Method for producing modified nucleic acid molecule-peptide complex, nucleic acid display method or ribosome display method

Abstract

The method of the present disclosure is a method for producing a modified nucleic acid molecule-peptide complex in which a functional molecule has been introduced into the peptide moiety of a nucleic acid molecule-peptide complex in which a nucleic acid molecule and a peptide encoded by the nucleic acid molecule are linked.

Description

Method for producing modified nucleic acid molecule-peptide complex, nucleic acid display method or ribosome display method

[0001] The present invention relates to a method for producing a modified nucleic acid molecule-peptide complex, and also to a nucleic acid display method or a ribosome display method comprising the method for producing the modified nucleic acid molecule-peptide complex.

[0002] Until now, small molecule drugs have been the main therapeutic agent. However, antibodies have attracted attention as molecularly targeted drugs, and the development of antibody drugs has rapidly increased in recent years. Among these, peptide drugs, which are medium-sized molecule drugs with molecular weights ranging from several thousand to tens of thousands, have attracted attention. Because peptide drugs can be produced as molecularly targeted drugs using directed evolutionary molecular engineering, research and development of peptide drugs using directed evolutionary molecular engineering has been actively conducted in recent years. Meanwhile, new functional peptides can also be created by introducing functional small molecule compounds into peptides. Therefore, by introducing such small molecule compounds into the random sequence library construction at the early stage of the directed evolutionary molecular engineering process and selecting from it, including the peptide sequence, it is possible to confer molecular recognition functions derived from the peptide sequence and functionality derived from the small molecule.

[0003] For example, Non-Patent Documents 1 and 2 disclose the selection of candidate molecules for peptide drugs by synthesizing functional- or functional compound-carrying molecular compound-carrying (misacylated) tRNA using organic synthesis methods and displaying it in a cell-free translation system. Non-Patent Document 3 discloses the selection of candidate molecules for peptides that recognize the target polysaccharide paramylon and emit fluorescence by synthesizing environmentally responsive fluorescent group-carrying (misacylated) tRNA using organic synthesis methods and displaying it in a cell-free translation system.

[0004] Med. Chem. Commun., 5 (9), 1400-1403 (2014)Bull. Chem. Soc. Jpn., 89, 444-446 (2016)Scientific Reports, 8, 8271 (2018)

[0005] In conventional directed evolutionary molecular engineering, new functional molecules, even if they are small compounds, have relatively large molecular weights and bulky three-dimensional structures that prevent them from passing through the ribosomal tunnel. Therefore, when constructing a library using conventional directed evolutionary molecular engineering, they could not be directly introduced into the library using a cell-free translation system.

[0006] One aspect of the present invention aims to realize a method that allows even compounds with relatively large molecular weights or bulky three-dimensional structures to be introduced into libraries used in nucleic acid display or ribosome display.

[0007] As a result of extensive research, the present inventors have discovered a method for introducing a compound with a relatively large molecular weight or a compound with a bulky three-dimensional structure into a specific reactive group of an amino acid residue constituting the peptide portion of a nucleic acid molecule-peptide complex by a chemical synthesis reaction in a water-soluble polar organic solvent. They have also discovered that this method can be used to introduce a compound with a relatively large molecular weight or a compound with a bulky three-dimensional structure into a library used in nucleic acid display or ribosome display, leading to the completion of the present invention.

[0008] In order to solve the above-mentioned problems, one embodiment of the present invention provides a method for producing a modified nucleic acid molecule-peptide complex, in which a functional molecule is introduced into the peptide portion of a nucleic acid molecule-peptide complex in which a nucleic acid molecule and a peptide encoded by the nucleic acid molecule are linked, and the method comprises the step of introducing the functional molecule into a reactive group of an amino acid residue constituting the peptide portion of a nucleic acid molecule-peptide complex synthesized using a cell-free protein synthesis system, by a chemical synthesis reaction in a water-soluble polar organic solvent.

[0009] According to one aspect of the present invention, even compounds with relatively large molecular weights or bulky three-dimensional structures can be introduced into libraries used for nucleic acid display or ribosome display.

[0010] FIG. 1 is a schematic diagram of ribosome display according to one embodiment of the present invention. FIG. 2 is a schematic diagram of ribosome display according to one embodiment of the present invention. FIG. 3 is a schematic diagram showing a click reaction in ribosome display according to one embodiment of the present invention. FIG. 4 is a schematic diagram of cDNA display according to one embodiment of the present invention. FIG. 5 is a diagram showing the fluorescence intensities of peptides 1 to 3. FIG. 6 is a diagram showing the results of SPR analysis of peptide 2. FIG. 7 is a diagram showing the results of observation of HT-1080 cells and MCF-704 cells using a confocal laser microscope. FIG. 8 is a diagram showing the intracellular fluorescence pattern of peptide 2 in T98G cells. FIG. 9 is a diagram showing the fluorescence profiles of heat-shocked T98G cells and untreated T98G cells stained with peptide 2.

[0011] One aspect of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the respective configurations described below, and various modifications are possible within the scope of the claims. Furthermore, embodiments and examples obtained by appropriately combining the technical means disclosed in the respective embodiments and examples are also included in the technical scope of the present invention. Furthermore, in this specification, "A to B" means A or more and B or less, unless otherwise specified.

[0012] [Method for Producing a Modified Nucleic Acid Molecule-Peptide Conjugate] A method for producing a modified nucleic acid molecule-peptide conjugate according to one aspect of the present invention is a method for producing a modified nucleic acid molecule-peptide conjugate by introducing a functional molecule into the peptide portion of the nucleic acid molecule-peptide conjugate. Hereinafter, this method may be referred to as the "method for producing the modified conjugate of this embodiment."

[0013] The method for producing a modified compound of this embodiment performs a chemical synthesis reaction in a water-soluble polar organic solvent, thereby enabling efficient introduction of functional molecules with relatively large molecular weights or bulky three-dimensional structures into the peptide portion of a nucleic acid molecule-peptide complex. A library containing the modified compound produced by the method for producing a modified compound of this embodiment can be used in the nucleic acid display method or ribosome display method described below.

[0014] (Nucleic Acid Molecule-Peptide Complex) Examples of nucleic acid molecules in nucleic acid molecule-peptide complexes include RNA molecules such as mRNA, DNA molecules such as cDNA, and RNA / DNA hybrid molecules (complementary double strands).

[0015] As used herein, the term "peptide" refers to a compound in which two or more amino acids are bound by peptide bonds. The number of amino acids is not limited and may be, for example, 2 to 1,000, preferably 3 to 200, more preferably 4 to 100, and even more preferably 5 to 50. Examples of the number of amino acids include 10 or more, 20 or more, and 30 or less, 40 or less, and the like. A peptide may be, for example, a fragment or full-length protein. In one example, the peptide may be an antibody (such as an scFv) or a fragment thereof.

[0016] The peptide of the nucleic acid molecule-peptide complex is a peptide encoded by the nucleic acid molecule and synthesized using a cell-free protein synthesis system, such as a cell-free protein synthesis system using an extract of Escherichia coli, wheat germ, rabbit reticulocytes, or the like, and a reconstituted cell-free protein synthesis system using Escherichia coli ribosomes in which factors required for translation are purified and mixed together.

[0017] A nucleic acid molecule-peptide complex can be obtained by linking a nucleic acid molecule to a peptide via a peptide acceptor molecule such as puromycin. Alternatively, a nucleic acid molecule-peptide complex can be obtained by linking a nucleic acid molecule to a peptide via a ribosome. Whether or not a peptide acceptor molecule is mediated, the nucleic acid molecule-peptide complex can be a nucleic acid molecule-peptide-ribosome complex.

[0018] (Functional Molecule) Examples of the functional molecule include fluorescent molecules such as carboxyfluorescein (FAM), fluorescein isothiocyanate (FITC), tetraphenylethylene (TPE), and N,N-dimethylamino-1,8-naphthalimide (DMN); inhibitory molecules such as immune checkpoint inhibitors; and ligand molecules such as folic acid, methotrexate (MTX), and folate receptor-binding ligands that bind to folate receptors. In a preferred example, the functional molecule is a bulky molecule that is difficult to directly incorporate into a peptide using a cell-free protein synthesis system (does not pass through the ribosomal tunnel).

[0019] The method for producing the modified product of this embodiment includes a step of introducing a functional molecule into a reactive group of an amino acid residue constituting the peptide portion of a nucleic acid molecule-peptide complex synthesized using a cell-free protein synthesis system, by a chemical synthesis reaction in a water-soluble polar organic solvent.

[0020] Examples of reactive groups include thiol groups, amino groups, and carboxyl groups. The reactive groups are bonded to functional molecules through a chemical synthesis reaction in a water-soluble polar organic solvent. An example of a thiol group is the thiol group of cysteine. An example of an amino group is the amino group of lysine or arginine. An example of a carboxyl group is the carboxyl group of glutamic acid or aspartic acid. An example of a functional group that reacts with a thiol group is a maleimide group. An example of a functional group that reacts with an amino group is a carboxyl group, N-hydroxylsuccinimide ester group, isothiocyanate group, and the like. An example of a functional group that reacts with a carboxyl group is an amino group.

[0021] Examples of reactive groups include bioorthogonal reactive groups. For example, one bioorthogonal reactive group linked to a functional molecule can be reacted with the other bioorthogonal reactive group of an amino acid residue constituting the peptide portion of a nucleic acid molecule-peptide complex by a bioorthogonal chemical click reaction or a strain-promoted inverse electron demand Diels-Alder reaction. This reaction allows the functional molecule to be introduced into the peptide portion of the nucleic acid molecule-peptide complex.

[0022] Examples of bioorthogonal reactive groups include azido groups such as an azidophenyl group, an N6-((prop-2-yn-1-yloxy)carbonyl) group, and a tetrazine group; alkyne groups such as an o-propargyloxy group, an N6-((prop-2-yn-1-yloxy)carbonyl) group, a bicyclo[6,1,0]nonyne group, and a dibenzocyclooctyne group; cyclooctene groups such as a transcyclooctene group and a norbornene group; and the like. Note that non-natural amino acids having these bioorthogonal reactive groups that can be incorporated into peptides using a cell-free protein synthesis system are known, so by using these non-natural amino acids, nucleic acid molecule-peptide complexes incorporating amino acids having bioorthogonal reactive groups can be prepared.

[0023] The water-soluble polar organic solvent used in the step of introducing the functional molecule is preferably an aprotic polar organic solvent, since it can suppress precipitation of the nucleic acid molecule. Examples of the aprotic polar organic solvent include dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetone, and acetonitrile.

[0024] The solvent used in the step of introducing the functional molecule may be a mixed solvent of an aqueous solvent and a water-soluble polar organic solvent. Examples of the aqueous solvent include water or a buffer solution. The concentration of the water-soluble polar organic solvent contained in the mixed solvent of the aqueous solvent and the water-soluble polar organic solvent is preferably 2% by mass or more, more preferably 5% by mass or more, and even more preferably 10% by mass or less, from the viewpoint of promoting the chemical synthesis reaction. Furthermore, from the viewpoint of suppressing precipitation of the nucleic acid molecule, it is preferably 50% by mass or less, more preferably 45% by mass or less, and even more preferably 40% by mass or less.

[0025] [Nucleic Acid Display Method] A nucleic acid display method according to one aspect of the present invention includes the method for producing a modified form of the present embodiment, and includes a step of selecting a desired modified form from the modified forms of the nucleic acid molecule-peptide complex. Examples of nucleic acid display methods include mRNA display and cDNA display.

[0026] An example of a nucleic acid display method according to one embodiment of the present invention will now be described with reference to Fig. 4c, which is a schematic diagram of a cDNA display method according to one embodiment of the present invention.

[0027] (Step 1: Preparation of a library of candidate DNA molecules) In step 1, a library of candidate DNA molecules is prepared, containing a plurality of different candidate DNA molecules, each containing a promoter region and a region downstream thereof that encodes a candidate peptide. Here, candidate DNA refers to a DNA sequence that may ultimately be selected as a peptide aptamer. Similarly, when the target is RNA or peptide, it is referred to as candidate RNA or candidate peptide.

[0028] As used herein, the term "library" refers to a collection of multiple (two or more) different molecules (e.g., multiple different DNA molecules, multiple different RNA molecules, multiple different DNA-peptide complexes, or multiple different RNA-peptide complexes). For example, a "library" refers to a collection of multiple different molecules that are classified into the same category (e.g., the category of DNA molecules, the category of RNA molecules, or the category of RNA-peptide complexes). It may also be referred to as a DNA library (a library of DNA molecules), an RNA library (a library of RNA molecules), a DNA-peptide complex library (a library of DNA-peptide complexes), an RNA-peptide complex library (a library of RNA-peptide complex molecules), etc., after the name of the molecular category. In the nucleic acid display method according to one aspect of the present invention, since selection starting from a large number of candidate molecules is facilitated as needed, the "library" in this embodiment is preferably a library of 10 ９ More than 10, more preferably 10 １０ 10 or more １１ More than 10 １２ more preferably 10 or more １３ It may contain two or more different molecules.

[0029] As used herein, "selection" refers to substantially separating a molecule from other molecules in a population. For example, "selection" refers to substantially separating a molecule from a collection of multiple different molecules that fall into the same category. As used herein, "selection" can enrich desired molecules by at least 2-fold, preferably 30-fold or more, more preferably 100-fold or more, and even more preferably 1000-fold or more, compared to non-desired molecules in the library after selection. As provided herein, the selection step can be repeated any number of times in a given manner, and different types of selection steps can be combined.

[0030] The candidate DNA molecule includes a region encoding the candidate peptide. The candidate DNA molecule also includes, as needed, a region for transcription using the antisense strand as a template (transcription control region). Examples of transcription control regions include promoter regions. These transcription control regions may be appropriately selected depending on the type of RNA polymerase used in the transcription reaction of step 2. In one example, the promoter region may be one recognized by T7 RNA polymerase, SP6 RNA polymerase, or T3 RNA polymerase (T7 promoter, SP6 promoter, or T3 promoter).

[0031] A candidate DNA molecule containing a promoter region and a region downstream thereof encoding a candidate peptide may be single-stranded, double-stranded, or a mixture of the two (partially double-stranded and the rest single-stranded), as long as a candidate RNA molecule encoding the peptide is produced by a transcription reaction.

[0032] Specifically, the promoter region in a candidate DNA molecule may be single-stranded, double-stranded, or a mixture of both, as long as a candidate RNA molecule encoding the candidate peptide is transcribed from a downstream region encoding the candidate peptide. For example, a portion of the promoter region may be single-stranded (sense or antisense strand), and the remaining portion may be double-stranded. Such a portion composed of a single strand (sense or antisense strand) is also included in the "promoter region" herein. If the promoter region exhibits promoter activity, the candidate RNA molecule encoding the candidate peptide will be transcribed. Promoter activity can be measured by methods known in the art. Furthermore, transcription of the candidate RNA molecule encoding the candidate peptide can be confirmed by detecting the RNA molecule or detecting the peptide produced by translation using methods known in the art.

[0033] Furthermore, the region encoding the candidate peptide in the candidate DNA molecule may be single-stranded, double-stranded, or a mixture of both, as long as a candidate RNA molecule encoding the candidate peptide is transcribed by the action of a promoter region located upstream. For example, the entire coding region may be single-stranded (sense strand or antisense strand). Since the candidate RNA molecule is transcribed using the sequence of the antisense strand as a template, the region encoding the candidate peptide preferably contains at least the antisense strand. Such regions that are partially or entirely single-stranded (sense strand or antisense strand) are also included in the "region encoding a peptide" herein.

[0034] If the reactive group is a thiol group, an amino group, or a carboxyl group, a library of DNA molecules is prepared so that the candidate peptides contain amino acids containing the reactive group.

[0035] (Step 2: Preparation of a library of candidate RNA molecules) In step 2, the candidate DNA molecules are transcribed as templates to prepare a library of corresponding candidate RNA molecules.

[0036] Transcription may be carried out by a known method. Typically, transcription is carried out in situ or in vitro, preferably in vitro. When transcription is carried out in vitro, the type of RNA polymerase is not particularly limited, and preferred examples include bacteriophage-derived RNA polymerases such as T7 RNA polymerase, SP6 RNA polymerase, and T3 RNA polymerase, with T7 RNA polymerase being more preferred.

[0037] (Step 3: Ligation of Peptide Acceptor Molecule to Candidate RNA Molecule) In step 3, a peptide acceptor molecule is ligated to the 3' end of the candidate RNA molecule. The peptide acceptor molecule is not particularly limited as long as it can be ligated to the translated peptide, and examples thereof include known molecules such as puromycin, puromycin derivatives, and oligo-RNA / amino acid complexes.

[0038] The 3' end of the candidate RNA molecule and puromycin are typically linked via a linker containing an oligonucleotide. The length of the oligonucleotide is not particularly limited and may be, for example, approximately 10 to 30 nucleotides, preferably 15 to 20 nucleotides. Examples of nucleotides in the oligonucleotide include DNA, RNA, PNA, and LNA. The linker may further contain other substances (e.g., polyethylene glycol (PEG)) in addition to the oligonucleotide. The length of the PEG is not particularly limited, but, for example, it is preferably formed by linking 3 to 10 PEGs each having a main chain of 6 to 18 atoms. For example, the linker may have a structure of 5'-(oligonucleotide)-(PEG)-(peptide acceptor molecule)-3'. Such linkers may be prepared by known methods.

[0039] The linker may contain biotin or the like as a molecule that forms a bond with the solid phase, which will be described later. Furthermore, the linker may contain a fluorescent group such as fluorescein isothiocyanate (FITC), which allows for easy detection of the presence or absence of binding to the linker.

[0040] (Step 4: Translation) In step 4, the candidate RNA molecules to which the peptide acceptor molecules obtained in step 3 are bound are translated in a cell-free protein synthesis system to prepare a library of first complexes of candidate RNA molecules and peptides, in which the RNA molecules and the peptides encoded by the RNA molecules are linked via the peptide acceptor molecules.

[0041] (Step 5: Reverse Transcription) In step 5, the candidate RNA molecules of the first complex obtained in step 4 are reverse transcribed using the other end of the linker as the reverse transcription initiation site to prepare a library of second complexes each consisting of the candidate RNA molecules, cDNA molecules corresponding to the candidate RNA molecules, and peptides. The candidate RNA molecules and the cDNA molecules corresponding to the candidate RNA molecules are complementary to each other, forming an RNA / DNA hybrid (double-stranded structure).

[0042] In the case of mRNA display, the reverse transcription step (preparation of the library of the second complex) is not essential, but it is preferable to perform the reverse transcription step to stabilize the RNA molecule linked to the peptide acceptor molecule. In the case of mRNA display, the cDNA generated by reverse transcription in the second complex is not linked to the peptide acceptor molecule.

[0043] In cDNA display, the cDNA molecules generated by reverse transcription are linked to a peptide acceptor molecule in a second complex.

[0044] (Step 6: Preparation of modified nucleic acid molecule-peptide complex) In step 6, a functional molecule (DMN in FIG. 4c) is introduced into a reactive group (thiol group of cysteine ​​in FIG. 4c) of an amino acid residue constituting the peptide portion of the second complex obtained in step 5 by a chemical synthesis reaction in a water-soluble polar organic solvent. Step 6 is carried out by the method for producing the modified compound of this embodiment described above.

[0045] (Step 7: Binding of modified nucleic acid molecule-peptide complexes to binding partners) In step 7, the library of nucleic acid molecule-peptide complexes is contacted with a binding partner (Hsp90α in the case of FIG. 4c) to carry out a binding reaction.

[0046] Binding reactions can be based on, for example, the binding between an antigen and an antibody, the binding between a protein receptor and a ligand, the binding between an adhesion molecule and a target molecule, the binding between an enzyme and a substrate, the binding between a nucleic acid and a protein that binds to it, the binding between proteins in a signal transduction system, the binding between a glycoprotein and a protein, or the binding between a sugar chain and a protein. The binding partner may be selected appropriately depending on the purpose of the selection. For example, the binding partner may be immobilized on a solid phase or may be labeled with a substance that can be captured by the solid phase. The solid phase may be any solid phase capable of binding to the binding partner, and may be shaped like a plate, rod, particle, or bead. The solid phase may be made of a material that is insoluble in water or an organic solvent, which is the medium used for screening. Examples of materials that can be used as the solid phase include plastic, glass, resins such as polystyrene, and metals such as gold thin film. Magnetic beads, etc. can also be used. One or more types of binding partners may be contained in a single reaction system.

[0047] The modified nucleic acid molecule-peptide complex that has not bound to the binding partner can be removed, for example, by washing with a buffer solution.

[0048] (Step 8: Recovery) In step 8, the modified nucleic acid molecule-peptide complex is dissociated from the binding partner, and the modified nucleic acid molecule-peptide complex is recovered. The dissociation method may be selected appropriately depending on the type of bond with the binding partner. For example, proteases such as ficin, papain, and trypsin, or antibody sequence-specific proteases such as IdeS and IdeZ can be used. Alternatively, denaturation can be performed using heat or a denaturing agent (urea or guanidine).

[0049] The modified nucleic acid molecule-peptide complex can be recovered, for example, by eluting (separating from) the substance bound to the binding partner.

[0050] By carrying out the above steps, a desired nucleic acid molecule-peptide complex is selected from a library of nucleic acid molecule-peptide complexes.

[0051] The selected nucleic acid molecule-peptide complex may then be analyzed for nucleic acid molecule and / or peptide sequences. Analysis can be performed using a conventional amino acid sequence sequencer, or by reverse transcribing RNA bound to such peptides to DNA and analyzing the nucleotide sequence of the resulting cDNA. Purification or quantification may also be performed as appropriate.

[0052] Alternatively, the cDNA obtained here can be used to carry out step 1 above. For example, a PCR reaction is carried out on the cDNA obtained here or its amplification product. Steps 2 to 8 are then carried out again using the DNA molecules obtained from this. By repeating the cycle of steps 1 to 8 multiple times in this manner, nucleic acid molecule-peptide complexes having the desired properties are enriched. These are then used for analysis.

[0053] [Ribosome display method] A ribosome display method according to one aspect of the present invention includes the method for producing a modified form of the present embodiment, and includes a step of selecting a desired modified form from the modified forms of the nucleic acid molecule-peptide complex described above. Hereinafter, an example of the ribosome display method according to one aspect of the present invention will be described with reference to Figure 1.

[0054] (Step A: Preparation of a library of candidate DNA molecules) A ​​library of candidate DNA molecules can be prepared in the same manner as in "Step 1: Preparation of a library of candidate DNA molecules" of the nucleic acid display method. In the ribosome display method, candidate DNA molecules lacking a codon that indicates translation termination are used.

[0055] (Step B: Preparation of a library of candidate RNA molecules) A ​​library of candidate RNA molecules can be prepared in the same manner as in "Step 2: Preparation of a library of candidate RNA molecules" of the nucleic acid display method.

[0056] (Step C: Translation) In step C, candidate RNA molecules are translated in a cell-free protein synthesis system in the presence of amino acids bearing reactive groups (bicyclo[6,1,0]nonyne (BCN) groups in Figure 1) for introducing functional molecules into the library. Because the candidate RNA molecules do not contain codons that terminate translation and contain ribosome arrest sequences (SecM), a library of complexes of candidate RNA molecules, peptides, and ribosomes (PRM complexes; peptide-ribosome-mRNA complexes) is produced by this translation.

[0057] (Step D: Linking of Reactive Group to Functional Molecule) In step D, a reactive group (tetrazine in FIG. 1) that reacts with the reactive group bound to the amino acid (BCN group in FIG. 1) is linked to the functional molecule. The linking may be performed via a linker by a known method.

[0058] (Step E: Preparation of modified nucleic acid molecule-peptide complex) In step E, a reactive group bound to an amino acid (BCN group in Figure 1) is reacted with a reactive group linked to a functional molecule (tetrazine in Figure 1) by a chemical synthesis reaction in a water-soluble polar organic solvent. This reaction introduces the functional molecule into the peptide portion of the PRM complex, producing a modified nucleic acid molecule-peptide complex. Step E is carried out by the method for producing the modified compound of this embodiment described above. In Figure 1, the functional molecule is introduced into the peptide portion of the PRM complex by a click reaction between the BCN group and tetrazine.

[0059] (Step F: Selection) In step F, the modified nucleic acid molecule-peptide complex obtained in step E is subjected to selection (e.g., affinity selection). From the selected modified complex, mRNA encoding the bound protein is recovered, and the target DNA is obtained by RT-PCR. Step F may be performed in the same manner as "Step 7: Binding of the modified nucleic acid molecule-peptide complex to a binding partner" and "Step 8: Recovery" in the nucleic acid display method.

[0060] By repeating steps A to F above, nucleic acid molecule-peptide complexes having desired properties are enriched.

[0061] The nucleic acid display method and ribosome display method according to one embodiment of the present invention can be used for identifying proteins that interact with target molecules in drug discovery, analyzing drug target proteins, obtaining or modifying antibody molecules, specifying antigen proteins recognized by antibodies and their antigenic sites, modifying various functional proteins and peptides, confirming the activity of vaccines, etc.

[0062] [Summary] The method according to aspect 1 of the present invention is a method for producing a modified nucleic acid molecule-peptide complex in which a functional molecule is introduced into the peptide portion of the nucleic acid molecule-peptide complex in which a nucleic acid molecule is linked to a peptide encoded by the nucleic acid molecule, and the method comprises the step of introducing the functional molecule into a reactive group of an amino acid residue constituting the peptide portion of a nucleic acid molecule-peptide complex synthesized using a cell-free protein synthesis system, by a chemical synthesis reaction in a water-soluble polar organic solvent.

[0063] In the method according to Aspect 2 of the present invention, in Aspect 1 of the present invention, the water-soluble polar organic solvent may be an aprotic polar organic solvent.

[0064] In the method according to aspect 3 of the present invention, in the method according to aspect 1 or 2 of the present invention, the functional molecule may be a fluorescent molecule, an inhibitor molecule, or a ligand molecule.

[0065] In the method according to Aspect 4 of the present invention, in any one of Aspects 1 to 3 of the present invention, the reactive group may be a thiol group, an amino group, or a carboxyl group.

[0066] A method according to aspect 5 of the present invention may be any of aspects 1 to 3 of the present invention, wherein the reactive group is a bioorthogonal reactive group.

[0067] In the method according to Aspect 6 of the present invention, in any one of Aspects 1 to 3 and 5 of the present invention, the nucleic acid molecule-peptide complex may be a nucleic acid molecule-peptide-ribosome complex to which a ribosome is further linked.

[0068] A method according to Aspect 7 of the present invention is a nucleic acid display method or a ribosome display method, which includes the method according to any one of Aspects 1 to 6 of the present invention and comprises a step of selecting a desired modified product from the modified products of the nucleic acid molecule-peptide complex.

[0069] A library containing a plurality of types of modified nucleic acid molecule-peptide complexes, produced by the method of any one of aspects 1 to 6 of the present invention, wherein the modified nucleic acid molecule-peptide complexes have the same introduced functional molecule but different amino acid sequences in the peptide moiety. 2 10 or more 3 10 or more 4 10 or more ９ More than 10, more preferably 10 １０ 10 or more １１ More than 10 １２ more preferably 10 or more １３ The library of modifications may contain one or more different modifications. The library of modifications is subjected to, for example, the method of Aspect 7 or 8 of the present invention, and a step of selecting desired modifications is carried out. In one example, the modifications of the nucleic acid molecule-peptide complexes constituting the library of modifications have substantially the same length of amino acid sequence of the peptide portion. "Substantially the same amino acid sequence length" means that the difference in length is 20% or less, 10% or less, 5% or less, or 0%. In one example, the library of modifications contains a water-soluble polar organic solvent as described in Aspect 1, etc., and in another example, does not contain a water-soluble polar organic solvent as described in Aspect 1, etc. In other words, the water-soluble polar organic solvent may be removed after the library of modifications is produced.

[0070] The following examples are provided to further explain the embodiments of the present invention. It goes without saying that the present invention is not limited to the following examples, and various modifications are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed herein are also included in the technical scope of the present invention. Furthermore, all of the documents described in this specification are incorporated by reference.

[0071] In the examples, % means % by mass unless otherwise specified.

[0072] [Evaluation Example 1] Stability of PRM Complexes First, we investigated whether peptide-ribosome-mRNA (PRM) complexes formed by ribosome display could remain stable in the presence of DMSO. First, we created a cell-free protein synthesis system (CellFree) from an extract of specialized E. coli (an E. coli strain lacking Amber (Amb, UAG codon) in the ΔRF1 and 94 genes). Using this CellFree, we translated RNA containing a FLAG gene lacking Amb downstream (FLAG mRNA, Figure 2, top panel) and, as a control, a portion of the GFP gene lacking Amb (GFP mRNA, Figure 2, bottom panel) in a 1:9 ratio.

[0073] The stability of the translated sample was assessed by passing it through a gel filtration column equilibrated with DMSO concentrations ranging from 0 to 100%. Specifically, the translated sample solution was converted to DMSO through the gel filtration column, and the PRM complex bound to anti-FLAG antibody-immobilized beads was recovered and mRNA was purified. The purified mRNA was reverse transcribed into cDNA and amplified by PCR to determine the intensity of the FLAG and GFP bands. After gel filtration, only the FLAG-derived band (650 bp) was detected in the sample selected with anti-FLAG antibody-immobilized beads, even when the DMSO concentration was varied from 0 to 100% (Table 1). On the other hand, RT-PCR of the untranslated sample (mRNA) detected GFP (850 bp) at approximately 10-fold higher intensity than FLAG, indicating no bias in the enrichment of the FLAG sequence by RT-PCR. These results demonstrate that the stability of the PRM complex formed during ribosome display is not affected by DMSO concentration.

[0074]

[0075] [Evaluation Example 2] Click Reaction Study To confirm whether a click reaction occurs in a cell-free translation solution, exoBCN-containing thioredoxin was expressed in a cell-free translation system, reacted with a tetrazine compound, and the resulting protein was purified using a FLAG tag that had been added separately to thioredoxin and subjected to MS analysis. Specifically, exoBCN-containing thioredoxin was translated for 2 hours in 100 μL of a cell-free translation system based on Escherichia coli lysate (0.4 mg / mL aminoacyl-tRNA synthetase, 4 μg / mL template, 5 mM exoBCN, 0.2 mg / mL tRNA). To 25 μL of the resulting translation solution, 25 μL of a solution of a tetrazine compound modified with either BMS1166 (a checkpoint inhibitor), methotrexate (MTX), or tetraphenylethylene (TPE) dissolved in DMSO to a final concentration of 5 mM was added. After 5 minutes of incubation at 37°C, thioredoxin was purified using the FLAG tag, eluted with TFA, and analyzed by MS. The resulting MS peaks showed the expected increase in mass upon modification with each small molecule, confirming that small molecules can be modified in a short time by the click reaction, even in the presence of contaminants associated with cell-free translation systems.

[0076]

[0077] [Evaluation Example 3] Study of target substance introduction in ribosome display The introduction of target substances was studied using the method shown in Figure 1. It has been previously shown that bicyclo[6.1.0]non-4-yne (BCN)-modified amino acids shown in Figure 1 can be introduced into a cell-free translation system. In this evaluation example, BCN-linked amino acids were used, and after preparing a peptide library, it was demonstrated that target substances could be introduced into the library by click reaction using a tetrazine compound in a water-soluble polar organic solvent.

[0078] Specifically, the FLAG gene without Amb downstream in Evaluation Example 1 was replaced with a FLAG gene with Amb downstream, and exoBCN and BCNRS / tRNA(CUA) were added as unnatural amino acids during translation, allowing the Amb codon to be translated as exoBCN. ExoBCN was incorporated into a peptide library as an unnatural amino acid, and a click reaction was carried out with a target inhibitor-modified tetrazine dissolved in DMSO to investigate whether the target substance could be incorporated into the ribosome display method (Figure 2). In this evaluation example, the fluorescent substance FAM was used as a model compound instead of the target substance.

[0079] Next, a peptide containing exoBCN (PRM complex) was mixed with FAM-tetrazine at various DMSO concentrations to induce a click reaction as shown in Figure 3. The resulting product was then recovered using anti-FAM antibody-immobilized beads to examine whether the desired FLAG sequence could be enriched.

[0080] The translated sample was passed through a gel filtration column equilibrated with a solution containing DMSO at concentrations ranging from 0 to 100%, and then reacted with a peptide containing FAM-tetrazine and exoBCN at various DMSO concentrations. To recover only the peptides that underwent the click reaction, the peptides were recovered using beads immobilized with anti-FAM antibodies and subjected to RT-PCR. The results are shown in Table 3. In Table 3, a "+" indicates that the FLAG band was enriched, and a "-" indicates that the FLAG band was not enriched.

[0081]

[0082] As shown in Table 3, only the target FLAG band was detected at DMSO concentrations between 2% and 50%. On the other hand, in the system using FAM-modified tetrazine, at DMSO concentrations above 50%, the target FLAG band was not enriched, as was the case with the amber-free FLAG gene RNA used as a negative control.

[0083] Evaluation Example 1 showed that even a 100% DMSO concentration did not adversely affect the stability of the PRM complex. Furthermore, Evaluation Example 3 demonstrated that selection using beads modified with FAM antibodies can be performed at DMSO concentrations of 2-50%, further expanding the range of options available.

[0084] [Evaluation Example 4] Preparation of a Target Library Using cDNA Display In this evaluation example, peptide aptamers were selected that specifically bind to the cancer marker Hsp90α and exhibit changes in fluorescence intensity upon binding. The selection was performed using cDNA display technology. cDNA display technology allows for modification of the peptide backbone with brominated 4-DMN (4-N,N-dimethylamino-1,8-naphthalimide) (4-DMN-Br), an environmentally responsive fluorescent substance, in organic solvent before the selection round. Due to its sequence homology with Hsp90α, Hsp70 was used for negative selection. Furthermore, human IgG and human albumin were also used for negative selection.

[0085] Hsp90α is a marker of interest to oncologists for early diagnosis. Several cancer markers are known to be specific to tissue types, including calcitonin-thyroid cancer and human chorionic gonadotropin-germ cell tumors. Carcinoembryonic antigen (CEA) is expressed in various malignant tissues, including breast, gastric, and lung cancers. Molecular chaperones have been reported to be involved in tumor growth and metastasis, and Hsp90 is being investigated for its potential as a nonspecific tumor marker. Hsp90 is known to play important roles in various cellular functions, including cell proliferation and differentiation, stress response, and protection of cellular proteins from stresses such as hyperthermia.

[0086] To employ cDNA display technology, we first designed a DNA library containing 13 random amino acids with a cysteine ​​at the eighth position. The cysteine ​​thiol group reacted with 4-DMN-Br (Figure 4a) to obtain fluorogenic peptide aptamer candidates. Previous studies have reported the selection of fluorogenic peptide aptamers for various target molecules. Many previous studies have employed tRNA modified with the relatively small fluorescent dye 7-nitrobenzofurazan (NBD) and ribosome display to prepare and select fluorogenic peptide aptamers. In this evaluation, we used DMN, which is bulkier than NBD, instead of NBD because its lower fluorescence intensity in hydrophilic environments is expected to provide a higher signal-to-noise ratio than NBD. cDNA display technology was used to incorporate DMN into peptide aptamer candidates. The cDNA display used in this evaluation also allows for the preparation of stable cDNA-peptide ternary complexes, allowing for peptide modification prior to selection.

[0087] Previously, we used ribosome display technology with tRNA-carrying environmentally sensitive fluorescent probes to select peptide aptamers whose fluorescence changes in response to various targets. The peptide aptamers were directly selected based on their binding ability and showed fluorescence changes in the presence of verotoxin.

[0088] After in vitro transcription, mRNA was ligated to a short biotin segment puromycin linker (SBS-Pu-linker). After in vitro translation, the peptide was transferred to the puromycin site of the SBS-Pu-linker. Thus, the C-terminus of the peptide was covalently fused to the cDNA encoding the peptide, forming a cDNA display complex (Fig. 4b).

[0089] The scheme for peptide selection using cDNA display used in this evaluation example is shown in FIG. 4c.

[0090] The cDNA display protocol used in this evaluation example will be described in detail below.

[0091] Protocol 1: Ligation of mRNA and SBS linker Ligation of mRNA and SBS linker (SBS-linker) was carried out using the reaction system shown in Table 4 below. The reaction times for the reaction system shown in Table 4 were (1) 2 minutes at 90°C, (2) 1 minute at 70°C, (3) 1 minute at 50°C, and (4) stop. After completion of the reaction, the mixture was maintained at 25°C for 30 minutes and then on ice to obtain the ligation product of mRNA and SBS linker (SBS-linker-mRNA).

[0092]

[0093] Protocol 2: Translation: 15.5 μL of reconstituted cell-free translation solution (containing ΔRF1, ribosomes, and RNase) was mixed with 10 μL of SBS-linker-mRNA and incubated at 37°C for 30 minutes. The reaction mixture was then transferred to room temperature (20-26°C) for 12 minutes to enhance binding. This translation was performed using a homemade pure system that did not contain RF1.

[0094] Next, 0.6 μL of 0.5 M EDTA (pH 8.0, RNase-free) was added to the reaction solution, and the mixture was incubated for 5 minutes.

[0095] Protocol 3: Preparation of DMN-modified peptide-cDNA. Concurrently with the translation in Protocol 2, 20 μL of Dynabeads Myone C1 (streptavidin, hereafter referred to as "beads C1") was washed twice with 200 μL of Solution A (100 mM NaOH, 50 mM NaCl) and then twice with 200 μL of Solution B (100 mM NaCl). After washing, 25 μL of the translation system prepared in Protocol 2 and 25 μL of 2× binding buffer (20 mM Tris-HCl (pH 8.0), 0.2 mM EDTA, 2 M NaCl, 0.2% Tween 20) were added to the washed beads C1, and the mixture was rotated at 65 rpm for 30 minutes at room temperature. The beads were then washed three times with 200 μL of 1× binding buffer and once with 100 μL of 1× ReverTraAce buffer (Toyobo).

[0096] Protocol 4: Reverse transcription and binding to DMN. To the prepared beads, 10 μL of 5x ReverTraAce buffer, 8 μL of 10 mM dnNTPs, 1 μL of ReverTraAce (100 U / μL), and 31 μL of RNase-free water were added and mixed. The 25 μL mixture was incubated at 42°C for 30 minutes to perform the reverse transcription reaction. The beads were then washed twice with 200 μL of 1x SBT (50 mM Tris-HCl (pH 7.6), 1 mM EDTA, 0.5 M NaCl, 0.05% Tween 20) and then twice with 200 μL of wash buffer (50 mM Tris, 150 mM NaCl).

[0097] Next, 0.2 μL of Tween 20 (0.1%), 40 μL of 5×DMN (1.5 mM in DMF), 20 μL of 10× reaction buffer (100 mM Tris pH 8, 1M NaCl, 2 mM TCEP), and 140 μL of RNase- and DNase-free water were added to the beads and mixed. The mixture was divided into two tubes, and the tubes were rotated at 65 rpm for 1 hour at 25°C to allow the DMN binding reaction. The beads were then washed twice with 200 μL of wash buffer and then twice with 200 μL of 1×SBT.

[0098] Protocol 5: Affinity Selection. After DMN binding, 31.5 μL of water (RNase-free), 10 μL of 5xSB (250 mM Tris-HCl (pH 7.6), 5 mM EDTA, 2.5 M NaCl), 5 μL of 0.1% BSA, 2.5 μL of Tween 20 (1%), and 1 μL of RNase T1 (1000 U / μL) were added to the beads and mixed. The 50 μL mixture was incubated at 37°C for 10 minutes. The supernatant was then collected.

[0099] (5.1 Preparation of Human Hsp70 and Hsp90 Beads) Human Hsp70 and Hsp90 beads were washed three times with 200 μL of PBS buffer (pH 7.4, 0.1% Tween 20) and then divided into four tubes.

[0100] (5.2 Preparation of Human IgG Beads) Human IgG beads were washed three times with 200 μL of PBS buffer (pH 7.4, 0.1% Tween 20) and then divided into four tubes.

[0101] (5.3 Preparation of Human Albumin Beads) Human albumin beads were washed three times with 200 μL of PBS buffer (pH 7.4, 0.1% Tween 20) and then divided into four tubes.

[0102] (5.4 Addition of Hsp70 beads) Five microliters of MyOne Carboxylic Acid beads (pre-immobilized Hsp70 beads) was added to the tube, washed three times with 200 μL of PBS buffer (pH 7.4, 0.1% Tween 20), and then divided into five tubes. The supernatant of 0096 (mRNA-peptide solution excised using RNAse T1) was added to one of the tubes, followed by the addition of 85 μL of 1.33× selection buffer. The mixture was then incubated at 65 rpm at 25°C for 5 minutes, and the supernatant was collected. The same procedure was performed on the remaining four tubes containing Hsp70-immobilized beads.

[0103] The collected supernatant was then exposed to IgG beads four times and then to human albumin beads four times to remove the DMN-modified peptide cDNA that bound to them. These steps correspond to negative selection, and the final supernatant was collected.

[0104] (5.5 Addition of HSP90α beads) Beads onto which HSP90α had been immobilized (HSP90α-immobilized beads) were washed three times with 200 μL of PBS buffer (pH 7.4, 0.1% Tween 20) and then mixed with the DMN-modified peptide cDNA that had undergone negative selection. The beads were then incubated at 60 rpm at 25°C for 30 minutes. Next, the beads were washed three times with 200 μL of 1× selection buffer (25°C).

[0105] Protocol 6: Elution: 40 μL of water was added to the HSP90α-immobilized beads and heated at 95°C for 5 minutes to elute the DMN-modified peptide-cDNA. Next, 10 μL of 10x buffer (TAKARA), 8 μL of dNTP mix, 4 μL of Trap-Fwd-44 (5 μM), 4 μL of Rev-ScDNA3 (5 μM), 73 μL of RNase-free water, and 1 μL of ExTaq enzyme (TAKARA) were added to 5 μL of the eluted template (i.e., DMN-modified peptide-cDNA) to prepare 100 μL of PCR solution, which was then subjected to PCR.

[0106] Of the 100 μL PCR solution, 2 μL was used for real-time PCR, and the remaining solution was used for PCR for the next round of selection.

[0107] Protocol 7: PCR Product Purification. PCR products were purified using NucleoSpin® Gel and PCR Clean-up (Takara). RNA was synthesized using the purified DNA. Specifically, 10 μL of purified DNA, 10 μL of RNase- and DNase-free water, 3.5 μL of rNTPs (4), 10 μL of 5x buffer (Promega), 1 μL of RNase inhibitor, and 5 μL of Promega Enzyme Mix were mixed and incubated overnight at 37°C.

[0108] Next, 2 μL of Turbo DNase (Thermo Fisher Scientific) was added and further incubated for 30 minutes at 37° C. to remove DNA. Then, RNA was purified using an RNA purification kit (Zymo research), and the above protocols 1 to 7 were repeated.

[0109] The conditions for each round are shown in Table 5.

[0110]

[0111] The amino acid sequences selected by cDNA display are shown in Table 6. As a result of employing the cDNA display technique, three peptide aptamer candidates (peptides 1 to 3 in Table 6) with high affinity to Hsp90α were selected, and further studies were conducted on the responsiveness of the fluorescence change to increasing concentrations of Hsp90α.

[0112]

[0113] Evaluation Example 5: Fluorescence Properties of Peptide Aptamers First, the fluorescence properties of peptides 1 to 3 selected in Evaluation Example 2 were examined. Peptide 2 showed a three-fold fluorescence enhancement in the presence of 6 μM commercially available recombinant Hsp90α, whereas no significant enhancement was observed with Hsp70 (Figures 5 and 6). However, peptide 1 showed similar fluorescence enhancement with Hsp70 and Hsp90α (Figures 5 and 6). This is likely due to peptide 1 recognizing the consensus sequence of Hsp90α and Hsp70. Peptide 3 did not show fluorescence enhancement with Hsp90α or Hsp70 (Figures 5 and 6). Since peptide 2 exhibited the desired properties, the specificity and selectivity of peptide 2 for Hsp90α were tested. SPR analysis of Hsp90α in the presence of various concentrations of peptide 2 was performed. As a result, the equilibrium dissociation constant (K d ) was 3.13±1.34 μM (Figure 7).

[0114] To examine selectivity, Hsp90α was replaced with BSA. Peptide 2 showed no fluorescence enhancement relative to BSA, even at a concentration of 8 μM, demonstrating its selectivity for Hsp90α. Furthermore, binding of peptide 2 to Hsp90α in the presence of 10% FBS was confirmed, demonstrating that peptide 2 exhibited fluorescence enhancement relative to Hsp90α even in the presence of FBS. Because peptide 2 does not interact with FBS components, it can be added to cell culture media to examine the behavior of Hsp90α within cells.

[0115] Evaluation Example 6: Detection of Cellular Hsp90α Using Peptide 2 Next, we analyzed the properties of peptide 2 at the cellular level. While peptide 2 can detect purified Hsp90α in solution, specific detection of cellular Hsp90α is challenging. To be suitable as an Hsp90α sensor, peptide 2 must specifically detect cellular Hsp90α. This requires high target selectivity and fluorescence that exceeds the cellular autofluorescence. In this evaluation example, we used T98G cells, a thermotolerant cell line derived from a glioma. It has previously been reported that heat-shocked T98G cells produce significantly more Hsp90α than other tumor cell lines (Kalamida et al.).

[0116] Figure 8 shows the results of confocal laser scanning microscopy of HT-1080 cells and MCF-7 cells stained with peptide 2 and anti-Hsp90α antibody. The green fluorescence from DMN of peptide 2 colocalized with the red fluorescence from the anti-Hsp90α antibody around the cell membrane. This is thought to be due to peptide 2 specifically recognizing Hsp90α. The bar in Figure 8 indicates 20 μm.

[0117] To examine the behavior of Hsp90α in cells through comparison with anti-Hsp90α antibody, 98G cells were fixed under normal conditions and after 2 h of heat shock and stained with anti-HSP90α antibody or peptide 2. Peptide 2 and anti-Hsp90α antibody colocalized in the cytoplasm and pericellular regions. T98G cells under normal conditions showed higher fluorescence in the cytoplasm (Fig. 9A). However, heat-shocked T98G cells showed higher fluorescence intensity in the nuclear region than in the cytoplasm (Fig. 9B). Figure 9B shows the quantitative analysis of total fluorescence in T98G cells stained with peptide 2 under heat-shocked (42°C) and normal (37°C) conditions. Quantitative analysis was performed by cytometry.

[0118] Next, we examined the fluorescence of peptide 2 in T98G cells. Heat-shocked T98G cells exhibited higher fluorescence intensity than T98G cells under normal conditions (Fig. 10, bar indicates 20 μm). Furthermore, similar to the monoclonal anti-Hsp90α antibody, the fluorescence intensity was higher in the nuclear region. In T98G cells under normal conditions, fluorescence was observed throughout the cells. These results confirmed the specific target binding of peptide 2.

[0119] To quantitatively examine the difference in fluorescence intensity, we analyzed cell images by cytometry. Cells that had been heat-shocked for 2 hours (42°C) and cells under normal conditions (37°C) were incubated with peptide 2, and the difference in fluorescence intensity was analyzed using image-based cell cytometry. As shown in Figure 9B, the fluorescence intensity of heat-shocked T98G cells was higher than that of cells under normal conditions.

[0120] Hsp90α recognizes various client proteins and participates in essential signaling cascades in living cells. However, some client proteins of Hsp90α are also known to be involved in cancer development by regulating tumor growth, adhesion, invasion, metastasis, angiogenesis, and apoptosis (J. Wu, T. Liu, Z. Rios, Q. Mei, X. Lin, S. Cao, Heat shock proteins and cancer., Trends Pharmacol. Sci., 38, 226-256, 2017). Suppression of Hsp90α has also been reported to suppress HIF-90α and NK-κB expression, leading to the inhibition of epithelial-mesenchymal transition, motility, and invasiveness in colon cancer cells (GP Nagaraju, TE Long, W. Park, JC Landry, L. Taliaferro-Smith, AB Farris, R. Diaz, BF El-Rayes, Heat shock protein 90 promotes epithelial-to-mesenchymal transition, invasion, and migration in colorectal cancer., Mol. Carcinog. 54, 1147-11458, 2015). Overexpression of Hsp90α has also been reported in various cancers, including pancreatic, ovarian, breast, lung, and endometrial cancer. Furthermore, a stress-inducible isoform of Hsp90α has been found primarily in the cytoplasm, and Hsp90α has also been reported to be secreted into the extracellular matrix via an unconventional exosome pathway. During stress, Hsp90α production is upregulated, and Hsp90α translocates to the plasma membrane and is either secreted or anchored to cell surface heparan sulfate proteoglycans. Secreted Hsp90α plays an important role in activating matrix metalloproteinases, indirectly promoting cancer cell migration and invasion. It has also been reported that plasma concentrations of Hsp90α are higher in lung cancer patients than in normal individuals, suggesting that Hsp90α may be useful as a diagnostic biomarker for lung cancer.Therefore, a biosensor capable of detecting Hsp90α may be effective and useful in the prognosis or diagnosis of cancer.

Claims

1. A method for producing a modified nucleic acid molecule-peptide complex by introducing a functional molecule into the peptide portion of a nucleic acid molecule-peptide complex in which a nucleic acid molecule is linked to a peptide encoded by the nucleic acid molecule, the method comprising the step of introducing a functional molecule into a reactive group of an amino acid residue constituting the peptide portion of a nucleic acid molecule-peptide complex synthesized using a cell-free protein synthesis system, by a chemical synthesis reaction in a water-soluble polar organic solvent.

2. The method of claim 1, wherein the water-soluble polar organic solvent is an aprotic polar organic solvent.

3. The method according to claim 1, wherein the functional molecule is a fluorescent molecule, an inhibitor molecule or a ligand molecule.

4. The method of claim 1, wherein the reactive group is a thiol group, an amino group, or a carboxyl group.

5. The method of claim 1, wherein the reactive group is a bioorthogonal reactive group.

6. The method of claim 1, wherein the nucleic acid molecule-peptide complex is a nucleic acid molecule-peptide-ribosome complex further comprising a ribosome linked thereto.

7. A nucleic acid display method or ribosome display method comprising the method according to any one of claims 1 to 6, further comprising a step of selecting a desired modified product from the modified products of the nucleic acid molecule-peptide complex.

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