[0003] Docket No.11164-018WO1 Table 2. Primer Sequences Name Sequence Vendor Oligo #1 / 5DBCON / AAATTCCA / i3Cyan / GCCGCCCC IDT Docket No.11164-018WO1 DARPin_NGS_ ACACTCTTTCCCTACACGACGCTCTTCCG Sigma F0 ATCT aa atctgggatccgacctgggt (SEQ ID NO 25) 5 0 5 / i3Cyan / : CynK / i5-TAMK / : Int 5-TAMRA / i3Cyan / : internal CNVK oligo is from Glen Research (https: / / www.glenresearch.com / 10- 4960.html) / 3Puro / : 3’ Puromycin / 5DBCON / : 5’ Dibenzocyclooctyl (DBCO) / 5AzideN / : 5’ Azide (NHS Ester)

[0004] Docket No.11164-018WO1 SEQUENCES 1. SEQ ID NO: 1 - DNA template encoding DARPin 3G86 atacgaaattaatacgactcactatagggagaccacaacggtttccctctagaaataattttgtttaactttaagaaggaggatatatccATG ggcagcagccaccatcaccaccatcatcatcacagcagcggcggagaacaaaaactgatcagcgaagaggatctgggatccgatctgg gtaagaaacttttggaggcggcccgtgccgggcaagacgacgaagtgcgcattttgatggctaacggggccgacgttaacgccttggac cgctttggtttaacgcctcttcacttggccgcacagcgcggccacttggagatcgtcgaagtcttactgaaatgtggcgctgacgtgaacgc cgcagacttgtggggacaaacgccacttcatttagcagccactgctggacacttggaaattgttgaggtgttacttaaatatggggcggatgt taatgcgcttgacttaatcgggaaaacaccactgcacctgacggcaatcgacggacatttagaaattgtggaggtgttgttaaaacatggtgc agatgtcaatgcacaggataagtttgggaagacggcgttcgatatctccattgataacgggaacgaggatttagcagaaatccttcaaggta ccggaggtcctactctcctcaaggaaggcaaaaggacggggggcggcgtggaataactagcataaccccttggggcctctaaacggg tcttgaggggttt T7 promoter BsaI recognition sequence Ribosome binding site ATG: start codon ML annealing site T7 terminator 2. SEQ ID NO: 2 - Amino acid sequence of eGFP MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWP TLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEG DTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGS VQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGM DELLEHHHHHH 6xHis tag 3. SEQ ID NO: 3 - Amino acid sequence of ClfA HHHHHHGTDITNQLTNVTVGIDSGTTVYPHQAGYVKLNYGFSVPNSAVKGDTFKITVP KELNLNGVTSTAKVPPIMAGDQVLANGVIDSDGNVIYTFTDYVNTKDDVKATLTMPAY IDPENVKKTGNVTLATGIGSTTANKTVLVDYEKYGKFYNLSIKGTIDQIDKTNNTYRQTI YVNPSGDNVIAPVLTGNLKPNTDSNALIDQQNTSIKVYKVDNAADLSESYFVNPENFED VTNSVNITFPNPNQYKVEFNTPDDQITTPYIVVVNGHIDPNSKGDLALRSTLYGYNSNII WRSMSWDNEVAFNNGSGSGDGIDKPVVP 6xHis tag Docket No.11164-018WO1 4. SEQ ID NO: 4 - Amino acid sequence of DARPin 1E5 MGSSHHHHHHSSGLVPRGSHMEQKLISEEDLGSDLGKKLLEAASAGQDDGVRILMANG ADVNAWSHVGVTPLHLAAAWGHLEVVEVLLKDGADVNAAIFIGHTPLHLAAARGHLE IVEVLLKNGADVNARTSAGNTPLHLAATLGHLEIVEVLLKYGADVNAQMLYGLTAFDI SIDNGNEDLAEILQ 6xHis tag Myc-tag 5. SEQ ID NO: 6 – P1E5 GKKLLEAASAGQDDGVRILMANGADVNA / WSHVGVTPLHLAAAWGHLEVVEVLLKD GADVNA / AIFIGHTPLHLAAARGHLEIVEVLLKNGADVNA / RTSAGNTPLHLAATLGHLE IVEVLLKYGADVNA / QMLYGLTAFDISIDNGNEDLAEILQ 6. SEQ ID NO: 7 – P1E10 GKKLLEAARAGQDDEARILMANGADVNA / YTVLGITPLHLAAFNGHLEIVEVLLNDGA DVNA / LSAGGVTPLHLAALCGHLEIVEVLLKHGADVNA / REWFGFTPLHLAATRGHLEI VEVLLKNGADVNA / QPPDGNTAFDIPIDNGNEDLAEILQ 7. SEQ ID NO: 8 – P1F8 GKKLLEAARAGQDDGARILMANGADVNA / YTVLGITPLHLAAFNGHLEIVEVLLNDGA DVNA / LSAGGVTPLHLAALCGHLEIVEVLLKHGADVNA / REWFGFTPLHLAATRGHLEI VEVLLKHGADVNA / QTPLGHTAFDISIDNGNEDLAEILQ 8. SEQ ID NO: 9 – P1G7 GKKLLEAARAGQDDEVHILMANGADVNA / YTVLGITPLHLAAFNGHLEIVEVLLKDGA DVNA / LSAGGVTPLHLAALCGHLEIVEVLLKHGADVNA / REWFGFTPLHLAATRGHLEI VEVLLKHGADVNA / QPAVGTTAFDISIGNGNEDLAEILQ 9. SEQ ID NO: 10 – P2D2 GKKLLEAARAGQDDEVRILMANGADVNA / YTVLGITPLHLAAFNGHLETVEVLLKDGA DVNA / LSAGGVTPLHLAALCGHLEIVEVLLKHGADVNA / REWFGFTPLHLAATRGHLEI VEVLLKNGADVNA / QPPDGNTAFDIPTDNGNEDLAEILQ Docket No.11164-018WO1 10. SEQ ID NO: 11 – P2G3 GKKLLEAARAGQDDEVRILMANGADVNA / YTVLGITPLHLAAFNGHLEIVEVLLKDGA DVNA / LSAGGVTPLHLAALCGHLEIVEVLLKHGADVNA / REWFGFTPLHLAATRGHLEI VEVLLKNGADVNA / QPPDGNTAFDISIDNGNEDLAEILQ 11. SEQ ID NO: 12 – P2E7 GKKLLEAARAGQDDEVRILMANGADVNA / YTVLGITPLHLAAFNGHLEIVEVLLKDGA DVNA / LSAGGVTPLHLAALCGHLEIVEVLLKHGADVNA / REWFGFTPLHLAATRGHLEI VEVLLKNGADVNA / QPPDGNTTFDISIDNGNEDLAEILQ 12. SEQ ID NO: 13 – Oligo #1 / 5DBCON / AAATTCCA / i3Cyan / GCCGCCCCCCGTCCT 13. SEQ ID NO: 14 – Oligo #2 / 5AzideN / AAAA / i5-TAMK / AAAAAAAAAAAAAAAAA AAAACC / 3Puro / 14. SEQ ID NO: 15 – 1892 (PHOSPHORYLATED)TTG AGG AGA GTA GGA CCT 15. SEQ ID NO: 16 – 2488 GTT TAA CTT TAA GAA GGA GGA TAT ATC CAT 16. SEQ ID NO: 17 – 1938 ATA CGA AAT TAA TAC GAC TCA CTA TAG GGA GAC CAC AAC GGT TTC CCT CTA GAA ATA ATT TTG TTT AAC 17. SEQ ID NO: 18 – 2643 AAA CCC CTC CGT TTA GAG AGG GGT TAT GCT AGT TAT TCC ACG CCG CCC CCC GTC CT 18. SEQ ID NO: 19 – AR-F2 AAAAAGGTCTCACGCCNNKNNKNNKNNKGGTNNKACGCCTCTTCACTTGGCCGCAN NKNNKGGCCACTTGGAGATCGTCGAAGTCTTACTGAAANNKGGCGCTGACGTGAAC Docket No.11164-018WO1 GCCNNKNNKNNKNNKGGANNKACGCCACTTCATTTAGCAGCCNNKNNKGGACACT TGGAAATTGTTGAGGTGTTACTTAAA “N” represents mixed nucleobases composed of adenine (A), thymine (T), cytosine (C), or guanine (G). “K” represents mixed nucleobases composed of cytosine (C) or thymine (T). 19. SEQ ID NO: 20 – AR-R2 TTTTTGGTCTCTCCGTMNNCCCMNNMNNMNNCTGTGCATTGACATCTGCACCMNNT TTTAACAACACCTCCACAATTTCTAAATGTCCMNNMNNTGCCGCCAGGTGCAGTGG TGTMNNCCCMNNMNNMNNMNNCGCATTAACATCCGCCCCMNNTTTAAGTAACAC CTCAACAATTTCCAAGTGTCC “N” represents mixed nucleobases composed of adenine (A), thymine (T), cytosine (C), or guanine (G). “M” represents mixed nucleobases composed of adenine (A) or cytosine (C). 20. SEQ ID NO: 21 – 2617 AAA AAC TCG AGA TAC GAA ATT AAT ACG ACT CAC TAT AGG GTG ACC ACA ACG GTT TCC CTC 21. SEQ ID NO: 22 – 2701 AAA AAG GTC TCA GGC GTT AAC GTC GGC CCC GTT AGC 22. SEQ ID NO: 23 – 2702 AAA AAG GTC TCA ACG GCG TTC GAT ATC TCC ATT GA 23. SEQ ID NO: 24 – 2581 AAA AAC TCG AGT TAT TCC ACG CCG CCC C 24. SEQ ID NO: 25 - DARPin_NGS_F0 ACACTCTTTCCCTACACGACGCTCTTCCGATCT aa atctgggatccgacctgggt 25. SEQ ID NO: 26 - DARPin_NGS_F1 ACACTCTTTCCCTACACGACGCTCTTCCGATCT ac atctgggatccgacctgggt Docket No.11164-018WO1 26. SEQ ID NO: 27 - DARPin_NGS_F2 ACACTCTTTCCCTACACGACGCTCTTCCGATCT at atctgggatccgacctgggt 27. SEQ ID NO: 28 - DARPin_NGS_F3 ACACTCTTTCCCTACACGACGCTCTTCCGATCT ag atctgggatccgacctgggt 28. SEQ ID NO: 29 - DARPin_NGS_R GACTGGAGTTCAGACGTGTGCTCTTCCGATCT GTA CCC GAG CTC GAT TGC AG 30. SEQ ID NO: 30 – mRNA Example AAAAGGACGGGGGGCGGCGUGGAA 31. SEQ ID NO: 31 – Cleaved Oligonucleotide #1 AAATTCCA / i3Cyan / GCCGCCCCCCGTCCT

Claims

Docket No.11164-018WO1 CLAIMS What is claimed is:

1. A modified linker comprising a first nucleic acid linked to a second nucleic acid, wherein the first nucleic acid comprises a ribosome binding molecule and a first click chemistry molecule, and wherein the second nucleic acid comprises a second click chemistry molecule and a crosslinking nucleotide.

2. The modified linker of claim 1, wherein the second nucleic acid comprises a sequence complementary to a target nucleic acid sequence.

3. The modified linker of claim 1 or 2, wherein the ribosome binding molecule comprises puromycin.

4. The modified linker of any one of claims 1-3, wherein the first click chemistry molecule comprises an alkyne compound.

5. The modified linker of claim 4, wherein the alkyne compound comprises a dibenzocyclooctyne (DBCO).

6. The modified linker of any one of claims 1-5, wherein the second click chemistry molecule comprises an azide (N3).

7. The modified linker of any one of claims 1-6, wherein the crosslinking nucleotide comprises a 3-cyanovinylcarbazole nucleotide (CnvK).

8. A method of preparing a modified linker, the method comprising: a. providing a first nucleic acid comprising a first click chemistry molecule and a ribosome binding molecule, b. providing a second nucleic acid comprising a second click chemistry molecule and a crosslinking nucleotide, and c. reacting the first and second click chemistry molecules to link the first nucleic acid to the second nucleic acid forming the modified linker.Docket No.11164-018WO1 9. The method of claim 8, wherein the first click chemistry molecule and the ribosome binding molecule are at opposite ends of the first nucleic acid.

10. The method of claim 8 or 9, wherein the second nucleic acid comprises a nucleic acid sequence complementary to a DNA template sequence.

11. The method of any one of claims 8-10, wherein the ribosome binding molecule comprises puromycin.

12. The method of any one of claims 8-11, wherein the first click chemistry molecule reacts with the second click chemistry molecule.

13. The method of any one of claims 8-12, wherein the first click chemistry molecule comprises an alkyne.

14. The method of claim 13, wherein the alkyne comprises a dibenzocyclooctyne (DBCO).

15. The method of any one of claims 8-14, wherein the second click chemistry molecule comprises an azide (N3).

16. The method of any one of claims 8-15, wherein the crosslinking nucleotide comprises a 3- cyanovinylcarbazole nucleotide (CnvK).

17. A method of preparing a complementary DNA (cDNA)-polypeptide complex, the method comprising: a. preparing the modified linker of any one of claims 1-7, b. providing a DNA template, c. transcribing the DNA template into an mRNA, d. crosslinking the modified linker to the mRNA, wherein a crosslinking nucleotide binds to the mRNA, e. degrading the DNA template, f. translating the mRNA into a polypeptide, wherein the polypeptide is transferred to the ribosome binding molecule, andDocket No.11164-018WO1 g. reverse transcribing the mRNA into a complementary DNA (cDNA), wherein the cDNA and polypeptide are bound by the modified linker forming a cDNA-polypeptide complex.

18. A method of sequentially inducing transcription, translation, and reverse transcription in one vessel, the method comprising: a. preparing the modified linker of any one of claims 1-7, b. providing a DNA template, c. transcribing the DNA template into an mRNA, d. crosslinking the modified linker to the mRNA, wherein the crosslinking nucleotide binds to the mRNA, e. degrading the DNA template, f. translating the mRNA into a polypeptide, wherein the polypeptide is transferred to a protein translation inhibitor, and g. reverse transcribing the mRNA into a complementary DNA (cDNA), wherein the cDNA and polypeptide are bound by the modified linker forming a cDNA-polypeptide complex.

19. A method of screening a panel of proteins, the method comprising: a. preparing the modified linker of any one of claims 1-7, b. providing a DNA template, c. transcribing the DNA template into an mRNA, d. crosslinking the modified linker to the mRNA, wherein the crosslinking nucleotide binds to the mRNA, e. degrading the DNA template, f. translating the mRNA into a polypeptide, wherein the polypeptide is transferred to the ribosome binding molecule, g. reverse transcribing the mRNA into a complementary DNA (cDNA), wherein the cDNA and polypeptide are bound by the modified linker forming a cDNA-polypeptide complex, and h. combining and incubating the cDNA-polypeptide complex with a panel of proteins to capture one or more proteins that bind to the polypeptide.

20. The method of any one of claims 17-19, wherein the second nucleic acid comprises a sequence complementary to the DNA template.Docket No.11164-018WO1 21. The method of any one of claims 17-20, wherein a light source enhances crosslinking of the modified linker to the mRNA.

22. The method of claim 21, wherein the light source comprises a wavelength of about 360nm or greater.

23. The method of claim 21 or 22, wherein the light source comprises an ultraviolet light source.

24. The method of any one of claims 17-23, wherein the DNA template is transcribed into the mRNA by one or more enzymes selected from RNA polymerases and transcription factors.

25. The method of any one of claims 17-24, wherein the mRNA is translated into the polypeptide by one or more biomolecules selected from a ribosome, a peptidyl transferase, and a transfer RNA synthetase.

26. The method of any one of claims 17-25, wherein the mRNA is reverse transcribed into the cDNA by one or more enzymes selected from a reverse transcriptase and a DNA polymerase.

27. The method of any one of claims 17-26, wherein the method is performed within 3 hours.

28. The method of claim 18, wherein the vessel comprises a tube, a cuvette, a dish, a microplate, or a partially enclosed or fully enclosed apparatus.

29. The method of claim 19, wherein the one or more proteins are identified using one or more protein detection techniques comprising enzyme-linked immunosorbent assay (ELISA), western blot, mass spectrometry, high performance liquid chromatography, protein immunoprecipitation, and protein immunostaining.

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