Method for selecting and detecting bound peptides

Abstract

Methods are provided for selecting, detecting, and / or enriching peptides that bind to a target.

Description

【Technical Field】 【0001】 The present invention pertains to the field of mRNA display technology. More specifically, the present invention relates to methods for selecting, detecting, and / or enriching peptides against cell-expressed or soluble peptides, and peptides having desired functional properties. 【Background Art】 【0002】 Messenger RNA (mRNA) display is a selection technology for soluble peptides and soluble proteins. (Szostak R. et al., RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl. Acad. Sci. 94, 12297-12302 (1997)). Messenger RNA display enables the construction of highly diverse peptide libraries that can contain natural and non-natural amino acids. The incorporation of non-natural amino acids is enabled by various approaches including amber codon suppression and the use of modified tRNAs (chemical conjugation of non-natural amino acids to tRNAs), or by highly mobile tRNA aminoacylating enzymes made by artificial ribozymes called flexizymes. (Niwa N. et al., A flexizyme that selectively charges amino acids activated by a water-friendly leaving group. Bioorganic & Med. Chem. Letters 19(14), 3892-3894 (2009)). 【0003】 The process of selecting peptides using mRNA display traditionally involves transcribing mRNA from a DNA variant pool and annealing the mRNA variant to a short DNA linker conjugated with puromycin at its 3' end (Ishizawa T. et al., TRAP Display: A High-Speed ​​Selection Method for the Generation of Functional Polypeptides. J.Am.Chem.Soc.135(14), 5433-5440(2013)). This creates an mRNA:DNA linker-puromycin library. Translation is initiated by adding bacterial or rabbit reticulocyte lysate or Pure Express®. 【0004】 During translation, ribosomes move along each mRNA molecule, translating the genetic information conserved within the mRNA into a peptide sequence. When ribosomes encounter an engineered TAG stop codon at the end of the peptide sequence, they stall in the absence of termination factor 1, allowing puromycin to enter ribosomal site A. As a result, puromycin forms a covalent bond between the polypeptide growth chain and the mRNA. Translation stops, and the mRNA hybridized to the DNA linker fused with puromycin is then ligated to the peptide. The mRNA is then reverse transcribed into cDNA to enable PCR amplification, producing a stable DNA:RNA double helix with reduced nonspecific binding during selection. The library is selected for the target of interest, nonspecific peptides are washed away, and the conjugates are eluted. Selection efficiency is monitored by quantitative PCR (qPCR). Next-generation sequencing is used to determine the peptide sequences enriched after multiple rounds of sequencing. 【0005】 While mRNA library selection has produced strongly specific conjugates for soluble peptides, mRNA display is not suitable for selection against cellular expression targets. Therefore, the need for a method for selection against cellular expression targets, such as proteins or protein analogs, remains. Furthermore, most conventional methods use qPCR to determine the ratio of output to input molecules (recovery rate) to determine efficiency. However, selected peptides contain both specific and nonspecific conjugates. Therefore, a method for selecting only specific conjugates is needed. [Overview of the Initiative] 【0006】 Accordingly, the present invention provides a method for selecting mRNA display libraries for cell-expressed and soluble peptides or peptide analogs. The present invention also provides means for quantitatively monitoring the enrichment of target-specific peptides in each selection round, which provides a more accurate measure of efficiency determination. In addition, the selection criteria of the present invention can be modified based on data obtained in real time for selection for soluble and cell-expressed targets. Furthermore, the present invention enables the incorporation of FACS for the selection of target-specific peptides for cell-expressed and recombinant proteins, enabling functional selection (selecting peptides with desired function in cell-based assays) and giving the ability to identify peptides that internalize within cells. 【0007】 Puromycin binds to a short DNA linker to obtain a puromycin-DNA linker. The fluorophore is then fused to the 5' end of the puromycin-DNA linker, resulting in a DNA linker fused to puromycin at its 3' end and to the fluorophore at its 5' end. As a result, each mRNA-DNA-peptide molecule in the library is automatically tagged with the fluorophore upon annealing to the DNA linker, enabling integration with mRNA display platforms for FACS and flow cytometry. A similar approach may involve incorporating a tag (such as a FLAG® tag) into the mRNA-DNA-peptide complex so that a fluorescent peptide, antibody, or other fluorescent molecule with specificity for the tag binds to the mRNA-DNA-peptide molecule, thereby enabling FACS and flow cytometry integration. 【0008】 This integration of FACS and flow cytometry provides a means to quantitatively monitor the enrichment of target-specific peptides in each selection round, whereas conventional methods use qPCR to determine the ratio of output to input molecules (recovery rate). The method of the present invention offers advantages over qPCR recovery rate determination, which selects both specific and nonspecific conjugates during each selection round. Thus, using qPCR recovery rate determination, both specific and nonspecific conjugates are measured in each selection round. However, enrichment and selection by flow cytometry and FACS largely enable the selection of specific conjugates. 【0009】 The method of the present invention is also advantageous because the selection criteria can be modified based on data obtained in real time. Furthermore, this approach allows for the incorporation of FACS to select target-specific peptides for cell expression and recombinant proteins, enabling functional selection and providing the ability to identify peptides that internalize within cells. [Modes for carrying out the invention] 【0010】 Accordingly, the present invention provides a method for detecting or selecting a target-bound peptide, wherein the method comprises detecting or selecting an mRNA-DNA-peptide complex conjugated to at least one detectable substance. In embodiments, the detectable substance is a fluorophore. In certain embodiments, the fluorophore is an organic dye, a biological fluorophore, or a quantum dot. In certain embodiments, the detectable substance is Alexa Fluor. In some embodiments, the detectable substance is conjugated to a puromycin linker. 【0011】 The present invention also provides a method for detecting or selecting a target-bound peptide, wherein the method comprises detecting or selecting an mRNA-DNA-peptide complex bound to at least one detectable substance, wherein the detectable substance is a tag. In embodiments, the tag comprises a natural or non-natural amino acid. In embodiments, the tag is a peptide tag. In certain embodiments, the peptide tag is a FLAG® tag. In embodiments, the tag is detected by a fluorescent molecule. In certain embodiments, the tag is detected by a fluorescent peptide. In other particular embodiments, the tag is detected by a fluorescent antibody. 【0012】 In embodiments, the method of the present invention includes a target immobilized on a sorting-enabled material. In certain embodiments, the sorting-enabled material is beads. In embodiments, the target is a peptide, protein, nucleic acid, sugar, lipid, mammalian cell, or mammalian cell extract. In certain embodiments, the target is a peptide or protein. 【0013】 In embodiments, the method of the present invention includes a conjugated peptide comprising at least one non-natural amino acid. 【0014】 In another embodiment, the conjugated peptide of the method of the present invention is detected or selected by a detectable substance. In some such embodiments, the conjugated peptide is detected or selected by flow cytometry or FACS. In other such embodiments, the conjugated peptide is detected or selected by ELISA, spectrophotometry, fluorescence spectroscopy, or microscopy. In a preferred embodiment, the conjugated peptide is detected by flow cytometry. In another preferred embodiment, the conjugated peptide is selected by FACS. In an embodiment, the conjugated peptide is detected and selected. In some such embodiments, the conjugated peptide is detected by flow cytometry and the conjugated peptide is selected by FACS. 【0015】 In some embodiments, the method of the present invention includes incorporating at least one detectable substance into an mRNA library to produce an mRNA-detectable substance complex. In further embodiments, the method of the present invention includes translating the mRNA-detectable substance complex to produce an mRNA-peptide-detectable substance complex. In even further embodiments, the method of the present invention includes reverse transcription of the mRNA-peptide-detectable substance complex to obtain an mRNA-DNA-peptide-detectable substance complex. In some specific embodiments, the method of the present invention includes incorporating at least one detectable substance into an mRNA library to produce an mRNA-detectable substance complex, translating the mRNA-detectable substance complex to produce an mRNA-peptide-detectable substance complex, and reverse transcription of the mRNA-peptide-detectable substance complex to obtain an mRNA-DNA-peptide-detectable substance complex. In some embodiments, the mRNA-DNA-peptide-detectable substance complex is detected by flow cytometry. In another embodiment, the mRNA-DNA-peptide-detectable substance complex is selected by FACS. 【0016】 In one embodiment, the method of the present invention includes transcribing a template DNA library to obtain an mRNA library. 【0017】 In some embodiments, the method of the present invention further includes removing mRNA-DNA-peptide complexes that are not bound to a target. 【0018】 In some embodiments, the method of the present invention includes removing an mRNA-DNA-peptide complex from a target. In certain embodiments, the mRNA-DNA-peptide complex is removed by adding acid or heat. In certain embodiments, the mRNA-DNA-peptide complex is removed by heat. In some embodiments, the method further includes amplifying the DNA of the removed mRNA-DNA-peptide complex by PCR. In some embodiments, the method further includes transcribing the amplified DNA to obtain an mRNA library. 【0019】 In embodiments, the method of the present invention includes determining the amino acid sequence of the binding peptide. In embodiments, the method of the present invention further includes determining the nucleic acid sequence of the binding peptide. 【0020】 In embodiments, the present invention provides bound peptides that are detected or selected by the method of the present invention. 【0021】 The present invention also provides mRNA-DNA-peptide complexes conjugated to at least one detectable substance. In embodiments, the present invention provides a library of mRNA-DNA-peptide complexes conjugated to at least one detectable substance. In embodiments, the detectable substance is a fluorophore. In embodiments, the fluorophore is an organic dye, a biological fluorophore, or a quantum dot. In certain embodiments, the detectable substance is Alexa Fluor. In embodiments, the detectable substance is conjugated to a puromycin linker. In other embodiments, the detectable substance is a tag. In some such embodiments, the tag contains a natural or non-natural amino acid. In embodiments, the tag is a peptide tag. In some such embodiments, the tag is a FLAG® tag. In embodiments, the tag is detected by a fluorescent molecule. In certain embodiments, the fluorescent molecule is a peptide or antibody conjugated to a fluorophore. 【0022】 The term "peptide" includes, but is not limited to, peptides, polypeptides, proteins, antibodies, antibody fragments, and antibody fusion proteins. "Peptide" may be used interchangeably with polypeptides, proteins, antibodies, antibody fragments, and antibody fusion proteins. As used herein, "bound peptide" refers to a peptide (including peptides, polypeptides, proteins, antibodies, antibody fragments, and antibody fusion proteins) that binds to a target. 【0023】 As used herein, "linker" refers to a reagent that can bind to a translation product. Linkers include, but are not limited to, amino acids, amino acid analogs, puromycin, and puromycin analogs. Preferably, the linker is an oligonucleotide fused at its 3' end to an amino nucleoside such as puromycin or a puromycin analog. 【0024】 As used herein, "target" refers to a molecule that can interact with a translation product. The target can be a peptide, protein, nucleic acid (DNA or RNA), sugar, lipid, mammalian cell, mammalian cell extract, or another molecule that has the ability to bind to a binding peptide. The target can be subjected to purification (including partial purification), insertion into the cell membrane, display on a phage, display on yeast, display on a baculovirus, or display on a cell. The target can also be a mammalian cell-expressed protein presented on a Nano disc, Amphipol, or liposome. Nano discs and Amphipols are used to stabilize membrane-bound proteins. 【0025】 A fluorophore bound (incorporated) to a DNA linker, or a tag incorporated into an mRNA-DNA-peptide complex, is an example of a "detectable substance" incorporated into the mRNA-DNA-peptide complex. "Bind" and "incorporate" (and their derivatives) can be used interchangeably herein when referring to a molecule containing a detectable substance or other such structure. 【0026】 One of ordinary skill in the art can recognize, for example, that the FLAG® tag is provided by the peptide sequence DYKDDDK. In addition to the FLAG® tag, examples of other peptide tags that can be used in the methods of the present invention include, but are not limited to, the HA tag, His tag, c-myc tag, and S tag. 【0027】 "Translation product" refers to the product of mRNA translation. Preferably, the translation product is a peptide that can bind to a target. 【0028】 As used herein, "select" and its derivatives refer to substantially isolating a particular molecule from other molecules in a library. After multiple rounds of selection, it is expected that molecules with the desired phenotype will be enriched in the library. 【0029】 As used herein, "natural amino acid" refers to the 20 types of amino acids that are α - aminocarboxylic acids (or substituted α - aminocarboxylic acids) used in standard translation. This includes alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), tryptophan (Trp), phenylalanine (Phe), methionine (Met), glycine (Gly), serine (Ser), threonine (Thr), tyrosine (Tyr), cysteine (Cys), glutamine (Gln), asparagine (Asn), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), and glutamic acid (Glu). 【0030】 As used herein, "unnatural amino acid" generally refers to an amino acid that has a structure different from the 20 types of natural amino acids used during natural translation. Unnatural amino acids can include naturally occurring amino acids with modified functional groups. This includes, but is not limited to, all unnatural amino acids or artificial amino acids such as D - amino acids, N - methyl amino acids, α - methyl amino acids, N - acyl amino acids, β - amino acids, γ - amino acids, and δ - amino acids. Delta amino acids are either natural amino acids whose side - chain structure is partially chemically changed / modified, or derivatives having a structure in which the amino group or carboxyl group on the amino - acid backbone is substituted. Peptides into which unnatural amino acids are introduced, or peptides referred to herein as "unnatural peptides", include polymers having these various unnatural amino acids as components. Unnatural peptides can be composed partially or completely of unnatural amino acids. Thus, unnatural peptides can have a backbone whose structure is different from the standard amide bond. 【0031】 As used herein, "library" refers to a group of molecules, such as multiple nucleic acids, translation products, product / linker / mRNA complexes, and translation product / linker / mRNA-cDNA complex molecules. Library diversity can be represented by different gene sequences. A DNA or RNA library refers to a DNA or RNA library that encodes a peptide library. [Examples] 【0032】 Example: Detection of mRNA-DNA-peptide complex binding to a target The peptide bound to the mRNA in the mRNA-DNA-peptide detectable complex (hereinafter referred to as "peptide A") is used to determine whether flow cytometry can be used to detect the binding of the peptide to its target (hereinafter referred to as "target A"). Target A may be a recombinant protein fused to an AVI tag (a tag recombinantly fused to the peptide of interest to promote its site-specific biotinylation in vivo by the biotin ligase (BirA) enzyme). 【0033】 The messenger RNA encoding each peptide is annealed to a fluorophore (e.g., Alexa Fluor 647) conjugated puromycin linker. In vitro translation (IVT) is then initiated using PDPS to reverse transcribe the mRNA and obtain an mRNA-DNA-peptide A complex. The mRNA-DNA-peptide A complex is added to the biotinylated target A-Avi tag for binding. Streptavidin-coated beads are added to the IVT mix (containing the target A-Avi tag), and the complex of the streptavidin-coated beads with the biotinylated target A bound to the mRNA-DNA-peptide complex is pulled down, washed, and diluted in FACS buffer before flow cytometry. Streptoavidin beads mixed with an in vitro transcription-translation mix (IVT mix) [IVT(-)] lacking amino acids and aminoacyl-tRNA synthetase (ARS) were used as a negative control to ensure that the binding observed by flow cytometry was specific to the target A-Avi tag-binding peptide. 【0034】 Essentially, in this embodiment, it is expected that strong binding of the mRNA-DNA-peptide complex to the target (target A) will be detected by following the procedure described above. 【0035】 Essentially, in this embodiment, strong binding of the mRNA-DNA-peptide complex to the target (target A) was detected by following the procedure described above. 【0036】 Example: Target-specific enrichment during mRNA library selection To determine whether flow cytometry can be used to monitor selection by detecting the enrichment of target-specific peptides, the selection round for recombinant target A-Avi conjugated on streptavidin beads can be examined. 【0037】 Chloroacetylphenylalanine is used to initiate translation and create a stable thioether bond with the C-terminally manipulated cysteine ​​residue. Selection is monitored by determining the recovery percentage by qPCR. Negative selection against streptavidin beads is incorporated into each round to eliminate nonspecific streptavidin conjugates. Increases in recovery percentage are expected to be most pronounced in later rounds, suggesting enrichment in the population of identified target A conjugates. 【0038】 Next, peptide sequences from subsequent rounds are determined using Illumina® next-generation sequencing. A small number of high-frequency peptides may demonstrate enrichment of those particular peptides. To determine whether the observed increase in recovery percentage can be attributed to the selection of nonspecific conjugates, peptide sequences from subsequent rounds are compared to those from previous rounds. Nonspecific conjugates are expected to be enriched in subsequent rounds using qPCR methods. These nonspecific conjugates may include peptides with shortened sequences consisting of one or two amino acids. 【0039】 Essentially following the procedure described above, a small number of high-frequency peptides showed enrichment of those specific peptides. Comparison of peptide sequences from later rounds with those from earlier rounds revealed that the highly enriched peptides were shortened sequences consisting of one or two amino acids. In most cases, nonspecific conjugates were enriched in later rounds as the number of selection rounds increased. qPCR quantifies the number of output DNA molecules indiscriminately and therefore cannot be used to differentiate between true peptides and nonspecific peptides. Thus, means other than qPCR should be used to improve the selection of specific conjugates. 【0040】 Flow cytometry is used to track the enrichment of target A-binding peptides from different selection rounds. DNA is isolated from the output of different rounds and amplified by PCR to reconstitute each peptide pool with a fluorophore-conjugated puromycin linker, and the binding of each pool to target A-AVI is evaluated by flow cytometry. A streptavidin + IVT(-) sample is used for gating and as a negative control. Despite the expected increase in recovery percentage by qPCR, the increase in binding from different rounds by flow cytometry is expected to be minimal, if present. This may be partly due to a reduction in the selection of nonspecific conjugates recovered from flow cytometry. 【0041】 Essentially following the procedure described above, the increased recovery percentage by qPCR suggests that later rounds should bind the target much more strongly. However, specific binding of the peptide pool to the target by flow cytometry was not observed. The lack of peptide pool binding to the target by flow cytometry supports the enrichment of shortened, nonspecific peptides in later selection rounds. 【0042】 Example: Use of FACS to isolate target-specific conjugates from mRNA libraries To determine whether libraries screened against target A-AVI can be sorted by FACS, DNA from the output of later rounds is transcribed and translated using a puromycin linker fused to a fluorophore such as Alexa Flour 647, and flow cytometry is performed to detect binding. Gating is performed around target A-AVI complexed with streptavidin beads, where a low percentage of peptides are expected to bind to target A (pre-sorting pool). The positive population is sorted by flow cytometry, and the binding of the peptide pool from the sorted rounds to peptide A-AVI is evaluated by flow cytometry. The results are expected to show a significant increase in binding in the sorted pool compared to the pre-sorting pool. The results are also expected to demonstrate that selection by FACS results in greater enrichment of target A-binding peptides compared to the use of traditional mRNA selection methods, and that selection by FACS is an efficient method for eliminating background conjugates and isolating specific peptides. 【0043】 Essentially following the procedure described above, a low percentage of peptides bound to target A (pre-sorting pool). The positive population was sorted by flow cytometry, and the binding of the peptide pool from the sorted rounds to target A-AVI was evaluated by flow cytometry. The results showed a significant increase in binding in the sorted pool compared to the pre-sorting pool. The results also demonstrated that selection by FACS results in greater enrichment of target A-specific binding peptides compared to the use of traditional recovery percentage methods, and that selection by FACS is an efficient method for eliminating background conjugates and isolating specific peptides. 【0044】 To further determine whether selection by recovery percentage can result in enrichment of both specific and nonspecific conjugates, the peptide sequences of sorted rounds are compared with the peptide sequences of the corresponding unsorted rounds. Some of the highest-frequency sequences in the sorted rounds are expected to differ from those in the unsorted rounds. In addition, the frequency of specific peptides is expected to increase after sorting by FACS. This suggests that selection of mRNA display libraries by FACS using a modified puromycin linker results in highly efficient isolation of peptides with strong binding affinity and specificity. 【0045】 Essentially following the procedure described above, some of the highest-frequency peptide sequences from the sorted rounds differed from those in the unsorted rounds. The frequency of specific peptides increased after sorting by FACS. This suggests that selection of mRNA display libraries by FACS using a modified puromycin linker results in highly efficient isolation of peptides with strong binding affinity and specificity. 【0046】 Example: Detection and selection of cell-expressed peptides Conventional mRNA display selection methods fail to efficiently detect cell-expressed peptides because high system-specific noise leads to nonspecific peptide selection. Four selection rounds were completed using PDPS against recombinant heterodimer cell-expressed protein (hereinafter referred to as "protein II"; biotinylated; bound to streptavidin beads). The selection was monitored by pulling down the complex of the peptide with protein II using streptavidin beads and determining the recovery percentage in each round. A significant increase in the recovery percentage was observed in round 4 (Table 1), and thus the rigor of selection was increased, partly by decreasing the target concentration to favor the isolation of stronger conjugates. 【0047】 [Table 1] 【0048】 To determine whether a cell-expressed protein can be used as a target by the method of the present invention, flow cytometry is used for either recombinant or cell-expressed protein II to monitor the enrichment of protein II conjugates during selection. DNA from the outputs of rounds 2–5 is transcribed, annealed to a modified puromycin linker, and translated to constitute a fluorophore library for each round. Binding of each mRNA-DNA-peptide pool to recombinant or cell-expressed protein II is detected by flow cytometry. 【0049】 Essentially following the procedure described above, the outputs of rounds 2 through 5, detected by flow cytometry, strongly bound to both recombinant and cell-expressed protein II (Table 2). These data demonstrate that the method of the present invention can be used to select both recombinant and cell-expressed peptides. 【0050】 [Table 2] 【0051】 Next, DNA from the Round 2 output was used for sorting of protein II conjugates using FACS. Since recombinant proteins were used in the initial selection round, sorting was performed on protein II expressed on CHO cells to isolate macrocyclic molecules that bind to both target types (recombinant or cellular expression). To determine whether FACS sorting resulted in protein II conjugate enrichment, the binding of the peptide pool to protein II before and after sorting was examined. The results showed enrichment of a population of specific conjugates determined by flow cytometry after sorting (4% of conjugates before sorting compared to 40% after sorting). Binding of the sorted pool to parental CHO cells lacking protein II expression was minimal. Next-generation sequencing of the Round 2 (sorted) peptides revealed the emergence of a novel family whose sequences were not present at detectable levels in the pre- and post-sorting selection rounds (performed by conventional mRNA display selection methods). 【0052】 To determine the binding and functional activity of the most concentrated peptides from the Round 2 (sorted) pool compared to Round 5, the peptides identified from the Round 2 (sorted) pool are chemically synthesized and tested in cell-based functional assays to determine whether the binding of protein II to its ligand can be blocked by the sorted peptides. The data are expected to demonstrate that the peptides present in the Round 2 (sorted) pool inhibit the interaction of protein II to its ligand. These results suggest that FACS sorting in the initial selection round leads to the isolation and identification of functionally active peptides that may be depleted from the library in later rounds due to performing multiple selection rounds. 【0053】 Essentially, following the procedure described above, the peptides present in the Round 2 (sorted) pool inhibited the interaction of protein II with its ligand. 【0054】 Example: Design and optimization of fluorophore-conjugated DNA linkers A fluorophore is conjugated to the 5' end of a puromycin linker to enable detection of mRNA-DNA-peptide complexes by flow cytometry. The fluorophore is conjugated to the oligo at its 5' end either directly using an NHS Ester linker or using spacers of varying lengths. Binding can occur, for example, by covalent bonding of puromycin to the 3' end of a short oligo linker via multiple hexaethylene glycol spacers. Increasing the spacer length is considered most effective when targeting multi-pass transmembrane proteins (GPCRs and ion channels) or when the peptide is bound to a cavity or groove in the protein. For example, the binding of mRNA-DNA-peptide to a multi-pass transmembrane cell expression protein (hereinafter referred to as "protein III") complex expressed on the cell surface of HEK cells is better detected by flow cytometry as the spacer length increases (Table 3). This may be due to the increased accessibility of the fluorophore with increasing linker length. However, the data in Table 3 also demonstrate that spacers may not be required for detection by flow cytometry. 【0055】 [Table 3]

Claims

[Claim 1] A method for detecting and selecting peptides or peptides bound to proteins expressed on the surface of mammalian cells, a) Incorporate at least one fluorophore into the mRNA library to produce an mRNA-fluorophore complex, b) The mRNA-fluorophore complex is translated to produce an mRNA-peptide-fluorophore complex, c) The mRNA-peptide-fluorophore complex is reverse transcribed to obtain an mRNA-DNA-peptide-fluorophore complex, d) The mRNA-DNA-peptide-fluorophore complex is added to a peptide or protein expressed on the surface of a mammalian cell to allow it to bind (the mRNA-DNA-peptide-fluorophore complex contains the binding peptide, and the binding peptide binds to the expressed peptide or protein), detected by flow cytometry, and then e) Selecting the binding peptide by fluorescence-activated cell sorting. Methods that include... [Claim 2] The method according to claim 1, wherein the fluorophore is an organic dye, a biological fluorophore, or a quantum dot. [Claim 3] The method according to claim 1 or 2, wherein the fluorophore is conjugated to a puromycin linker. [Claim 4] The method according to any one of claims 1 to 3, wherein the bound peptide comprises at least one non-natural amino acid. [Claim 5] The method according to any one of claims 1 to 4, further comprising transcribing a template DNA library to obtain an mRNA library. [Claim 6] The method according to any one of claims 1 to 5, further comprising removing the mRNA-DNA-peptide-fluorophore complex that is not bound to the expressed peptide or protein. [Claim 7] The method according to any one of claims 1 to 6, further comprising removing the mRNA-DNA-peptide-fluorophore complex from the expressed peptide or protein. [Claim 8] The method according to claim 7, wherein the mRNA-DNA-peptide-fluorophore complex is removed by heat. [Claim 9] The method according to claim 7 or 8, further comprising amplifying the DNA of the mRNA-DNA-peptide-fluorophore complex removed from the expressed peptide or protein by PCR. [Claim 10] The method according to claim 9, further comprising transcribing the DNA to obtain an mRNA library. [Claim 11] The method according to any one of claims 1 to 10, further comprising determining the amino acid sequence of the binding peptide. [Claim 12] The method according to any one of claims 1 to 10, further comprising determining the nucleic acid sequence encoding the binding peptide.

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