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CN-121986174-A - Methods for screening peptides using multiple libraries

CN121986174ACN 121986174 ACN121986174 ACN 121986174ACN-121986174-A

Abstract

The present invention provides a method for screening candidate peptides capable of binding to a target molecule, comprising the steps of (1) preparing a plurality of nucleic acid display libraries comprising barcoded peptide- (nucleic acid) complexes, wherein the barcoded peptide- (nucleic acid) complexes comprise a nucleic acid portion and a peptide portion, the nucleic acid portion comprising a barcode sequence and a nucleic acid sequence encoding the peptide, and the plurality of nucleic acid display libraries are generated by translation using a cell-free translation system, respectively, (2) mixing the plurality of nucleic acid display libraries to prepare a mixed nucleic acid display library, (3) contacting the mixed nucleic acid display library with a target molecule, and (4) amplifying nucleic acids corresponding to the nucleic acid portion in the barcoded peptide- (nucleic acid) complexes that have bound to the target molecule using a barcode primer.

Inventors

  • Chongjin Hangyang
  • Nitta Takeo
  • Shallow Wheel Taiyun
  • Song Weidu

Assignees

  • 中外制药株式会社

Dates

Publication Date
20260505
Application Date
20241010
Priority Date
20231011

Claims (15)

  1. 1. A method for screening candidate peptides for binding to a target molecule, the method comprising the steps of: (1) Preparing a plurality of nucleic acid display libraries comprising barcoded peptide-nucleic acid complexes, wherein the barcoded peptide-nucleic acid complexes comprise a nucleic acid portion and a peptide portion, the nucleic acid portion comprising a barcode sequence and a nucleic acid sequence encoding the peptide, and each of the plurality of nucleic acid display libraries is a nucleic acid display library that is independently generated by translation using a cell-free translation system; (2) Mixing the plurality of nucleic acid display libraries to prepare a mixed nucleic acid display library; (3) Contacting the mixed nucleic acid display library with the target molecule, and (4) Amplifying a nucleic acid corresponding to the nucleic acid portion of the barcoded peptide-nucleic acid complex bound to the target molecule using a barcoded primer.
  2. 2. The method of claim 1, wherein step (1) comprises translating a nucleic acid encoding a peptide to produce a peptide and ligating the peptide to the nucleic acid to produce a peptide-nucleic acid complex.
  3. 3. The method of claim 1 or 2, wherein step (1) comprises barcoding the peptide-nucleic acid complex to produce a barcoded peptide-nucleic acid complex.
  4. 4. The method of any one of claims 1 to 3, wherein the barcoding is reverse transcription of a nucleic acid portion of the peptide-nucleic acid complex.
  5. 5. The method of any one of claims 1-4, wherein the barcoding is reverse transcription of a nucleic acid portion of the peptide-nucleic acid complex with a first primer comprising a barcode sequence.
  6. 6. The method of any one of claims 1 to 5, wherein the plurality of nucleic acid display libraries has a different barcode sequence for each nucleic acid display library.
  7. 7. The method of any one of claims 1-6, wherein each of the plurality of nucleic acid display libraries is a nucleic acid display library that is independently generated by translation using cell-free translation systems that are different from each other.
  8. 8. The method of any one of claims 1 to 7, wherein in step (2), two or more nucleic acid display libraries are mixed.
  9. 9. The method according to any one of claims 1 to 8, comprising, between step (3) and step (4), a step (3A) of eluting the nucleic acid portion from the barcoded peptide-nucleic acid complex bound to the target molecule.
  10. 10. The method according to any one of claims 1 to 9, further comprising, between step (3) and step (4), a step (3B) of identifying the amino acid sequence of the peptide portion of the barcoded peptide-nucleic acid complex that binds to the target molecule.
  11. 11. The method of any one of claims 1 to 10, wherein in step (4), nucleic acids of the barcoded peptide-nucleic acid complex having the same barcode sequence as the barcode sequence of the barcode primer are selectively amplified.
  12. 12. The method of any one of claims 1-11, wherein the barcode primer is a second primer comprising a barcode sequence.
  13. 13. The method according to any one of claims 1 to 12, wherein steps (1) to (4) constitute one cycle, and the cycle is repeated a plurality of times.
  14. 14. The method of any one of claims 1 to 13, further comprising step (5) of further amplifying the nucleic acid amplified in step (4) with a primer that does not contain a barcode sequence after step (4) or before starting the next cycle.
  15. 15. The method of any one of claims 1-14, wherein the nucleic acid display library is a library having a diversity of 10 4 or greater.

Description

Methods for screening peptides using multiple libraries Technical Field The present invention relates to a method for screening peptides using a plurality of libraries. Background MRNA display technology using a recombinant cell-free translation system of Escherichia coli (E.coli) has been used to synthesize a peptide library containing unnatural amino acids and use it to screen peptides with high binding ability (patent document 1). In order to find useful peptides efficiently in the screening, the diversity of the library is very important. For this reason, it is important to increase the diversity of constituent units (amino acids) of peptides contained in the library. However, when synthesizing peptides by translation, the types of amino acids that can be incorporated into a codon table are limited, and thus the number of available amino acids is limited. Therefore, it is being studied to create highly diverse libraries using different cell-free translation systems (non-patent documents 1 and 2). [ Quotation list ] [ Patent literature ] [ Patent document 1] International publication No. WO 2013/100132 [ Non-patent literature ] Non-patent document 1 d.e. hacker et al ACS chem. Biol., 2017, 12, 795-804. Non-patent document 2 m.e. brousseau et al Cell chem. Biol., 2022, 29, 249-258. Disclosure of Invention [ Technical problem ] Theoretically, when a plurality of cell-free translation systems are used to individually generate a peptide-nucleic acid complex library in which phenotypes and genotypes are correlated, the diversity of peptides as phenotypes can be increased as a whole. However, in this case, since genotypes are common in libraries, it is necessary to independently process the libraries according to each cell-free translation system, and thus the time and effort required for panning increases according to the number of libraries, which is a problem. Thus, it is conceivable to use a mixed library and a method of panning to more easily select candidate peptides that can bind to a target molecule. However, when mixing and panning against a target molecule pair, the association between genotype (nucleic acid) and phenotype (peptide) can fail and it is difficult to identify from which library the enriched peptide (specifically, the peptide that is the phenotype corresponding to the enriched nucleic acid) is derived. Furthermore, when libraries are mixed and panned, if there is bias, such as enrichment of peptides that bind easily to target molecules in a particular library, peptides derived from that library are easily enriched, while peptides derived from other libraries are relatively difficult to enrich (resulting in low enrichment), and thus it may be difficult to obtain hit peptides with various structures. The present invention has been made in view of such circumstances, and an object of the present invention is to provide a method of efficiently screening a plurality of peptide-nucleic acid complex libraries. [ Solution to the problem ] The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result have found a method capable of identifying from which library a peptide binding to a target molecule is derived by imparting a peptide-nucleic acid complex nucleic acid barcode (barcoding) contained in the library, even when a plurality of libraries are mixed, and have finally completed the present invention. The present invention provides the following [ A1] to [ A67], [ B1] to [ B3], [ C1] to [ C5] or [ D1] to [ D9]. [A1] a method for screening candidate peptides for binding to a target molecule, the method comprising the steps of: (1) Preparing a plurality of nucleic acid display libraries comprising barcoded peptide-nucleic acid complexes, wherein the barcoded peptide-nucleic acid complexes comprise a nucleic acid portion and a peptide portion, the nucleic acid portion comprising a barcode sequence and a nucleic acid sequence encoding the peptide, and the plurality of nucleic acid display libraries are nucleic acid display libraries each of which is independently generated by translation using a cell-free translation system; (2) Mixing the plurality of nucleic acid display libraries to prepare a mixed nucleic acid display library; (3) Contacting the mixed nucleic acid display library with the target molecule, and (4) A nucleic acid corresponding to the nucleic acid portion of the barcoded peptide-nucleic acid complex bound to the target molecule is amplified using a barcoded primer. [A2] the method of [ A1], wherein the step (1) comprises independently translating a plurality of nucleic acid libraries using a cell-free translation system to produce a plurality of peptide-nucleic acid complexes. [A3] the method of [ A1] or [ A2], wherein the step (1) comprises translating a nucleic acid encoding a peptide to produce a peptide and ligating the peptide to the nucleic acid to produce a peptide-nucleic acid complex. [A4] The method of any one of