Method for screening sequences that modulate the display efficiency of polynucleotide display methods

Abstract

The present invention addresses the problem of providing a method that can more easily screen for, with a higher precision, a sequence that regulates the presentation efficiency in a polynucleotide presentation method. The problem is solved by a method for screening for a sequence that regulates the presentation efficiency in a polynucleotide presentation method, the method comprising: (a) a step for binding a polynucleotide-polypeptide complex, obtained through translation by an acellular translation system from a polynucleotide including a polypeptide encoding sequence and random sequences, to a solid phase via a modification substance on the polypeptide in the polynucleotide-polypeptide complex; and (b) a step for selecting a random sequence by using, as an index, the ratio (concentration factor) of the emergence frequency of each of the random sequences in the polynucleotide-polypeptide complex bonded to the solid phase with respect to the emergence frequency of each of the random sequences in the polynucleotide.

Description

[Technical field]

[0001] The present invention relates to a method for screening a sequence that regulates the display efficiency of a polynucleotide display method, and the like. [Background technology]

[0002] Functional molecules that have high affinity and specificity for a target are used as detection tools and candidate therapeutic drugs. One method for creating such new functional molecules (peptides and proteins) is evolutionary molecular engineering, which obtains functional molecules from libraries with high diversity. In evolutionary molecular engineering, the greater the diversity of the initial library used, the higher the probability of obtaining functional molecules with high affinity, so diversity is an important factor in evolutionary molecular engineering.

[0003] The diversity of libraries varies greatly depending on the technique used. The phage display technique, which is widely used as a directed evolution technique, requires transformation, so the diversity of libraries is limited to 10 9 ~10 11 On the other hand, in the case of a method using a cell-free translation system such as the mRNA display method (this method and similar methods are sometimes collectively referred to as the "polynucleotide display method" in this specification), since it does not involve transformation of E. coli, the amount of translation can be limited to about 10 13 It is possible to construct libraries with a wide range of diversity.

[0004] In the mRNA display method, a phenotype protein is displayed on the genotype mRNA via puromycin linker (PuL). However, since PuL needs to be covalently bound to mRNA before the translation reaction, the multi-step reactions of transcription to mRNA, ligation with PuL, and translation reaction must be performed separately. Therefore, the inventors have improved the mRNA display method and developed the TRAP display method, which enables simple and rapid selection. In the TRAP display method, an mRNA-protein complex can be automatically generated from a DNA template using a reconstituted cell-free translation system that does not include RF1, called the TRAP reaction system. This allows the above multi-step reactions to be performed continuously, which successfully shortens the time required for selection.

[0005] Diversity in the TRAP display method is expressed as the product of mRNA concentration, translation reaction volume, and efficiency of protein display on mRNA. The mRNA concentration is limited by the ribosome concentration in the translation reaction solution (approximately 1 μM), and the translation reaction solution is expensive and there is a limit to the amount that can be used. Therefore, in order to increase diversity, it is necessary to improve the display efficiency.

[0006] Non-Patent Document 1 reports an attempt to obtain N- and C-terminal sequences that improve the presentation efficiency in the mRNA display method. In this prior study, a library in which both the N- and C-terminal sequences of VHH antibodies were randomized was prepared, and then transcription to mRNA and protein presentation on mRNA in a cell-free translation system were performed. From there, only the mRNA-protein complex was collected by gel purification, and N- and C-terminal sequences with high presentation efficiency were enriched. As a result of performing this selection for several cycles, a combination of N- and C-terminal sequences that slightly increased the presentation efficiency (1.2 times) in the mRNA display method was obtained. In Non-Patent Document 2, as in Non-Patent Document 1, a library in which the C-terminal sequence of scFv was randomized was prepared, and then transcription to mRNA and protein presentation on mRNA in a cell-free translation system were performed. From this library, only the mRNA-protein complex was collected using beads on which the target of scFv, which is a phenotype, was used to enrich C-terminal sequences with high presentation efficiency. As a result, a C-terminal sequence that improved the presentation efficiency by about 3 times was obtained. However, these methods require repeated selection until a sequence with high display efficiency is obtained, and their versatility is unknown. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] K. Takahashi, M. Sunohara, T. Terai, S. Kumachi, N. Nemoto, Enhanced mRNA-protein fusion efficiency of a single-domain antibody by selection of mRNA display with additional random sequences in the terminal translated regions. Biophysics and Physicobiology 14, 23-28 (2017). [Non-Patent Document 2] Y. Nagumo, K. Fujiwara, K. Horisawa, H. Yanagawa, N. Doi, PURE mRNA display for in vitro selection of single-chain antibodies. Journal of Biochemistry 159, 519-526 (2016). Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present invention is to provide a method for more easily and more accurately screening for sequences that regulate the display efficiency of polynucleotide display methods. [Means for solving the problem]

[0009] In view of the above problems, the present inventors have conducted intensive research and found that the above problems can be solved by a method for screening a sequence that regulates the presentation efficiency of a polynucleotide display method, the method comprising: (a) binding a polynucleotide-polypeptide complex obtained by translating a polynucleotide containing a polypeptide coding sequence and a random sequence in a cell-free translation system to a solid phase via a modifying substance on the polypeptide in the polynucleotide-polypeptide complex; and (b) selecting a random sequence using as an index the ratio (enrichment rate) of the frequency of occurrence of each random sequence in the polynucleotide-polypeptide complex bound to the solid phase relative to the frequency of occurrence of each random sequence in the polynucleotide. Then, using the screening method, a sequence that improves the presentation efficiency of the polynucleotide display method, independent of the polypeptide coding sequence, was found. Based on these findings, the present inventors conducted further research and completed the present invention. That is, the present invention encompasses the following aspects.

[0010] Item 1. (a) A step of binding a polynucleotide-polypeptide complex obtained by translating a polynucleotide containing a polypeptide coding sequence and a random sequence in a cell-free translation system to a solid phase via a modifying substance on the polypeptide in the polynucleotide-polypeptide complex; and (b) selecting a random sequence based on the ratio (enrichment factor) of the frequency of occurrence of each random sequence in the polynucleotide-polypeptide complex bound to the solid phase to the frequency of occurrence of each random sequence in the polynucleotide; A method for screening for a sequence that regulates the display efficiency of a polynucleotide display method, comprising:

[0011] Item 2. The screening method according to Item 1, wherein in the step (b), a random sequence having a high enrichment rate is selected as a sequence that improves display efficiency.

[0012] Item 3. The screening method according to Item 1 or 2, wherein in the step (b), a random sequence having an enrichment rate in the top 10% is selected as a sequence that improves the display efficiency.

[0013] Item 4. The screening method according to any one of Items 1 to 3, wherein the modifying substance is at least one selected from the group consisting of biotin, a peptide tag, and a substance containing a readily reactive group.

[0014] Item 5. The screening method according to any one of Items 1 to 4, wherein the random sequence is located downstream of the polypeptide-encoding sequence.

[0015] Item 6. The screening method according to Item 5, wherein a linker sequence is disposed between the random sequence and the polypeptide-encoding sequence.

[0016] Item 7. The screening method according to Item 5 or 6, wherein a stop codon is located adjacent to or within the random sequence.

[0017] Item 8. A polynucleotide comprising a polypeptide coding sequence or a site for inserting a polypeptide coding sequence, sequence 1 which is 3 bases long, sequence 2 which is 3 bases long, and a stop codon, which are arranged in the following order from upstream: a polypeptide coding sequence or a site for inserting a polypeptide coding sequence, sequence 1, sequence 2, and the stop codon, wherein sequence 1 and sequence 2 are adjacent to each other, and sequence 1 is GGC, CGT, GGT, or AAA, and / or sequence 2 is CGA, AGC, AGA, ACA, CGT, AGT, GCA, AGG, GGT, CGC, GCT, GGC, CGG, ACT, GAT, GAC, ATG, CCA, CCG, CCT, CTA, or CTG.

[0018] Item 9. The polynucleotide according to Item 8, wherein sequence 1 is GGC, CGT, GGT, or AAA, and sequence 2 is CGA, AGC, AGA, ACA, CGT, AGT, GCA, AGG, GGT, CGC, GCT, GGC, CGG, ACT, GAT, GAC, ATG, CCA, CCG, CCT, CTA, or CTG.

[0019] Item 10. The polynucleotide according to Item 8 or 9, wherein the consecutive sequences of sequence 1 and sequence 2 are GGCCGA, GGCAGA, GGCGCA, GGTAGA, GGCAGT, GGCAGC, GGCACA, GGCCGT, GGCGCT, GGCGAT, GGCACT, GGTCGA, GGCAGG, GGCACG, GGTGAC, GGTAGG, CGTCGA, GGTAGT, GGTCGT, GGCGGT, GGACGA, GGTGGT, GGTACA, GGTCGC, AATCGA, GGTAGC, or GCGCGA.

[0020] Item 11. The polynucleotide according to any one of Items 8 to 10, wherein the sequence 2 and the termination codon are adjacent to each other.

[0021] Item 12. A reagent comprising the polynucleotide according to any one of Items 8 to 11.

[0022] Item 13. The reagent according to Item 12, which is a reagent for a polynucleotide display method. Effect of the Invention

[0023] According to the present invention, a method for screening sequences that regulate the display efficiency of polynucleotide display method can be provided more simply and with higher accuracy. Furthermore, according to the present invention, a polynucleotide useful as a reagent for polynucleotide display method can be provided, which includes a sequence that improves the display efficiency of polynucleotide display method, independent of a polypeptide coding sequence. In addition, since the enrichment rate obtained from the N-terminal library reflects the efficiency of translation initiation, which is important in protein expression, it is believed that the amount of protein expression can be controlled by using this information. This is useful for the production of useful substances using cells and for highly efficient production of proteins. [Brief description of the drawings]

[0024] [Figure 1] A schematic diagram of the screening method for the C-terminal random sequence in Test Example 1 is shown. A: The design of the library used is shown. A random sequence was added to the C-terminus of DNA encoding Monobody or Anticalin. The random sequence was composed of Codon 1, Codon 2, and Base 1, and two types of mixed libraries, I and II, were created. B: The procedure of the screening method used in this study is shown. After creating a DNA library by PCR, it was transcribed into mRNA, annealed with PuL, and translated in a TRAP reaction system. A portion of the library was recovered using biotin-streptavidin interaction. The libraries before and after recovery were sequenced, and the ratio of the frequency of occurrence in each library was calculated. This value was taken as the enrichment rate and used as an index for predicting the display efficiency. [Figure 2A] The data analysis and evaluation of the results of screening for the C-terminal random sequence in Test Example 1 are shown. A: The enrichment rate of each C-terminal sequence is plotted in the Monobody library and the Anticalin library. The lower part shows a distribution diagram of each. The position of the sequence used in B is shown in the distribution diagram. [Figure 2B]Data analysis and evaluation of the results of screening for C-terminal random sequences in Test Example 1 are shown. A: The display efficiency of monobodies with the 19 types of C-terminal sequences listed in Table 1 added was measured. The graph on the bottom left shows the results of plotting the display efficiency against the enrichment rate. Error bars represent standard deviation (N = 3). The method for calculating the display efficiency is shown in the bottom right. [Diagram 3] The results of measuring the display efficiency when three types of C-terminal sequences were added to the upstream sequence other than T Monobody are shown. c1 is the C-terminal sequence with the highest enrichment rate, c16 is in the top 2.75%, and c13 is 34.69%. Error bars represent standard deviation (N = 3). A: Monobody library. B: Anticalin. C: Macrocyclic peptide library. [Figure 4] 1 shows a heat map (Test Example 1) in which the enrichment rate of each C-terminal sequence in the Monobody library is represented by color. [Diagram 5] FIG. 1 shows a schematic diagram of the screening method for N-terminal random sequences in Test Example 3. [Figure 6] The data analysis of the results of screening for the N-terminal random sequence in Test Example 3 is shown. A: To confirm reliability, two independent experiments were repeated using the Monobody library, and the enrichment rate of each N-terminal sequence of the obtained data was plotted. B: The enrichment rate of each N-terminal sequence was plotted for the values ​​of each N-terminal sequence of the data obtained for two types of sequences, Monobody and Anticalin. C: The enrichment rate of each N-terminal sequence was plotted for the values ​​of each N-terminal sequence of the data obtained for two types of sequences including the SD sequence. [Figure 7] 1 shows a heat map (Test Example 4) in which the enrichment rate of each C-terminal sequence in the monobody library is represented by color. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0026] 1. Screening Method In one aspect, the present invention relates to a method for screening a sequence that regulates the display efficiency of a polynucleotide display method, comprising steps (a) and (b) (also referred to herein as the "screening method of the present invention"). This will be described below.

[0027] Step (a) is a step of binding a polynucleotide-polypeptide complex obtained by translation in a cell-free translation system from a polynucleotide comprising a polypeptide coding sequence and a random sequence (sometimes referred to as a "polynucleotide for screening" in this specification) to a solid phase via a modifying substance on the polypeptide in the polynucleotide-polypeptide complex.

[0028] The polypeptide coding sequence contained in the screening polynucleotide is not particularly limited as long as it is a nucleotide sequence that encodes a polypeptide, such as an antibody, a monobody, an enzyme, a ligand, a receptor, a structural protein, a linear polypeptide, an oligopeptide, or a fragment thereof.

[0029] The random sequence contained in the polynucleotide for screening is a sequence to be screened. The polynucleotide for screening is a mixture containing multiple types of polynucleotides with different random sequences. The number of types of polynucleotides for screening (the number of types of random sequences) is, for example, 10 1 ~10 11 The lower limit of this number is, for example, 10 2 , 10 3 The upper limit of the number can be, for example, 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 105 , 10 4 It can be.

[0030] The position of the random sequence is not particularly limited. The random sequence may be located upstream of the polypeptide coding sequence (i.e., on the N-terminus side when translated into a polypeptide) or downstream of the polypeptide coding sequence (i.e., on the C-terminus side when translated into a polypeptide). In a preferred embodiment of the present invention, the random sequence is located downstream of the polypeptide coding sequence. This allows screening for a sequence that can regulate the display efficiency regardless of the type or sequence of the polypeptide encoded by the upstream polypeptide coding sequence.

[0031] In the screening polynucleotide, the random sequence may be present at only one position, or may be present at two or more positions that are not adjacent to each other. In one embodiment, the random sequence contains a stop codon. In this case, the random sequence 1, the stop codon, and the random sequence 2 are arranged in this order.

[0032] The length of the random sequence (base length: when the random sequence is present at two or more positions, the total length of all positions) is not particularly limited, and is, for example, 3 to 200, preferably 3 to 100, more preferably 3 to 50, even more preferably 4 to 30, still more preferably 5 to 20, and particularly preferably 5 to 10.

[0033] As used herein, the term "stop codon" refers to a codon that does not include a tRNA having a corresponding anticodon in the reaction system, and is not particularly limited thereto. Specific examples of stop codons include codons to which no amino acid is assigned in the codon table, such as TAA, TAG, and TGA, as well as codons corresponding to the anticodon of a tRNA that is not added to the reaction system, and codons containing modified bases.

[0034] The configuration of the screening polynucleotide is not particularly limited as long as it can be translated in a cell-free translation system to synthesize a polypeptide encoded by the polypeptide coding sequence. When the polypeptide coding sequence does not have an initiation codon and / or a stop codon, the screening polynucleotide has an initiation codon upstream of the polypeptide coding sequence and / or a stop codon downstream of the polypeptide coding sequence.

[0035] When the random sequence is located downstream of the polypeptide coding sequence in the screening polynucleotide, it is preferable that another sequence is located between the polypeptide coding sequence and the random sequence. The other sequence can be preferably a linker sequence. The linker sequence is not particularly limited as long as it has a flexible chain structure, and examples thereof include linker sequences containing glycine and / or serine. The length (base length) of the other sequence is, for example, 3 or more, preferably 4 or more. The upper limit of the length is not particularly limited, and can be, for example, 100, 50, 30, or 20.

[0036] The screening polynucleotide can be used as a cell-free translation system, either a prokaryotic translation system or a eukaryotic translation system. From the viewpoint of simplicity, it is preferable to use a prokaryotic translation system as a cell-free translation system. When the screening polynucleotide uses a prokaryotic translation system, it can preferably have a Shine-Dalgarno sequence (SD sequence) upstream of the start codon. When the screening polynucleotide uses a eukaryotic translation system, it is preferable that the screening polynucleotide has a Kozak sequence, a 5' cap, and an internal ribosome entry site (IRES).

[0037] A preferred embodiment of the screening polynucleotide is a polynucleotide comprising a sequence consisting of a polypeptide coding sequence, a random sequence, and a stop codon, in that order from upstream. In this embodiment, the random sequence and the stop codon are preferably adjacent to each other, and a linker sequence is preferably disposed between the polypeptide coding sequence and the random sequence.

[0038] Another preferred embodiment of the screening polynucleotide is a polynucleotide comprising a sequence arranged in this order from upstream: an initiation codon, a random sequence, a polypeptide coding sequence, and a termination codon. In this embodiment, it is preferred that the initiation codon and the random sequence are adjacent to each other.

[0039] The screening polynucleotide may include a peptide tag coding sequence adjacent to the polypeptide coding sequence. In one embodiment, the peptide tag encoded by the peptide tag coding sequence may be used as a modifier on the polypeptide described below, and may be bound to a solid phase. Examples of peptide tags include His tag, FLAG tag, Halo tag, GST tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, etc.

[0040] The type of nucleotide constituting the screening polynucleotide is not particularly limited as long as it is capable of synthesizing a polypeptide in a translation system. The screening polynucleotide may preferably be RNA (mRNA).

[0041] The screening polynucleotide can be obtained according to or in accordance with a known method, for example, by transcription in a cell-free transcription system from a DNA vector encoding the screening polynucleotide.

[0042] The polynucleotide-polypeptide complex is obtained by translating the screening polynucleotide in a cell-free translation system. The polynucleotide-polypeptide complex is a complex of the screening polynucleotide and / or its complementary strand (e.g., cDNA, etc.) and a polypeptide encoded by the polypeptide coding sequence in the screening polynucleotide. The complex can be obtained according to or in accordance with a known method. For example, this is performed by binding puromycin downstream of the stop codon of the screening polynucleotide. Puromycin may be bound to the screening polynucleotide via a linker composed of a peptide or nucleic acid. By binding puromycin to the downstream region of the stop codon of the screening polynucleotide, a ribosome that has translated the polypeptide coding sequence of the screening polynucleotide takes up puromycin, and a complex of the screening polynucleotide and the polypeptide is formed.

[0043] As used herein, the term "cell-free translation system" refers to a translation system that does not contain cells. Examples of cell-free translation systems that can be used include Escherichia coli extract, wheat germ extract, rabbit red blood cell extract, and insect cell extract. In addition, a reconstituted cell-free translation system constructed by reconstituting purified ribosomal proteins, aminoacyl-tRNA synthetases (aaRS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation initiation factors (IFs), elongation factors (EFs), release factors (RFs), and ribosome recycling factors (RRFs), as well as other factors necessary for translation, may also be used. A system containing RNA polymerase may also be used to perform transcription from DNA. Commercially available cell-free translation systems that can be used include RTS-100 (registered trademark) from Roche Diagnostics, an Escherichia coli-derived system, PURESYSTEM (registered trademark) from PGI, and PURExpressR In Vitro Protein Synthesis Kit from New England BioLabs, as well as systems using wheat germ extract from Zoigene and Cell Free Sciences.As a system using Escherichia coli ribosomes, for example, the techniques described in the following documents are known: H. F. Kung et al., 1977, The Journal of Biological Chemistry Vol. 252, No. 19, 6889-6894; M. C. Gonza et al., 1985, Proceeding of National Academy of Sciences of the United States of America Vol. 82, 1648-1652; M. Y. Pavlov and M. Ehrenberg, 1996, Archives of Biochemistry and Biophysics Vol. 328, No. 1, 9-16; Y. Shimizu et al., 2001, Nature Biotechnology Vol. 19, No. 8, 751-755; H. Ohashi et al., 2007, Biochemical and Biophysical Research Communications Vol. 352, No. 1, 270-276. According to the cell-free translation system, an expression product can be obtained in a highly pure form without purification. The cell-free translation system of the present invention may be used not only for translation but also for transcription by adding factors necessary for transcription.

[0044] The polynucleotide-polypeptide complex has a modifying substance on the polypeptide for binding to the solid phase. The modifying substance is not particularly limited as long as it can be used for binding to the solid phase, and examples thereof include biotin, peptide tags, and substances containing easily reactive groups. Biotin can be bound to a solid phase modified with avidins (e.g., streptavidin, etc.). Peptide tags can be bound to a solid phase modified with a substance having affinity for it depending on the type of the tag (e.g., anti-HA antibodies in the case of HA tags, metals such as nickel in the case of His tags, anti-FLAG antibodies in the case of FLAG tags, and glutathione in the case of GST tags). Examples of easily reactive groups include ethynyl groups, vinyl groups, azide groups, epoxy groups, aldehyde groups, and oxylamino groups. It is known that an ethynyl group forms a 1,2,3-triazole ring by 1,3-dipolar cycloaddition reaction with an azide group. A vinyl group reacts with a thiol group to form a bond. An epoxy group reacts with an amino group or a thiol group to form a bond. Aldehyde groups react with amino groups to form Schiff bases, which can be reduced to form bonds. Oxylamino groups react with ketone and aldehyde groups to form oximes. Azide groups are known to undergo 1,3-dipolar cycloaddition with ethynyl groups to form 1,2,3-triazole rings. These reactions can be used to bond easily reactive groups to solid phases modified with the corresponding functional groups.

[0045] The modifying substance can be bound to the polypeptide according to or in accordance with a known method. For example, in the case of a peptide tag, as described above, the polypeptide can be modified with the peptide tag by arranging the peptide tag coding sequence adjacent to the polypeptide coding sequence in the screening polynucleotide. In addition, in the case of a substance containing biotin or a reactive group, the polypeptide can be modified with these substances by introducing the tRNA obtained by acylation of any tRNA with an amino acid containing these substances into a cell-free translation system.

[0046] The above tRNA can be prepared by using flexizyme. Flexizyme is an artificial aminoacylation RNA catalyst that can acylate any tRNA with any amino acid or hydroxy acid. By using flexizyme instead of the aminoacyl-tRNA synthesized by natural aminoacyl-tRNA synthetase, the genetic code table can be rewritten by associating a desired amino acid or hydroxy acid with any codon. This is called codon reallocation. For codon reallocation, a translation system can be used in which the components of the translation system can be freely removed according to the purpose and only the necessary components are reconstituted. For example, when a translation system in which a specific amino acid has been removed is reconstituted, the codon corresponding to the amino acid becomes a vacant codon that does not code for any amino acid. Therefore, by using flexizyme or the like to link any amino acid to a tRNA having an anticodon complementary to the vacant codon and adding this to perform translation, the arbitrary amino acid will be coded by that codon, and a peptide in which the arbitrary amino acid has been introduced in place of the removed amino acid will be translated.

[0047] The solid phase material is not particularly limited, and can be selected from, for example, organic polymer compounds, inorganic compounds, biopolymers, etc. Examples of organic polymer compounds include latex, polystyrene, polypropylene, etc. Examples of inorganic compounds include magnetic materials (iron oxide, chromium oxide, ferrite, etc.), silica, alumina, glass, etc. Examples of biopolymers include insoluble agarose, insoluble dextran, gelatin, cellulose, etc. The solid phase material can be one type alone or two or more types in combination.

[0048] The shape of the solid phase is not particularly limited, and examples thereof include particles, microplates, microtubes, test tubes, and membranes.

[0049] The binding of the polynucleotide-polypeptide complex to the solid phase can be carried out according to or in accordance with a known method depending on the type of modifier on the polypeptide, the type of modifier on the solid phase, etc. Specifically, the polynucleotide-polypeptide complex and the solid phase can be bound to each other by contacting them in a solution of appropriate composition. After binding, it is preferable to wash the polynucleotide-polypeptide complex and the solid phase with a solution or solvent that does not dissociate them.

[0050] Step (b) is a step of selecting a random sequence based on the ratio (enrichment rate) of the frequency of occurrence of each random sequence in the polynucleotide-polypeptide complex bound to the solid phase to the frequency of occurrence of each random sequence in the screening polynucleotide.

[0051] The occurrence frequency (occurrence frequency X) of each random sequence in the screening polynucleotide is, for example, (X1) The number of molecules of the screening polynucleotide before translation in the cell-free translation system; (X2) The number of molecules of the screening polynucleotide in a mixture containing a polynucleotide-polypeptide complex before binding to a solid phase (which also contains a screening polynucleotide in which the polypeptide has not been translated), or (X3) The number of molecules of the screening polynucleotide in the mixture that has not been subjected to solid-liquid separation after being subjected to binding to the solid phase (which also includes the screening polynucleotide in which the polypeptide has not been translated), For example, the ratio of the number of screening polynucleotides having each random sequence to the number of screening polynucleotides having each random sequence. 4 A mixture containing a variety of polynucleotides, and the number of polynucleotide molecules having a certain random sequence (random sequence A) in the mixture is 10 2 If there are 10, the occurrence frequency X of random sequence A is 1% (=(10 2 / 10 4 )×100 (%).

[0052] The frequency of occurrence (frequency of occurrence Y) of each random sequence in the polynucleotide-polypeptide complex bound to the solid phase can be expressed as, for example, (Y1) the number of molecules of the screening polynucleotide in the polynucleotide-polypeptide complex obtained by binding to the solid phase and then performing solid-liquid separation, or (Y2) the number of molecules of the screening polynucleotide in the polynucleotide-polypeptide complex obtained by binding to the solid phase, followed by solid-liquid separation and washing; The ratio of the number of molecules of the screening polynucleotide having each random sequence to the number of molecules of the screening polynucleotide having each random sequence.

[0053] The occurrence frequency of each random sequence can be measured and calculated according to or in accordance with a known method. The occurrence frequency can be measured and calculated, for example, by sequence analysis using a next-generation sequencer. In this case, the above-mentioned "number of molecules" can be replaced with the "number of reads" by the next-generation sequencer.

[0054] In step (b), a random sequence is selected using the ratio (enrichment rate) of occurrence frequency Y to occurrence frequency X as an index. In the present invention, it has been found that the enrichment rate correlates with the display efficiency of the polynucleotide display method, and based on this, it has been found that sequences that regulate the display efficiency can be screened in step (b).

[0055] The polynucleotide display method is a technique for artificially evolving molecules by in vitro selection using a cell-free translation system. Examples of polynucleotide display methods include mRNA display, cDNA display, ribosome display, and TRAP display. The display efficiency can be measured according to or in accordance with a known method, for example, by electrophoresing a reaction product after translation in a cell-free translation system from mRNA encoding a protein to be displayed, and measuring the band intensity according to the formula shown in FIG. 2B described below.

[0056] In step (b), for example, a random sequence with a high enrichment rate can be selected to screen for a sequence that improves the presentation efficiency. Here, "high enrichment rate" refers to a sequence with a high enrichment rate when all random sequences are arranged in order of enrichment rate. In a preferred embodiment of the present invention, a random sequence with an enrichment rate in the top 30% (preferably 20%, more preferably 10%, even more preferably 5%, and even more preferably 3%) can be selected as a sequence that improves the presentation efficiency. In another preferred embodiment of the present invention, a random sequence with an enrichment rate of 1.5 or more (preferably 2 or more, more preferably 2.5 or more, and even more preferably 3 or more) can be selected as a sequence that improves the presentation efficiency.

[0057] 2. Polynucleotides and Reagents In one aspect, the present invention relates to a polynucleotide (sometimes referred to as the "polynucleotide of the present invention" in this specification) that includes a polypeptide coding sequence or a site for inserting a polypeptide coding sequence, sequence 1 having a length of 3 bases, sequence 2 having a length of 3 bases, and a stop codon, and is arranged in the order of a polypeptide coding sequence or a site for inserting a polypeptide coding sequence, sequence 1, sequence 2, and a stop codon from upstream, and sequence 1 and sequence 2 are adjacent to each other, and sequence 1 is GGC, CGT, GGT, or AAA, and / or sequence 2 is CGA, AGC, AGA, ACA, CGT, AGT, GCA, AGG, GGT, CGC, GCT, GGC, CGG, ACT, GAT, GAC, ATG, CCA, CCG, CCT, CTA, or CTG. In another aspect, the present invention relates to a reagent (sometimes referred to as the "reagent of the present invention" in this specification) that includes the polynucleotide of the present invention. These will be described below.

[0058] In a more preferred embodiment of the invention, said sequence 1 is GGC, CGT, GGT, or AAA (or GGC, CGT, or GGT), and said sequence 2 is CGA, AGC, AGA, ACA, CGT, AGT, GCA, AGG, GGT, CGC, GCT, GGC, CGG, ACT, GAT, GAC, ATG, CCA, CCG, CCT, CTA, or CTG (or GA, AGC, AGA, ACA, CGT, AGT, GCA, AGG, GGT, CGC, GCT, GGC, CGG, ACT, GAT, or GAC).

[0059] In a more preferred embodiment of the present invention, the contiguous sequences of sequence 1 and sequence 2 are GGCCGA, GGCAGA, GGCGCA, GGTAGA, GGCAGT, GGCAGC, GGCACA, GGCCGT, GGCGCT, GGCGAT, GGCACT, GGTCGA, GGCAGG, GGCACG, GGTGAC, GGTAGG, CGTCGA, GGTAGT, GGTCGT, GGCGGT, GGACGA, GGTGGT, GGTACA, GGTCGC, AATCGA, GGTAGC, GCGCGA.

[0060] In an even more preferred embodiment of the invention, the contiguous sequences of SEQ ID NO:1 and SEQ ID NO:2 are GGCCGA, GGCAGA, GGTAGA, GGCACA, GGCCGT, GGCAGT, GGCAGC, GGCAGG, GGTCGA, GGCGCT, GGCACT, GGCGCA, GGTAGG.

[0061] In particularly preferred embodiments of the invention, the contiguous sequences of SEQ ID NO:1 and SEQ ID NO:2 are GGCCGA, GGCAGA, GGTAGA, GGCCGT, GGCAGT, GGCAGC, GGTCGA, GGCGCT, GGCACT.

[0062] In the polynucleotide of the present invention, sequence 2 is preferably located relatively close to the stop codon, and the base length between sequence 2 and the stop codon is preferably 0 to 6, more preferably 0 to 3, and even more preferably 0 (i.e., sequence 2 and the stop codon are adjacent to each other). This can further improve the display efficiency.

[0063] In the polynucleotide of the present invention, the 1 base adjacent to the downstream side of the termination codon is more preferably T or C.

[0064] The site for inserting a polypeptide coding sequence can be, for example, a restriction enzyme site, and preferably, the site for inserting a polypeptide coding sequence can be a multicloning site containing multiple restriction enzyme sites.

[0065] A polynucleotide of the invention can be, for example, an mRNA or a vector for expressing said mRNA.

[0066] Regarding the configuration of the polynucleotide of the present invention other than the above, the configuration of the polynucleotide for screening described above can be adopted.

[0067] The reagent of the present invention is preferably a reagent for a polynucleotide display method. The polynucleotide display method is as described above.

[0068] In the reagent of the present invention, the polynucleotide of the present invention can be in the form of a composition containing it. The composition may contain other components as necessary. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0069] The reagent of the present invention can be in the form of a kit. In this case, the reagent of the present invention (reagent kit) may contain instruments, reagents, etc. that can be used in the polynucleotide display method. Examples of the instruments include test tubes, microplates, particles, latex particles, purification columns, epoxy-coated slide glasses, gold colloid-coated slide glasses, etc. Examples of the reagents include buffer solutions, cell-free transcription reagents, cell-free translation reagents, etc. EXAMPLES

[0070] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0071] Materials and Methods (1) Oligonucleotides and DNA sequences used The sequence numbers of the oligonucleotides and DNA sequences used in this study are shown in Table 1.

[0072] [Table 1]

[0073] (2) Preparation of a DNA library with random C-terminal sequences A PCR reaction solution (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM MgSO4, 0.2 mM each dNTP, 0.375 μM SD8-MQANSGS-MonoS(H).F61, 0.375 μM MonoS(H)-GGG.R32, 0.2 nM MonoS(H)SSS-HA, 2 nM Pfu-S DNA polymerase) was prepared and 10 cycles of PCR reaction were performed. This was designated Template 1. Next, PCR reaction solution (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM MgSO4, 0.2 mM each dNTP, 0.375 μM T7SD8M2.F44, 0.276 μM MonoS(H)-VVN2N1.R49 or 0.031 μM MonoS(H)-VVN1N2.R50, 0.375 nM Template 1, 2 nM Pfu-S DNA polymerase) was prepared and PCR reaction was performed for 9 cycles. After PCR, DNA was treated with phenol-chloroform and precipitated with isopropanol. This was named MonobodyVVNDNA. MonobodyNNNDNA was prepared in a similar manner to that described above, using MonoS(H)-NNN3.R53 instead of MonoS(H)-VVN2N1.R49.

[0074] A PCR reaction solution (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM MgSO4, 0.2 mM each dNTP, 0.375 μM SD8-Lcn.F62, 0.375 μM Lcn-GGG.R33, 0.375 nM pQ106-Ant-wt, 2 nM Pfu-S DNA polymerase) was prepared and PCR reaction was performed for 18 cycles. This was designated Template 2. Next, PCR reaction solution (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM MgSO4, 0.2 mM each dNTP, 0.375 μM T7SD8M2.F44, 0.276 μM Lcn-VVN2N1.R49 or 0.031 μM Lcn-VVN1N2.R50, 0.375 nM Template 2, 2 nM Pfu-S DNA polymerase) was prepared and PCR reaction was performed for 9 cycles. After PCR, DNA was treated with phenol-chloroform and precipitated with isopropanol. This was named AnticalinVVNDNA.

[0075] (3) Screening for C-terminal random sequences MonobodyVVNDNA, MonobodyNNNDNA, and AnticalinVVNDNA were transcribed into mRNA using T7 RNA polymerase, and then treated with phenol-chloroform and was precipitated with isopropanol twice. These were designated as MonobodyVVNmRNA, MonobodyNNNmRNA, and AnticalinVVNmRNA, respectively. The annealing solution (6.67 μM MonobodyVVNmRNA or MonobodyNNNmRNA or AnticalinVVNmRNA, 4 μM Hex-PuL-an21-3, 25 mM HEPES-K pH7.8, 200 mM AcOK) was incubated at 95°C for 3 minutes and at 25°C for 5 minutes to prepare mRNA-PuL complexes. This was then used in a reconstituted cell-free translation system (16 μM Biotin-Phe-tRNA except for RF1 and Formyl donor). fMet CAU (containing 1 μM of mRNA-PuL complex: fc) and incubated for 30 minutes at 37° C. To the reaction solution (4 μL), 0.8 μL of 100 mM EDTA pH 8.0 was added.

[0076] 0.2 pmol of the mRNA-protein complex prepared above was added to 20 μL of Dynabeads M-280 streptavidin (Thermo Fisher Scientific) and mixed by rotation at 25°C for 5 minutes. The mRNA-protein complex was then collected and washed with 50 μL of HBST (50 mM Hepes-KOH (pH 7.5), 300 mM NaCl, 0.05% (v / v) Tween 20) for 60 seconds and with 50 μL of HBS (50 mM Hepes-KOH (pH 7.5), 300 mM NaCl) for 60 seconds.

[0077] RTprimer annealing solution (5 μM an21-3.R21, 25 mM HEPES-K pH7.8, 200 mM AcOK) was added to 0.02 pmol of unrecovered mRNA-protein complex (before recovery) and the total amount of recovered mRNA-protein complex (after recovery) to make a total of 10 μL, and incubated at 95°C for 2 minutes and at 25°C for 30 seconds. After recovery, only the supernatant after centrifugation was collected. Then, 10 μL of 2xRT mix (124 mM Tris-HCl (pH 8.4), 62.2 mM MgCl2, 186 mM KCl, 13.7 mM dithiothreitol (DTT), 1.24 mM dNTPs, 0.8% (v / v) in-house moloney murine leukemia virus reverse transcriptase (HMLV)) was added and reverse transcription reaction was carried out at 42°C for 30 minutes.

[0078] 180 μL of 1xPCR dNTPs (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2 mM MgSO4, 0.22 mM dNTPs) was added to the reverse transcription product. 85 μL of the mixture was taken, and an equal volume of 2xPCR reaction solution (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 4% (v / v) DMSO, 2 mM MgSO4, 0.2 mM each dNTP, 4 nM Pfu-S DNA polymerase) and 1.28 μL of primer mix [(25 μM MonoMidF22, 25 μM MonoMidF22+2, 50 μM an21-3barcode11 or an21-3barcode12) or (25 μM LcnMidF22, 25 μM LcnMidF22+2, 50 μM an21-3barcode13 or an21-3barcode14)] were added, and 12 cycles of PCR reaction (13 cycles only after recovery of Anticalin) were performed. After PCR, the DNA was treated with phenol-chloroform and precipitated with isopropanol. Sequence analysis of the DNA was performed using a next-generation sequencer (Macrogen Japan).

[0079] (4) Calculation of concentration rate The DNA read by sequence analysis was clustered by C-terminal sequence, and the total number of reads was counted. The percentage of the number of reads of each C-terminal sequence relative to the number of reads in the entire library was calculated as the frequency of occurrence, and the ratio of the frequency of occurrence after recovery to the frequency of occurrence before recovery was calculated as the enrichment rate. In addition, a distribution of the enrichment rates of C-terminal sequences was created.

[0080] (5) Preparation of mRNA with added C-terminal sequence The combinations of primers, template DNA, and cycle numbers used below are shown in Table 2.

[0081] [Table 2]

[0082] A PCR reaction solution (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM MgSO4, 0.2 mM each dNTP, 0.375 μM Forward primer A, 0.375 μM Reverse primer A, 0.375 nM Template A, 2 nM Pfu-S DNA polymerase) was prepared and PCR reaction was performed for W cycles. Next, a PCR reaction solution (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM MgSO4, 0.2 mM each dNTP, 0.375 μM Forward primer B, 0.375 μM Reverse primer B, 0.375 nM Template B, 2 nM Pfu-S DNA polymerase) was prepared and a Z-cycle PCR reaction was performed. After PCR, DNA was treated with phenol-chloroform and precipitated with isopropanol.

[0083] This was transcribed into mRNA using T7 RNA polymerase, treated with phenol / chloroform, and precipitated with isopropanol twice, then gel purified, treated with phenol / chloroform, and precipitated with isopropanol again.

[0084] (6) Presentation efficiency measurement The annealing solution (4.8 μM mRNA, 4 μM Hex-PuL-an21-3, 25 mM HEPES-K pH7.8, 200 mM AcOK) was incubated at 95°C for 3 minutes and then at 25°C for 5 minutes to prepare an mRNA-PuL complex. This was added to the reconstituted cell-free translation system (except for RF1) (mRNA-PuL complex: fc 1 μM) and incubated at 37°C for 30 minutes. However, for the macrocyclic peptide library, 10 μM N-Chloroacetyl-L-Phe-tRNA was added except for RF1 and Formyl donor. fMet CAU A reconstituted cell-free translation system was used, in which the following had been newly added: The translation reaction for the monobody library was carried out at 37°C for 10 minutes. When measuring the display efficiency in the DNA start, DNA (fc 5 nM) and Hex-PuL-an21-3 (fc 1 μM) were added to the reconstituted cell-free translation system (excluding RF1, but containing T7 RNA polymerase) and incubated at 37°C for 30 minutes.

[0085] 11 μL of gel loading buffer (62.5 mM Tris-HCl pH 6.8, 5 mM DTT, 0.05% (w / v) SDS, 10 mM MgCl2, 20% (v / v) Glycerol) was added to 1 μL of the sample before and after the translation reaction, and 2 μL of the sample was electrophoresed on 8% PAGE (0.375 M Tris-HCl pH 8.8, 6 M Urea, 0.05% SDS). For the macrocyclic peptide library, 8% PAGE (0.45 M Tris-HCl pH 8.8, 6 M Urea, 0.05% SDS) was used. Bands were confirmed by HEX fluorescence observation using a ChemiDoc MP Imaging System (Bio-Rad). Display efficiency was calculated from the band intensity in the lane after the translation reaction as described in Figure 2B.

[0086] (7) Preparation of DNA library with N-terminal random sequences A PCR reaction solution (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM MgSO4, 0.2 mM each dNTP, 0.375 μM MonoS(H)SSS.F26, 0.375 μM HATag-G4S.R48, 0.2 nM MonoS(H)SSS-HA, 2 nM Pfu-S DNA polymerase) was prepared and 10 cycles of PCR reaction were performed. This was designated Template 3. Next, a PCR reaction solution (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM MgSO4, 0.2 mM each dNTP, 0.375 μM MonoS(H)NNW3SSS.F58, 0.375 μM G5S-4Gan21-3.R42, 0.375 nM Template 3, 2 nM Pfu-S DNA polymerase) was prepared and PCR reaction was performed for 9 cycles. This was designated Template 4. Next, a PCR reaction solution (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM MgSO4, 0.2 mM each dNTP, 0.375 μM T7SD8M2.F44, 0.375 μM G5S-4Gan21-3.R42, 0.375 nM Template 4, 2 nM Pfu-S DNA polymerase) was prepared, and 10 cycles of PCR reaction were performed. After PCR, the DNA was treated with phenol-chloroform and precipitated with isopropanol. This was used as MonobodyNNWDNA. AnticalinNNWDNA was also prepared in a similar manner.

[0087] A PCR reaction solution (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM MgSO4, 0.2 mM each dNTP, 0.375 μM catMonoS(H)NNW3SSS.F60, 0.375 μM G5S-4Gan21-3.R42, 0.375 nM Template 3, 2 nM Pfu-S DNA polymerase) was prepared and PCR reaction was performed for 9 cycles. This was designated Template 5. Next, a PCR reaction solution (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM MgSO4, 0.2 mM each dNTP, 0.375 μM T7SDCATM.F46, 0.375 μM G5S-4Gan21-3.R42, 0.375 nM Template 4, 2 nM Pfu-S DNA polymerase) was prepared and 10 cycles of PCR reaction were performed. After PCR, the DNA was treated with phenol-chloroform and precipitated with isopropanol. This was named catMonobodyNNWDNA.

[0088] (8) Screening for N-terminal random sequences MonobodyNNWDNA, catMonobodyNNWDNA, and AnticalinNNWDNA were transcribed into mRNA using T7 RNA polymerase, and then treated with phenol-chloroform and was precipitated with isopropanol twice. These were named MonobodyNNWmRNA, catMonobodyNNWmRNA, and AnticalinNNWDNA, respectively. The annealing solution (6.67 μM MonobodyNNWmRNA or catMonobodyNNWmRNA or AnticalinNNWDNA, 4 μM Hex-PuL-an21-3, 25 mM HEPES-K pH7.8, 200 mM AcOK) was incubated at 95°C for 3 minutes and at 25°C for 5 minutes to prepare mRNA-PuL complexes. This was added to the reconstituted cell-free translation system (excluding RF1) (mRNA-PuL complex: fc 1 μM) and incubated at 37°C for 30 minutes. 0.8 μL of 100 mM EDTA pH 8.0 was added to the reaction solution (4 μL). 2.4 μL of 3xRT mix (150 mM Tris-HCl (pH 8.4), 75 mM MgCl2, 225 mM KCl, 16.5 mM dithiothreitol (DTT), 1.5 mM dNTPs, 0.12% (v / v) in-house moloney murine leukemia virus reverse transcriptase (HMLV), 7.5 μM G4S.R19) was added and reverse transcription reaction was carried out at 42°C for 15 minutes. After that, the reaction solution was incubated at 42°C for 15 minutes in a Zeba buffer equilibrated with HBST (50 mM Hepes-KOH (pH 7.5), 300 mM NaCl, 0.05% (v / v) Tween 20). TM Buffer exchange was performed by filtration using Spin Desalting Columns 7K MWCO (Thermo Scientific).

[0089] The mRNA-protein complex after reverse transcription (3.2 pmol) prepared above was mixed with 1 μL of Anti-HA tag mAb (TANA2) (Medical & Biological Laboratories Co., Ltd.) and incubated at 25°C for 30 minutes. TM Protein G for immunoprecipitation (Thermo Fisher Scientific, 20 μL) was added and mixed by rotation at 25°C for 10 minutes. The mRNA-protein complex was then collected and washed with 10 μL of HBST (50 mM Hepes-KOH (pH 7.5), 300 mM NaCl, 0.05% (v / v) Tween 20) for 10 seconds.

[0090] 1x PCR dNTPs (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2 mM MgSO4, 0.22 mM dNTPs) was added to 0.4 pmol of unrecovered mRNA-protein complex (before recovery) and the total amount of recovered mRNA-protein complex (after recovery) to make a total of 1000 μL. Take 85 μL from there, add equal volume of 2x PCR reaction solution (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton SD8barcode2, 12.5 μM HATagR23+3, 12.5 μM HATagR23+2, 12.5 μM HATagR23+1, 12.5 μM HATagR23) or (50 μM SDcatbarcode9 or SDcatbarcode10, 12.5 μM HATagR23+3, 12.5 μM HATagR23+2, 12.5 μM HATagR23+1, 12.5 μM HATagR23)] was added to the PCR reaction mixture (1.275 μL) and 8 cycles were performed. After PCR, DNA was treated with phenol-chloroform and precipitated with isopropanol. Sequence analysis of the DNA was performed using a next-generation sequencer (Macrogen Japan).

[0091] Test Example 1. Data analysis and evaluation of screening results for C-terminal random sequences 1 As described in Fig. 1A, a mixed DNA library was created by adding random C-terminal sequences of type I (VVNVVNTAG) and type II (VVNVVNTAGN) to the upstream sequence (Monobody or Anticalin). The DNA library was used as a template to transcribe mRNA, and an mRNA-protein complex was created in a TRAP reaction system. The complex can be recovered with streptavidin beads using biotin modified at the N-terminus of the protein. Reverse transcription to cDNA was performed on both the pre- and post-recovery libraries, and sequences were analyzed using a next-generation sequencer. As a result of sequence analysis, more than 20 million reads were obtained for each library. Theoretically, there are 7056 types of sequences in the mixed library, but at least one read was obtained for all sequences. Of these, sequences with more than 100 reads both before and after recovery were considered to be valid for future analysis (more than 99.9% of the total except for four types for Monobody, and more than 99.8% of the total except for nine types for Anticalin).

[0092] First, the enrichment rate was calculated. Figure 4 shows a heat map in which the enrichment rate of each C-terminal sequence in the Monobody library is represented by color. A diagram was created in which the enrichment rate of each C-terminal sequence in Monobody and Anticalin was plotted (Figure 2A). The enrichment rate of each C-terminal sequence was strongly correlated between the library in which the upstream sequence was Monobody and the library in which the upstream sequence was Anticalin (R 2 = 0.83). This result suggested the possibility of obtaining a C-terminal sequence that exhibits versatile high display efficiency.

[0093] To confirm the correlation between enrichment rate and display efficiency, 19 types of C-terminal sequences that showed various enrichment rates were selected as targets for display efficiency measurements (Table 3). DNA was prepared by introducing these sequences into the C-terminus of a monobody, and the sequences were transcribed into mRNA (named McX). The transcribed mRNA was reacted in a TRAP reaction system, and the display efficiency was measured. As a result, it was found that the enrichment rate in the library with a monobody as the upstream sequence was strongly correlated with the display efficiency (R 2= 0.83) (Figure 2B). In other words, the enrichment rate calculated by this method can be used to indirectly evaluate the display efficiency of the C-terminal sequence.

[0094] [Table 3]

[0095] In addition, the codon 1 sequences with an enrichment rate of 3.2 or higher in at least one of the monobody and anticalin were GGC, CGT, and GGT, and the codon 2 sequences with an enrichment rate of 3.2 or higher in at least one of the monobody and anticalin were CGA, AGC, AGA, ACA, CGT, AGT, GCA, AGG, GGT, CGC, GCT, GGC, CGG, ACT, GAT, and GAC. The codon 1-codon 2 sequences with an enrichment ratio of 2.8 or higher in both the monobody and anticalin were GGCCGA, GGCAGA, GGCGCA, GGTAGA, GGCAGT, GGCAGC, GGCACA, GGCCGT, GGCGCT, GGCGAT, GGCACT, GGTCGA, GGCAGG, GGCACG, GGTGAC, GGTAGG, CGTCGA, GGTAGT, GGTCGT, GGCGGT, GGACGA, GGTGGT, GGTACA, GGTCGC, AATCGA, GGTAGC, and GCGCGA. The codon 1-codon 2 sequences with an enrichment ratio of 3.0 or higher in both the monobody and anticalin were GGCCGA, GGCAGA, GGTAGA, GGCACA, GGCCGT, GGCAGT, GGCAGC, GGCAGG, GGTCGA, GGCGCT, GGCACT, GGCGCA, and GGTAGG. The Codon 1-Codon 2 sequences with an enrichment rate of 3.2 or more for both Monobody and Anticalin were GGCCGA, GGCAGA, GGTAGA, GGCCGT, GGCAGT, GGCAGC, GGTCGA, GGCGCT, and GGCACT. A polynucleotide in which a polypeptide coding sequence or a polypeptide coding sequence insertion site, the above Codon 1, the above Codon 2, and a stop codon are arranged in this order, and in which the Codon 1 and the Codon 2 are adjacent to each other, can exhibit high display efficiency in a polynucleotide display method.

[0096] Test Example 2. Evaluation of the versatile high display efficiency of the obtained C-terminal sequence In previous experiments, we used Monobody WT, whose entire sequence is constant. However, when actually obtaining functional molecules using evolutionary molecular engineering techniques, we use a Monobody library with large diversity by randomizing the two loop structures that are the binding sites. Therefore, it cannot be said that an effective sequence has been obtained unless it shows high display efficiency not only for Monobody WT but also for the Monobody library containing random sequences. Therefore, we decided to confirm whether the obtained C-terminal sequence can be applied to the Monobody library. The Monobody library used in this study was designed and prepared with reference to previous studies. The Monobody library has random sequences in the two loops, and the random sequences are designed not to contain a stop codon. We prepared DNA that introduced c1, which has the highest enrichment rate, c16 at position 194, and c13 at position 2448 to the C-terminus of the Monobody library, and transcribed it into mRNA (named MRcX). The transcribed mRNA was reacted in a TRAP reaction system for an optimized time, and the display efficiency was measured. As a result, MRc1 and MRc16, which are in the top 3% of enrichment rate, had significantly higher display efficiency than MRc13 (MRc1: 14.6%, MRc16: 14.1%, MRc13: 6.91%) (Fig. 3A). Therefore, it can be said that if a C-terminal sequence with an enrichment rate within the top 3% is selected, a high display efficiency will be achieved even for libraries with random sequences at the binding site.

[0097] However, the high display efficiency against the monobody library may be due to the 78-84% homology with Monobody WT. Therefore, we prepared DNA with the same three types of C-terminal sequences introduced into the C-terminus of Anticalin, another artificial antibody scaffold with no sequence homology, and transcribed it into mRNA (named LcX). The transcribed mRNA was reacted in a TRAP reaction system and the display efficiency was measured. As a result, Lc1 and Lc16, which are included in the top 3% of enrichment rates, had significantly higher display efficiency than Lc13 (Lc1: 18.0%, Lc16: 17.4%, Lc13: 4.85%) (Figure 3B). Therefore, if a C-terminal sequence with an enrichment rate within the top 3% is selected, it can be said that a high display efficiency will be shown even against other artificial antibody scaffolds with completely different sequences.

[0098] Next, we decided to confirm whether the C-terminal sequences obtained in this screening could be applied to macrocyclic peptide libraries, which are expected to be a new drug discovery modality. The macrocyclic peptide library used in this study has a random sequence of 15 residues, and N-chloroacetyl-L-phenylalanine and cysteine ​​at both ends are designed to cause a cyclization reaction. The random sequence was designed not to contain a stop codon. We created DNA with the same three types of C-terminal sequences introduced into the C-terminus of the macrocyclic peptide library as above, and transcribed it into mRNA (named PRcX). The transcribed mRNA was reacted in a TRAP reaction system, and the display efficiency was measured. As a result, PRc1 and PRc16 had significantly higher display efficiency than PRc13 (PRc1: 16.5%, PRc16: 14.0%, PRc13: 5.79%) (Figure 3C). Therefore, it can be said that if a C-terminal sequence with an enrichment rate within the top 3% is selected, it will also show high display efficiency for the macrocyclic peptide library.

[0099] Test Example 3. Screening for N-terminal random sequence The enrichment rate was also measured for the N-terminal random sequence adjacent to AUG in the same manner as for the C-terminal random sequence (Figures 5 and 6). As a result, as with the screening for the C-terminal random sequence, it was found that the enrichment rate differed depending on the sequence, and that there were sequences with high enrichment rates. In addition, when two types of Monobodies with different upstream sequences containing SD sequences were compared, it was found that the enrichment rate depended on the combination of the SD sequence and the N-terminal random sequence. Furthermore, when a Monobody and Anticain, which have upstream sequences containing the same SD sequence, were compared, it was found that the enrichment rate depended on the combination of the protein and the N-terminal random sequence. The enrichment rate here reflects the efficiency of translation initiation, which is important for protein expression. Therefore, by obtaining the enrichment efficiency for any protein using this method, it is possible to obtain a group of sequences that can control the expression of any protein. This information will enable precise control of protein expression and is thought to be useful for highly efficient protein production.

[0100] Test Example 4. Data analysis and evaluation of screening results for C-terminal random sequences 2 A mixed DNA library was created in which a C-terminal random sequence of type I (NNNNNNTAG) was added to the upstream sequence (Monobody). Using this library, screening was performed in the same manner as in Test Example 1, and the enrichment rate was calculated. Figure 7 shows a heat map in which the enrichment rate of each C-terminal sequence in the Monobody library is expressed by color. The correspondence between color and enrichment rate is the same as in Figure 4.

[0101] Codon 1 sequences with relatively high enrichment rates were AAA, GGC, CGT, and GGT, and codon 2 sequences with relatively high enrichment rates were ACA, AGA, AGC, AGG, AGT, ATG, CCA, CCG, CCT, CGA, CGC, CGT, CTA, and CTG.

Claims

1. (a) binding a polynucleotide-polypeptide complex obtained by translating a polynucleotide comprising a polypeptide coding sequence and a random sequence in a cell-free translation system to a solid phase via a modifying substance on the polypeptide in the polynucleotide-polypeptide complex; (b) measuring and calculating the ratio (enrichment rate) of the frequency of occurrence of each random sequence in the polynucleotide-polypeptide complex bound to the solid phase to the frequency of occurrence of each random sequence in the polynucleotide, and selecting a random sequence using the obtained enrichment rate as an index; A method for screening for a sequence that regulates the display efficiency of a polynucleotide display method, comprising:

2. The screening method according to claim 1 , wherein in the step (b), a random sequence having a high enrichment rate is selected as a sequence that improves display efficiency.

3. The screening method according to claim 1, wherein in step (b), random sequences having the top 10% enrichment rate are selected as sequences that improve the display efficiency.

4. The method of claim 1, wherein the modifying substance is at least one selected from the group consisting of biotin, a peptide tag, and a substance containing a reactive group.

5. The screening method according to claim 1, wherein the random sequence is located downstream of the polypeptide coding sequence.

6. The screening method according to claim 5 , wherein a linker sequence is disposed between the random sequence and the polypeptide coding sequence.

7. The screening method according to claim 5 or 6, wherein a stop codon is located adjacent to or within the random sequence.

8. A reagent for a polynucleotide display method comprising a polynucleotide, the polynucleotide comprises a polypeptide coding sequence or a site for inserting a polypeptide coding sequence, sequence 1 which is 3 bases long, sequence 2 which is 3 bases long, and a stop codon, which are arranged in the following order from upstream: a polypeptide coding sequence or a site for inserting a polypeptide coding sequence, sequence 1, sequence 2, and the stop codon, sequence 1 and sequence 2 are adjacent to each other, sequence 2 and the stop codon are adjacent to each other, and sequence 1 is GGC, CGT, GGT, or AAA, and / or sequence 2 is CGA, AGC, AGA, ACA, CGT, AGT, GCA, AGG, GGT, CGC, GCT, GGC, CGG, ACT, GAT, GAC, ATG, CCA, CCG, CCT, CTA, or CTG, and The polynucleotide display method is a polynucleotide display method using a cell-free translation system that is a prokaryotic cell translation system. reagent.

9. The reagent of claim 8, wherein sequence 1 is GGC, CGT, GGT, or AAA, and sequence 2 is CGA, AGC, AGA, ACA, CGT, AGT, GCA, AGG, GGT, CGC, GCT, GGC, CGG, ACT, GAT, GAC, ATG, CCA, CCG, CCT, CTA, or CTG.

10. The reagent according to claim 8 or 9, wherein the consecutive sequences of sequence 1 and sequence 2 are GGCCGA, GGCAGA, GGCGCA, GGTAGA, GGCAGT, GGCAGC, GGCACA, GGCCGT, GGCGCT, GGCGAT, GGCACT, GGTCGA, GGCAGG, GGCACG, GGTGAC, GGTAGG, CGTCGA, GGTAGT, GGTCGT, GGCGGT, GGACGA, GGTGGT, GGTACA, GGTCGC, AATCGA, GGTAGC, or GCGCGA.

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