JP2008099700A - Method for forming stable complex of transcription product and translation product of DNA encoding arbitrary polypeptide, nucleic acid construct used in the method, complex formed by the method, and functionality utilizing the method Screening of protein and mRNA or DNA encoding the protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for screening functional proteins from a group of amino acid random sequences without limiting diversity of the sequences. <P>SOLUTION: A method, by which a stable linkage between a genotype and a phenotype in a cell-free system is successfully achieved by using interaction between an RNA-binding protein and RNA, interaction between a DNA-binding protein and DNA, or a protein that inactivates a ribosome, is provided. Furthermore, a method for screening functional proteins by using the stable linkage is provided, and nucleic acid construct usable for the stable linkage is provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method of forming a complex of a transcription product and a translation product of a DNA encoding an arbitrary polypeptide, a nucleic acid construct used in the method, a complex formed by the method, and a function using the method The present invention relates to screening of sex protein and mRNA or DNA encoding the protein.

  Methods for selecting and identifying functional proteins from a random sequence of a group of amino acids have become increasingly important in recent years. Many screening systems that are effective in selecting specific functional proteins, such as phage display and two-hybrid methods, use living cells (Fields, S. et al. Nature 1989, 340, 245-246 (non-patent literature). 1). Harada, K. et al. Nature 1996, 380, 175-179 (Non-Patent Document 2). Moore, JC et al. Nature Biotech. 1996, 14, 458-467 (Non-patent Document 3). Schatz, PJ et al. Methods Enzymol 1996, 267, 171-191 (Non-patent Document 4). Boder, ET et al. Nature Biotech. 1997, 15, 553-557 (Non-patent Document 5). Smith, GP et al. Chem, Rev. 1997, 97, 391- 410 (non-patent document 6).). In such a selection system, sequence information (genotype) encoding the selected protein (phenotype) is determined from the DNA introduced into each cell presenting the appropriate phenotype. Genotype-phenotype linking is an important determinant in selecting functional proteins from random sequences.

  However, as long as such methods depend on living cells, the diversity of sequence libraries is limited. For example, the transfection efficiency is low, the size of the displayed protein is limited, and the nature of the protein of interest is limited due to the inability to select proteins that are harmful to cells This limits the diversity of the library.

To overcome these problems, several methods for selecting proteins in cell-free systems have been developed (Mattheakis, LC et al. Proc. Natl, Acad. Sci. USA 1994, 91, 9022-9026 ( Non-patent literature 7). Mattheakis, LC et al. Methods Enzymol. 1996, 267, 195-207 (non-patent literature 8). Hanes, J. et al. Proc. Natl. Acad. Sci. USA 1997, 94, 4937-4942 He, M. et al. Nuclelic Acids Res. 1997, 25, 5132-5134 (Non-patent Document 10). Nemoto, N. et al. FEES Lett. 1997, 414, 405-408 (Non-patent Document 11). Roberts, RW et al. Proc. Natl. Acad. Sci. USA 1997, 94, 12297-12302 (Non-Patent Document 12). Hanes, J. et al. Proc. Natl. Acad. Sci. USA 1998, 95, 14130-14135 Tawfik, DS et al. Nature Biotech. 1998, 16, 652-656 (Non-patent Document 14). Doi, N. et al. FEBS Lett. 1999, 457, 227-230 (Non-patent Document 15). Hanes, J. et al. FEES Lett. 1999, 450, 105-110 (Non-patent Document 16). Makeyev, EV et al. FEBS Lett. 1999, 444, 177-180 (Non-patent Document 17). He, M. et al. Immunol. Methods 1999, 231, 105-117 (Non-patent Document 18). Schaffitzel, C. et al. J. Immunol. Methods 1999, 231, 119-135 (Non-patent Document 19). Hanes, J. et al. Nat. Biotechnol. 2000, 18, 1287-1292 (Non-Patent Document 20). Hanes, J. et al. Methods Enzymol. 2000, 328, 404-430 (Non-Patent Document 21).
Specific examples include a ribosome display method, a protein-mRNA covalent fusion method, and a micelle method. However, currently available cell-free systems include operations that reduce diversity and are generally difficult to put into practical use.

  For example, in the ribosome display method, a relatively stable complex between a translated protein and mRNA encoding the protein is formed by the ribosome. This is because the release of the stop codon slows the release of the protein from the complex. Under such conditions, the termination factor cannot efficiently induce the release of the protein from the ribosome complex. However, in this method, whether protein selection is possible depends on the half-life of the complex (release of the protein in the absence of a stop codon is only delayed to a limited extent), so selection is short. Must end with In fact, maintaining an intact mRNA-ribosome-protein complex is not easy.

  Ribosome display using E. coli is, for example, Jermutus L, et al. Tailoring in vitro evolution for protein affinity or stability. Proc Natl Acad Sci US A. 2001 Jan 2; 98 (1): 75-80 (Non-patent Document 22 Hanes J, et al. Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat Biotechnol. 2000 Dec; 18 (12): 1287-92 (Non-Patent Document 20); Hanes J, et al Selecting and evolving functional proteins in vitro by ribosome display. Methods Enzymol. 2000; 328: 404-30 (Non-Patent Document 21); and Schaffitzel C, et al. Ribosome display: an in vitro method for selection and evolution of antibodies from libraries. J Immunol Methods. 1999 Dec 10; 231 (1-2): 119-35 (Non-Patent Document 19). Ribosome display using a rabbit reticulocyte system is, for example, He M, et al. Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display J Immunol Methods. 1999 Dec 10; 231 (1-2): 105-17 (Non-Patent Document 18); and Bieberich E, et al. Protein-ribosome-mRNA display: affinity isolation of enzyme-ribosome-mRNA complexes and Anal Biochem. 2000 Dec 15; 287 (2): 294-8 (Non-patent Document 23). Ribosome display using wheat germ system is described in Gersuk GM, et al. High-affinity peptide ligands to prostate-specific antigen identified by polysome selection. Biochem Biophys Res Commun. 1997 Mar 17; 232 (2): 578-82 (Non-patent Document 24).

  In the protein-mRNA covalent fusion method, the translated protein is covalently bound to each mRNA whose 3 'end is labeled with puromycin. This method usually requires chemical synthesis to bind puromycin to the mRNA, but this step reduces the size of the available library. References on protein-mRNA covalent fusion methods include Keefe AD, et al. Functional proteins from a random-sequence library. Nature. 2001 Apr 5; 410 (6829): 715-8 (Non-patent Document 25); Wilson DS The use of mRNA display to select high-affinity protein-binding peptides.Proc Natl Acad Sci US A. 2001 Mar 27; 98 (7): 3750-5 (Non-Patent Document 26); Liu R, et al Optimized synthesis of RNA-protein fusions for in vitro protein selection.Methods Enzymol. 2000; 318: 268-93 (Non-Patent Document 27); and Cho G, et al. Constructing high complexity synthetic libraries of long ORFs using in vitro selection. J Mol Biol. 2000 Mar 24; 297 (2): 309-19 (non-patent document 28).

In the micelle method, each chemically modified individual DNA molecule is packaged one by one into a colloidal micelle for transcription and translation, but the modified DNA is encoded in the lumen of the micelle. Bind to proteins. However, by diluting the reaction mixture by designing a single molecule of DNA to be packaged in individual micelles, the diversity of the sequence library is reduced. Thus, while cell-free systems have the advantage of being able to test more sequences compared to systems using live cells, the currently available cell-free systems fully explore a significant portion of the pool of sequences. Not.
Fields, S. et al. Nature 1989, 340, 245-246. Harada, K. et al. Nature 1996, 380, 175-179. Moore, JC et al. Nature Biotech. 1996, 14, 458-467. Schatz, PJ et al. Methods Enzymol. 1996, 267, 171-191. Boder, ET et al. Nature Biotech. 1997, 15, 553-557. Smith, GP et al. Chem, Rev. 1997, 97, 391-410. Mattheakis, LC et al. Proc. Natl, Acad. Sci. USA 1994, 91, 9022-9026. Mattheakis, LC et al. Methods Enzymol. 1996, 267, 195-207. Hanes, J. et al. Proc. Natl. Acad. Sci. USA 1997, 94, 4937-4942. He, M. et al. Nuclelic Acids Res. 1997, 25, 5132-5134. Nemoto, N. and others FEES Lett. 1997, 414, 405-408. Roberts, RW et al. Proc. Natl. Acad. Sci. USA 1997, 94, 12297-12302. Hanes, J. et al. Proc. Natl. Acad. Sci. USA 1998, 95, 14130-14135. Tawfik, DS and others Nature Biotech. 1998, 16, 652-656. Doi, N. and others FEBS Lett. 1999, 457, 227-230. Hanes, J. et al. FEES Lett. 1999, 450, 105-110. Makeyev, EV, etc.FEBS Lett. 1999, 444, 177-180. He M, et al. Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display.J Immunol Methods. 1999 Dec 10; 231 (1-2): 105-17 Schaffitzel C, et al. Ribosome display: an in vitro method for selection and evolution of antibodies from libraries.J Immunol Methods. 1999 Dec 10; 231 (1-2): 119-35 Hanes J, et al. Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat Biotechnol. 2000 Dec; 18 (12): 1287-92 Hanes J, et al. Selecting and evolving functional proteins in vitro by ribosome display.Methods Enzymol. 2000; 328: 404-30 Jermutus L, et al. Tailoring in vitro evolution for protein affinity or stability.Proc Natl Acad Sci US A. 2001 Jan 2; 98 (1): 75-80 Bieberich E, et al. Protein-ribosome-mRNA display: affinity isolation of enzyme-ribosome-mRNA complexes and cDNA cloning in a single-tube reaction. Anal Biochem. 2000 Dec 15; 287 (2): 294-8 Gersuk GM, et al. High-affinity peptide ligands to prostate-specific antigen identified by polysome selection. Biochem Biophys Res Commun. 1997 Mar 17; 232 (2): 578-82 Keefe AD, et al. Functional proteins from a random-sequence library.Nature. 2001 Apr 5; 410 (6829): 715-8 Wilson DS, et al. The use of mRNA display to select high-affinity protein-binding peptides. Proc Natl Acad Sci US A. 2001 Mar 27; 98 (7): 3750-5 Liu R, et al. Optimized synthesis of RNA-protein fusions for in vitro protein selection.Methods Enzymol. 2000; 318: 268-93 Cho G, et al. Constructing high complexity synthetic libraries of long ORFs using in vitro selection.J Mol Biol. 2000 Mar 24; 297 (2): 309-19

  The present invention has been made in view of such circumstances, and an object thereof is to provide a method capable of selecting a functional protein from a random sequence of a group of amino acids without reducing sequence diversity. There is. Another object of the present invention is to provide a method capable of stably linking genotype and phenotype even in a cell-free system in order to enable selection of such a functional protein. . Furthermore, an object of the present invention is to provide a nucleic acid construct that can be used for the stable linkage of such a genotype and a phenotype.

In order to solve the above-mentioned problems, the present inventor tried to stably link a genotype and a phenotype in a cell-free system.
The first technique uses a strong interaction between RNA-binding protein and RNA for stable linkage between genotype and phenotype. As an example, an aptamer that binds to the Tat protein of HIV-1 with stronger intensity than RNA that interacts with Tat (trans activation) protein known as TAR (trans activation response region) (Referred to as Tat aptamer) was selected (Yamamoto, R. et al. Genes Cells 2000, 5, 371-388). The interaction between a Tat aptamer and a peptide derived from Tat (RE peptide, 38 amino acids, or CQ peptide, 36 amino acids) is very strong, and its dissociation constant (Kd) is in the nM or less range. In order to link genotype and phenotype using this interaction, the present inventors prepared a DNA construct in which DNA encoding a Tat aptamer and a peptide derived from Tat was linked to a DHFR gene, and DHFR We examined whether gene transcripts (mRNA) and translation products (proteins) can form stable complexes.

  As a result, it was found that the interaction between the Tat aptamer and the Tat-derived peptide is useful for linking the DHFR gene genotype and phenotype, that is, the transcription product and the translation product, unless a network structure is formed. Increasing the number of aptamers and peptide motifs in the DNA construct and modifying the DNA construct such that the transcript lacks a stop codon were effective in improving the stability of the complex.

  As another example, we tried to link the genotype and phenotype using the interaction between the MS2 coat protein dimer and the corresponding specific binding motif RNA sequence. A complex of the transcription product and translation product of the protein gene could be formed.

  Furthermore, the present inventors considered the use of the interaction between the DNA-binding protein and DNA in place of the above-described interaction between the RNA-binding protein and RNA in order to stably link the genotype and the phenotype. It was. That is, a nucleic acid construct that is a complex of an mRNA encoding a chimeric peptide of a DNA binding protein and an arbitrary peptide and a DNA containing a region to which the DNA binding protein binds can be prepared, and the mRNA in the nucleic acid construct can be translated. For example, it was thought that the translation product could bind to the DNA in the nucleic acid construct, thereby forming a stable complex of the translation product and the nucleic acid construct.

  The second technique uses ribosome inactivation for stable linkage of genotype and phenotype. For example, ricin A chain was selected for ribosome inactivation. Lysine is a cytotoxic protein derived from castor and stops protein synthesis by inactivating the ribosome. The subunit ricin A chain hydrolyzes the N-glucoside bond at the α-salicine site present in the 28S rRNA very specifically, and inactivates the ribosome. In rat 28S rRNA, the 4324th adenosine undergoes hydrolysis. Ribosomes inactivated by lysine stop on the mRNA strand. In order to link the genotype and the phenotype by utilizing the ribosome interaction by the ricin A chain, the present inventor linked the ricin A chain DNA to the DHFR gene, GST gene, or streptavidin gene. DNA constructs were prepared, and it was verified whether the transcription product (mRNA) and translation product (protein) of these genes could form a stable complex.

  As a result, we found that inactivation of ribosomes by ricin A chain is effective in stabilizing the complex of transcription and translation products of these genes. Insertion of the spacer peptide into the translation product to avoid steric hindrance was effective in inactivating the ribosome.

  In addition, complexes prepared using the above DNA constructs containing DHFR gene, GST gene, or streptavidin gene selectively bound to MTX ligand, glutathione-sepharose or biotin-agarose, respectively. This fact uses a molecule that can interact with the phenotype of a specific gene as a target substance, and screens the functional protein that binds to the target substance and the mRNA that encodes it from the complex formed by the above method. It shows that it is possible to do. The technique of the present invention enables efficient selection of a functional protein and the mRNA encoded by it without reducing the diversity of the amino acid sequence of the peptide to be screened. In addition, the complex containing a transcription product and a translation product is stabilized by genetic manipulation of fusion of the DNA encoding the transcription product and the translation product with the DNA that contributes to the stabilization, and a series of selection steps is complicated. The chemical synthesis process is not included, and it is a further advantage of this method that it can be easily operated.

That is, the present invention provides the following inventions.
(1) A DNA construct comprising the following DNAs (i) and (ii), and wherein the DNAs (i) and (ii) below are joined to express a fused transcription product and translation product: .
(I) DNA encoding any polypeptide
(Ii) DNA encoding a polypeptide having a function of stabilizing a complex comprising a transcription product and a translation product of the DNA of (i)
(2) The DNA construct according to (1), wherein the DNA of (ii) is a DNA encoding a polypeptide having a function of inactivating a ribosome.
(3) The DNA construct according to (2), wherein a DNA encoding a spacer peptide is bound downstream of a DNA encoding a polypeptide having a function of inactivating a ribosome.
(4) The DNA construct according to (2), wherein a DNA encoding a linker peptide is inserted between a DNA encoding an arbitrary polypeptide and a DNA encoding a polypeptide having a function of inactivating a ribosome. .
(5) The DNA construct according to (1), wherein the DNA of (ii) is a combination of a DNA encoding an RNA binding protein and a DNA encoding an RNA to which the RNA binding protein binds.
(6) The DNA construct according to (5), wherein the transcript of the fused DNA of (i) and (ii) is modified so as to lack a stop codon.
(7) The DNA construct according to (5), wherein a plurality of DNAs encoding RNA binding proteins are juxtaposed and a plurality of DNAs encoding RNAs to which the RNA binding protein binds are juxtaposed.
(8) A DNA encoding a plurality of juxtaposed RNA binding proteins is a DNA encoding a plurality of different RNA binding proteins, and a DNA encoding an RNA to which a plurality of juxtaposed RNA binding proteins bind The nucleic acid construct according to (7), which is a DNA encoding RNA to which a plurality of RNA binding proteins different from each other bind.
(9) A nucleic acid construct which is a complex of the mRNA of the following (i) and the DNA of the following (ii).
(I) Fusion mRNA of RNA encoding a DNA binding protein and RNA encoding an arbitrary polypeptide
(Ii) DNA comprising a DNA region to which the translation product of the fusion mRNA of (i) binds
(10) The nucleic acid construct according to (9), wherein a complex is formed by hybridization of the mRNA of (i) and the DNA of (ii).
(11) The nucleic acid construct according to (9), wherein the 3 ′ side region of the mRNA of (i) and the 3 ′ side region of the DNA of (ii) are hybridized.
(12) A DNA construct of the following (i) or (ii) for use in the preparation of the DNA construct according to (1).
(I) a DNA construct comprising DNA having a function of stabilizing a complex of a transcription product and a translation product of a DNA encoding an arbitrary polypeptide (ii) a transcription product and a translation product of a DNA encoding an arbitrary polypeptide A DNA construct comprising a DNA having a function of stabilizing the complex and a cloning site of a DNA encoding the arbitrary polypeptide (13) (i) and (ii) DNA in the DNA construct according to (1) (I) and (ii) A method for producing a complex comprising a transcription product and a translation product of DNA.
(14) The translation product characterized by translating the mRNA of (i) in the nucleic acid construct according to (9) and binding the translation product to the DNA of (ii) in the nucleic acid construct according to (9) And a method for producing a complex of the nucleic acid construct according to (9).
(15) The method according to (14), wherein a nucleic acid construct in which a complex is formed by hybridization of the mRNA of (i) and the DNA of (ii).
(16) The method according to (14), wherein a nucleic acid construct in which the 3 ′ region of the mRNA of (i) and the 3 ′ region of the DNA of (ii) are hybridized is used.
(17) A method for extending the DNA of (ii) in the complex produced by the method according to (15), wherein the complex is contacted with a reverse transcriptase, and the mRNA of (i) is used as a template. Performing a reverse transcription reaction.
(18) A method of extending the 3 ′ region of the DNA of (ii) in the complex produced by the method according to (16), wherein the complex is contacted with a reverse transcriptase, and (i) And (ii) performing a reverse transcription reaction using the mRNA of (ii) as a template.
(19) The method according to any one of (13) to (18), which is performed in a cell-free system.
(20) A composite produced by the method according to (13).
(21) A composite produced by the method according to any one of (14) to (18).
(22) A method for screening a polypeptide that binds to a specific target substance or mRNA encoding the polypeptide, the method comprising the following steps (a) to (c):
(A) A step of expressing a DNA of (i) and (ii) in the DNA construct according to (1) to form a complex containing the transcription product and translation product of the DNA of (i) and (ii) (B) contacting the specific target substance with the complex formed in step (a) (c) recovering the complex bound to the target substance (23) binding to the specific target substance A method for screening a polypeptide or mRNA encoding the polypeptide, comprising the following steps (a) to (c):
(A) by translating the mRNA of (i) in the nucleic acid construct described in (9) and binding the translation product to the DNA described in (ii) in the nucleic acid construct described in (9), (B) forming a complex containing the nucleic acid construct according to (9), contacting the specific target substance with the complex formed in step (a), and (c) binding to the target substance Step of recovering complex (24) A method of screening a polypeptide that binds to a specific target substance or mRNA or DNA encoding the polypeptide, comprising the following steps (a) to (d):
(A) by translating the mRNA of (i) in the nucleic acid construct described in (10) and binding the translation product to the DNA described in (ii) of the nucleic acid construct described in (10), (B) forming a complex containing the nucleic acid construct according to (10), contacting the complex formed in step (a) with a reverse transcriptase, and using the mRNA of (i) in the complex as a template (C) a step of extending the DNA of (ii) in the complex (c) by contacting the specific target substance with the complex formed in the step (b) (d) Recovering the complex bound to the target substance (25) A method for screening a polypeptide that binds to a specific target substance or mRNA or DNA encoding the polypeptide, comprising the following (a) to (d) Comprising the steps of:
(A) by translating the mRNA of (i) in the nucleic acid construct described in (11) and binding the translation product to the DNA described in (ii) of the nucleic acid construct described in (11), (B) forming a complex comprising the nucleic acid construct according to (11), contacting the complex formed in step (a) with a reverse transcriptase, and using the mRNA of (i) in the complex as a template And (c) extending the 3 ′ region of the DNA of (ii) by performing a reverse transcription reaction using the DNA of (ii) in the complex as a primer, and (c) the specific target substance, Contacting the complex formed in step (b) (d) recovering the complex bound to the target substance (26) polypeptide binding to a specific target substance or mRNA encoding the polypeptide Or screening DNA A method, the method comprising the following (a) of step (d).
(A) by translating the mRNA of (i) in the nucleic acid construct described in (10) and binding the translation product to the DNA described in (ii) of the nucleic acid construct described in (10), (B) forming a complex comprising the nucleic acid construct according to (10), contacting the specific target substance with the complex formed in step (a), and (c) binding to the target substance. A step of extending the DNA of (ii) in the complex by contacting a reverse transcriptase with the complex and performing a reverse transcription reaction using the mRNA of (i) in the complex as a template (27) A method for screening a polypeptide that binds to a specific target substance, or mRNA or DNA encoding the polypeptide, comprising the following (a) to (d): Comprising the steps of:
(A) by translating the mRNA of (i) in the nucleic acid construct described in (11) and binding the translation product to the DNA described in (ii) of the nucleic acid construct described in (11), (B) forming a complex containing the nucleic acid construct according to (11), contacting the specific target substance with the complex formed in step (a), and (c) binding to the target substance A reverse transcriptase is brought into contact with the complex, and a reverse transcription reaction is performed using the mRNA of (i) in the complex as a template and the DNA of (ii) in the complex as a primer. (ii) extending the 3′-side region of the DNA of (ii) recovering the complex bound to the target substance (28), wherein the target substance is immobilized on a carrier (22) to (27 ) Any one of the methods.
(29) A kit for use in the screening method according to (21), comprising the DNA construct according to (1) or (12) or the complex according to (20).
(30) A kit for use in the screening method according to any one of (23) to (27), comprising the nucleic acid construct according to (9) or the complex according to (21).

Hereinafter, the implementation method and embodiment of this invention are demonstrated in detail.
(1) Nucleic acid construct The DNA construct of the present invention comprises (i) a DNA encoding an arbitrary polypeptide, and (ii) a DNA having a function of stabilizing a complex of a transcription product and a translation product of the DNA. . In the DNA construct, the above DNAs (i) and (ii) are linked so as to express the fused transcription product and translation product.

  “DNA encoding an arbitrary polypeptide” means a desired DNA for which a complex of a transcription product and a translation product is to be formed. In screening for functional proteins, for example, a cDNA library or a random library can be suitably used.

  The kind of DNA having a function of stabilizing the complex of the transcription product and the translation product is not particularly limited. A first example of suitable DNA is DNA encoding a polypeptide having a function of inactivating ribosomes.

  As used herein, the term “polypeptide having a function of inactivating ribosome” refers not only to a polypeptide that destroys the ribosome itself and inactivates it, but also by stopping the ribosome on mRNA. Also included are polypeptides capable of stopping the translation function.

  Specific examples of such polypeptides include N-glycosylase or RNase. Specific examples of N-glycosylase include Pokeweed antiviral protein (PAP) (Phytolacca americana), Momordin (Momordica charantia), Luffin (Luffa cylindrica), Bryodin (Bryonia dioica), Dianthin (Dianthus caryophyllus), Trichosanthin (Trichosanthes angu) a-Momorcharin (Momordia charantia), Saporin (Saponaria officinalis), Ricin A-chain (RTA) (ricinus communis), Abrin A-chain (Abrus precatorius), Mistletoe lectin I (MLI) (Viscum album), Shiga toxin AI, Another example is Diphtheria toxin (DT) A-chain. A specific example of RNase is α-Sarcin.

  For ricin, The RNA N-glycosidase activity of ricin A-chain.The characteristics of the synthetic activity of ricin A-chain with ribosomes and with rRNA.J Biol Chem. 1988 Jun 25; 263 (18): 8735- As for α-sarcin, The ribonuclease activity of the cytotoxin alpha-sarcin.The characteristics of the enzymatic activity of alpha-sarcin with ribosomes and ribonucleic acids as substrates.J Biol Chem. 1983 Feb 25; 258 (4 ): 2662-7, and for Pokeweed antiviral protein (PAP), X-ray crystallographic analysis of the structural basis for the interaction of pokeweed antiviral protein with guanine residues of ribosomal RNA.Protein Sci. 1999 Nov; 8 (11 ): 2399-405.

  In one preferred embodiment of the DNA construct of the present invention, a DNA encoding a spacer peptide is bound downstream of a DNA encoding a polypeptide having a function of inactivating a ribosome. This “spacer peptide” is a peptide that enables a polypeptide having a function of inactivating a ribosome to attack the ribosome with cis before translation by the ribosome is completed. The spacer peptide is preferably a peptide that is not itself folded. The spacer peptide has a chain length that can ensure that the translated polypeptide having the function of inactivating the ribosome attacks the ribosome with cis. The chain length is usually 50 to 200 amino acids, preferably 100 to 140 amino acids.

  In another preferred embodiment of the DNA construct of the present invention, a linker peptide is used between a DNA encoding an arbitrary polypeptide on the DNA construct of the present invention and a DNA encoding a polypeptide having a function of inactivating a ribosome. DNA encoding is inserted. This “linker peptide” refers to a fused product in a translation product (fusion protein) from the DNA construct of the present invention, in order to avoid the fusion of the fused polypeptides with each other due to steric hindrance. A polypeptide that is inserted between peptides. The chain length of the linker peptide is not particularly limited, but generally a peptide of about 10 to 30 amino acids is used.

  As a suitable second example of DNA having a function of stabilizing a complex of a transcription product and a translation product, a combination of a DNA encoding an RNA binding protein and a DNA encoding an RNA to which the RNA binding protein binds. Can be mentioned. When a combination of a DNA sequence encoding such an RNA binding protein and a DNA sequence encoding an RNA to which the RNA binding protein binds is used, a plurality of DNAs encoding the RNA binding protein are juxtaposed in the DNA construct. It is preferable that a plurality of DNAs encoding RNA to which the RNA binding protein binds are juxtaposed. Thereby, the interaction between RNA-binding protein and RNA can be enhanced.

  More preferably, as DNA encoding a plurality of RNA binding proteins, DNA encoding a plurality of different RNA binding proteins is used, and as a DNA encoding RNA to which a plurality of the RNA binding proteins are bound, DNAs encoding different RNAs to which a plurality of different RNA binding proteins are bound are used. By using such a plurality of different DNAs, formation of network structures between different complexes can be prevented.

  Specific examples of the combination of RNA binding protein and RNA to which the RNA binding protein binds include Tat protein and TAR RNA or Tat aptamer, Rev protein and RRE RNA or Rev aptamer, U1A spliceosomal protein and U1A spliceosomal protein aptamer or U1A Sn RNA , Zinc finger proteins or variants thereof and the RNA they recognize. Moreover, it is also possible to use the RNA sequence of MS2 coat protein and the specific binding motif corresponding to it for this invention.

  For Tat-TAR or Tat aptamer, A novel RNA motif that binds efficiently and specifically to the Ttatprotein of HIV and inhibits the trans-activation by Tat of transcription in vitro and in vivo.Genes Cells. 2000 May; 5 (5 ): 371-88, and for Rev-RRE complex, A structural model for the HIV-1 Rev-RRE complex deduced from altered-specificity rev variants isolated by a rapid genetic strategy.Cell. 1996 Oct 4; 87 (1): 115-25, for U1A spliceosomal protein, Crystal structure at 1.92 A resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin.Nature. 1994 Dec 1; 372 (6505) : 432-8.

  Moreover, as literature regarding the interaction between Zn Finger protein and RNA, Friesen WJ, et al. Specific RNA binding proteins constructed from zinc fingers. Nat Struct Biol. 1998 Jul; 5 (7): 543-6; McColl DJ, et al. Structure-based design of an RNA-binding zinc finger.Proc Natl Acad Sci US A. 1999 Aug 17; 96 (17): 9521-6; Friesen WJ, et al. Phage display of RNA binding zinc fingers from transcription factor IIIA. J Biol Chem. 1997 Apr 25; 272 (17): 10994-7; Theunissen O, et al. RNA and DNA binding zinc fingers in Xenopus TFIIIA. Cell. 1992 Nov 13; 71 (4): 679-90; Clemens KR, et al. Molecular basis for specific recognition of both RNA and DNA by a zinc finger protein.Science. 1993 Apr 23; 260 (5107): 530-3; and Joho KE, et al. A finger protein structurally similar to TFIIIA that binds exclusively to 5S RNA in Xenopus. Cell. 1990 Apr 20; 61 (2): 293-300.

  In another preferred embodiment of the DNA construct of the present invention, the DNA construct is modified such that the fused transcript expressed from the DNA construct lacks a stop codon. This delays the release of the ribosome from the complex containing the transcription product and translation product, thereby stabilizing the complex.

  The DNA construct of the present invention can be prepared, for example, by inserting a DNA having a function of stabilizing a complex of a transcription product and a translation product into a vector containing a DNA encoding an arbitrary polypeptide. In addition, a DNA encoding an arbitrary polypeptide is introduced into the cloning site of a vector comprising a DNA having a function of stabilizing a complex of a transcription product and a translation product and a cloning site of the DNA encoding the arbitrary polypeptide. It can also be prepared by insertion.

  The present invention also includes DNA having a function of stabilizing a complex of a transcription product and a translation product of DNA encoding <1> an arbitrary polypeptide, which is used for preparing the DNA construct as described above. DNA comprising a DNA construct, <2> DNA having a function of stabilizing a complex of a transcription product and a translation product of a DNA encoding an arbitrary polypeptide, and a cloning site of the DNA encoding the arbitrary polypeptide Providing a construct.

  In the present invention, a nucleic acid construct comprising mRNA and DNA comprises (i) a fusion mRNA of RNA encoding a DNA binding protein and an RNA encoding an arbitrary polypeptide, and (ii) translation of the fusion mRNA of (i). A complex with DNA containing a DNA region to which the product binds. The mRNA of (i) and the DNA of (ii) in the nucleic acid construct preferably form a complex by hybridizing (hydrogen bonding). The number of base pairs to hybridize is not particularly limited as long as the mRNA of (i) and the DNA of (ii) in the nucleic acid construct can be stably bound, but is preferably 10 base pairs or more.

  If reverse transcription reaction is performed using the mRNA of (i) in the nucleic acid construct as a template, DNA corresponding to the mRNA can be synthesized. Thereby, in addition to the transcription product and the translation product, the DNA encoding them can be used as a component of the complex.

  When the nucleic acid construct is prepared as a nucleic acid construct in which the 3 ′ region of the mRNA of (i) and the 3 ′ region of the DNA of (ii) are hybridized, the mRNA of (i) is used as a template. It is possible to carry out a reverse transcription reaction using the DNA of (ii) as a primer.

  When other primers are used without using the DNA of (ii) as a reverse transcription reaction primer, the 3′-side region of the mRNA of (i) and the DNA of (ii) must be hybridized in the nucleic acid construct. No, the 5′-side regions may hybridize. In this case, in order to perform the reverse transcription reaction, it is necessary to add a primer (an oligonucleotide that specifically hybridizes to the mRNA of (i)) to the reaction system.

  See the literature (FEBS Lett 2001 Nov 23; 508 (3): 309-12) for the binding of mRNA and DNA for the preparation of nucleic acid constructs comprising mRNA and DNA of the present invention.

(2) Method for producing complex of transcription product and translation product The present invention also provides a method for producing the complex of transcription product and translation product described above. In one embodiment of this production method, by adding or introducing the DNA construct into an appropriate transcription / translation system, (i) DNA encoding an arbitrary polypeptide and (ii) transcription product and translation of the DNA. It is characterized by expressing a fusion DNA with a DNA having a function of stabilizing the complex of the product. The transcription product and translation product resulting from the expression of this fusion DNA can be produced by the action of a polypeptide that inactivates the ribosome contained in the translation product, or the RNA binding protein contained in the translation product and its binding site contained in the transcription product. A stable complex is formed by interaction with.

  In another embodiment of this production method, the nucleic acid construct comprising RNA and DNA is added or introduced into an appropriate transcription / translation system to translate (i) mRNA in the nucleic acid construct, and the translation product thereof. Is bound to the DNA of (ii) in the nucleic acid construct. A stable complex is formed by the interaction between the DNA-binding protein contained in the translation product and the DNA of (ii) in the nucleic acid construct. By performing reverse transcription using the mRNA of (i) in the complex as a template and extending the DNA of (ii), DNA corresponding to the mRNA can be synthesized. When a nucleic acid construct in which the 3 'region of the mRNA of (i) and the 3' region of the DNA of (ii) are hybridized is used, a reverse transcription reaction is performed using the DNA of (ii) as a primer. Can do.

  Examples of the transcription / translation system used in the present method include a cell-free transcription / translation system or living cells. Cell-free transcription / translation system or living cells, etc., by adding or introducing the nucleic acid construct of the present invention to express the transcription product and translation product from the nucleic acid construct (when using a nucleic acid construct comprising mRNA and DNA) Is not limited as long as it guarantees the expression of the translation product. In the present method, a cell-free transcription / translation system, particularly preferably a transcription / translation system composed of a cell extract can be used.

  As the cell-free transcription / translation system, cell-free transcription / translation systems composed of prokaryotic or eukaryotic extracts such as Escherichia coli, rabbit reticulocyte, wheat germ extract and the like can be used. As a living cell translation system, prokaryotic or eukaryotic organisms such as cells of Escherichia coli can be used.

  Complexes comprising a transcription product and a translation product obtained by this method are also within the scope of the present invention. Such a complex can exist stably, and can be used for screening of a polypeptide that interacts with a specific target substance, mRNA or DNA encoding the same, as described later.

(3) Method for Screening Polypeptide that Binds to Specific Target Substance or mRNA or DNA that Encodes the Polypeptide The present invention also provides a polypeptide that binds to a specific target substance or mRNA or DNA that encodes the polypeptide A method for screening is provided.

  The first embodiment of the method of the present invention is an embodiment using the DNA construct of the present invention. In this embodiment, first, a fusion DNA of (i) a DNA encoding an arbitrary polypeptide in the DNA construct and (ii) a DNA having a function of stabilizing a complex of a transcription product and a translation product of the DNA is prepared. And a complex containing the transcription product and translation product of the fusion DNA is formed (step (a)), and then a specific target substance is contacted with the complex formed in step (a) (step (B)) Next, the complex bound to the target substance is recovered (step (c)).

  In the second to fourth aspects of the method of the present invention, a nucleic acid construct comprising the mRNA and DNA of the present invention is used.

  The second aspect is an aspect that does not include a step of performing a reverse transcription reaction. In the second embodiment, first, the translation product is bound to the DNA described in (ii) of the nucleic acid construct by translating the mRNA of (i) in the nucleic acid construct comprising the mRNA and DNA of the present invention, A complex containing the translation product and the nucleic acid construct is formed (step (a)), and then the specific target substance is contacted with the complex formed in step (a) (step (b)). Subsequently, the complex bound to the target substance is recovered (step (c)).

  The third and fourth aspects of the method of the present invention are aspects including a step of performing a reverse transcription reaction. The third aspect is an aspect in which a reverse transcription reaction is performed before the target substance is bound to the complex, and the fourth aspect is an aspect in which the reverse transcription reaction is performed after the target substance is bound to the complex. .

  In the third embodiment of the method of the present invention, first, the mRNA of (i) in the nucleic acid construct comprising the mRNA of the present invention and DNA is translated, and the translation product is converted into the DNA described in (ii) of the nucleic acid construct. By binding, a complex containing the translation product and the nucleic acid construct is formed (step (a)), and then the complex formed in step (a) is contacted with a reverse transcriptase to form the complex. Using the mRNA of (i) in the body as a template, a reverse transcription reaction is performed to extend the DNA of (ii) in the complex (step (b)), and then the specific target substance and step (b) ) Is contacted (step (c)), and then the complex bound to the target substance is recovered (step (d)).

  In the fourth aspect of the method of the present invention, the mRNA of (i) in the nucleic acid construct comprising the mRNA of the present invention and DNA is translated, and the translation product is bound to the DNA described in (ii) of the nucleic acid construct. Thus, a complex containing the translation product and the nucleic acid construct is formed (step (a)), and then the specific target substance is brought into contact with the complex formed in step (a) (step (B)) Next, a reverse transcriptase is brought into contact with the complex bound to the target substance, and a reverse transcription reaction is performed using the mRNA of (i) in the complex as a template. ) Is extended (step (c)), and then the complex bound to the target substance is recovered (step (d)).

  In the third or fourth embodiment of the method of the present invention, when a nucleic acid construct in which the 3 ′ region of the mRNA of (i) and the 3 ′ region of the DNA of (ii) are hybridized is used, The reverse transcription reaction can be easily performed using the DNA of (ii) as a primer.

  The target substance in the method of the present invention is not particularly limited, and various target substances can be used depending on the purpose of screening. Specific examples of the target substance include peptides, proteins, low molecular compounds, ligands, enzymes, antibodies, environmental pollutants, and the like. For example, when a ligand is used as a target substance, a receptor that binds to the ligand can be screened, which is useful for identification of an orphan receptor ligand. In order to facilitate the recovery of the complex, it is preferable to fix the target substance on a carrier. Depending on the purpose, screening may be performed using a substrate on which a plurality of types of target substances are arranged.

  The following method can be mentioned as an example of the screening of the present invention. After transcription and translation of the nucleic acid construct of the present invention (after translation in the case of using a nucleic acid construct comprising mRNA and DNA), a suspension of a target substance bound to a solid phase carrier such as agarose is added, and a certain period of time is added. By holding, the target substance and the product of the target gene are bound. The supernatant containing unbound mRNA and protein is removed from the mixture, and the solid phase carrier is washed with an appropriate buffer. MRNA (or DNA) bound to a solid phase carrier can be isolated by elution with a solution containing urea at high temperature.

  The present invention also provides a kit for use in the screening of the present invention. The kit for screening using the DNA construct of the present invention includes, for example, <1> a DNA construct of the present invention, <2> (i) an arbitrary polypeptide for preparing the DNA construct of the present invention. A DNA construct containing a DNA having a function of stabilizing a complex of a transcription product and a translation product of the DNA to be synthesized or (ii) a function of stabilizing a complex of a transcription product and a translation product of a DNA encoding any polypeptide A DNA construct comprising a DNA having DNA and a cloning site of a DNA encoding the arbitrary polypeptide, or <3> a complex formed by expression of the DNA construct of the present invention. be able to.

  The kit for screening using the nucleic acid construct comprising the mRNA and DNA of the present invention includes, for example, <1> a nucleic acid construct comprising the mRNA and DNA of the present invention, and <2> the mRNA and DNA of the present invention. Any one or both of the complexes formed by the expression of the nucleic acid construct consisting of (including those in which the DNA corresponding to the mRNA in the complex was synthesized by performing a reverse transcription reaction) Can be included.

The present invention is specifically described by the following examples, but the present invention is not limited by the following examples.
[Example 1]
A. Method
(A-1) Preparation of three kinds of proteins [DHFR- (RE) _n ]
In preparing DHFR- (RE) _1-3 fusion protein, plasmid pTZDHFR20-derived (Blakley, RL In Folates and Pterins; Blakley, RL; Benkovic, S. J ,, Eds .; Wiley: New York, 1985; pp. 191 The gene of DHFR having a PstI site at its 3 ′ end was inserted into plasmid pET30a (Novagen, Madison, Wis.). Sense and antisense oligonucleotides encoding RE peptide (38 amino acids; RKKRR QRRRP PQGSQ TBQVS LSKQP TSQSR GDPTG PKE (SEQ ID NO: 3)) were synthesized, annealed, and rTaq polymerase (Takara Shuzo Co., Kyoto, Japan) Used to elongate. The obtained dsDNA was cleaved at the PstI site and EcoT22I site, and inserted into the 3 ′ side of the DHFR gene at the PstI site. Since the restriction sites of PstI and EcoT22I are ligated and the ligated site is not cleaved by either PstI or EcoT22I, the sequence encoding the RE peptide is continuously linked to the PstI site at the 3 ′ end of the DHFR gene, pDHFR- (RE) ₁ to pDHFR- (RE) ₃ were obtained. These plasmids were used to transfect E. coli NovaBlue (DE3) (Novagen), and the resulting bacteria were incubated at 500C in 500 ml of LB medium. In the logarithmic growth phase, 1 mM IPTG (isopropyl-1-thio-β-D-galactoside) was added and incubated at 30 ° C. for 4 hours. The protein was expressed as a polypeptide to which a polyhistidine tag was added. Proteins were purified using HisTrap ^™ chelating kit (Amersham Pharmacia Biotech Uppsala, Sweden) according to the instructions and low molecular weight material was removed using HiTrap ^™ desalting system (Amersham Pharmacia Biotech). Furthermore, HiTrap ^™ SP (Amersham Pharmacia Biotech) cation exchange HPLC was performed to remove the degraded protein. Protein purity was examined by SDS-PAGE. The results are shown in FIG. The concentration of the purified protein was measured with a BCA assay kit (Pierce, Rockford, IL).

(A-2) Preparation of three kinds of tandem RNA aptamers [(Apt) _n ] For the preparation of tandem Tat aptamers [(Apt) _1-3 ], sense and antisense oligonucleotides (5′-AAA AAA CGA AGC TTG ATC CCG TTT GCC GGT CGA TCG CTT CG-3 ′ (SEQ ID NO: 4) and 5′-TTT TTT CGA AGC GAT CGA CCG GCA AAC GGG ATC AAG CTT C-3 ′ (SEQ ID NO: 5)) were synthesized. These oligonucleotides were annealed and ligated using DNA ligation kit version 2 (Takara Shuzo Co.) to obtain Tat aptamer [(Apt) _1-3 ] linked to tandem. Ligated DNA sequences encoding 1-3 Tat aptamers were isolated after cloning (TA Clonign Kit, Invitrogen Carlsbad, CA). DNA encoding 1-3 aptamers was transcribed using the T7 Ampliscribe kit (Epicentre Technologies, Madison, Wis.).

(A-3) Measurement of dissociation constant (K _d ) For gel mobility shift assay, each transcription aptamer [(Apt) _1-3 ] was isolated by PAGE with 15% polyacrylamide denaturing gel, and alkaline phosphatase (E. coli A19; Takara Syuzo Co., Kyoto, Japan) was treated at 37 ° C. for 1 hour to dephosphorylate the 5 ′ end. Thereafter, T4 polynucleotide kinase was allowed to act at 37 ° C. for 1 hour, and the aptamer was labeled with ³² P and purified by PAGE (15% polyacrylamide). 40 nM tRNA was added to a fixed concentration of 5 ′ end labeled aptamer, and Tat binding buffer [10 mM Tris-HCl (pH 8.0), 70 mM NaCl, 2 mM EDTA, and 0.1% Nonidet P-40] Denature by treatment at 95 ° C. for 3 minutes, leave to regenerate at room temperature, followed by 10 μl total volume of DHFR- (RE) _1-3 in excess concentration range in Tat binding buffer And treated at 30 ° C. for over 1 hour. Free RNA, protein-bound RNA, or peptide-bound RNA is electrophoresed in 0.5 × TBE (Tris-Borate / EDTA) buffer using 5-15% non-denaturing polyacrylamide gels Were separated and analyzed.

(A-4) Construction of pDHFR- (RE) _3- (Apt) ₃ (wild type), pMuDHFR- (RE) _3- (Apt) ₃ (mutant), pDHFR- (RE) ₃ (control)
In order to introduce site-specific mutations in the core region of DHFR, four oligonucleotides (5'-ACA ATT CCC CTC TAG AAA TA-3 '(SEQ ID NO: 6), 5'-GTG GTG GTG CTC GAG AAT TC- 3 '(SEQ ID NO: 7), 5'-CCT GCC GCC CTC GCC TGG GCC AAA CGC AAC ACC TTA AAT AAA-3' (SEQ ID NO: 8), and 5'-GCG TTT GGC CCA GGC GAG GGC GGC AGG CAG GTT CCA CGG-3 ′ (SEQ ID NO: 9)) was prepared. Using these oligonucleotides and the megaprimer PCR mutagenesis method, pMuDHFR- (RE) ₃ was constructed by introducing two mutations into the DHFR gene. Next, the three aptamer sequences (Apt) ₃ of tandem were introduced into the Eco RI site downstream of the RE peptide in pDHFR- (RE) ₃ and pMuDHFR- (RE) ₃ , and pDHFR- (RE) ₃ − (Apt) ₃ (wild type) and pMuDHFR- (RE) _3- (Apt) ₃ (mutant) were obtained.

(A-5) Genotype-phenotype linkage by interaction of (Apt) ₃ and (RE) ₃
^In order to obtain capped (cap structure analog; New England Bio Labs, Beverly, MA) mRNA labeled with ³² P, the linear plasmid [pDHFR- (RE) _3- (Apt) ₃ (wild type), pMuDHFR -(RE) _3- (Apt) ₃ (mutant), pDHFR- (RE) ₃ (control)] using T7Ampliscribe ^TM kit (Epicentre Technologies, Madison, WI) for 2 hours at 37 ° C, Transcribed in vitro. Labeled mRNA was purified on a NICK ^™ column (Amersham Pharmacia Biotech, Uppsala, Sweden). 1 μg of capped mRNA encoding DHFR- (RE) ₃ (wild type) or MuDHFR- (RE) ₃ (mutant) was translated in 20 μl of rabbit reticulocyte lysate for 20 minutes at 30 ° C (Rabbit Reticulocyte Lysate System; Promega, Madison, WI). As a control, mRNA containing no aptamer was translated (control). Each protein was labeled with ³⁵ S. After translation, binding buffer [10 mM sodium phosphate buffer (pH 7.5), 70 mM NaCl, 10 μl tRNA] 180 μl, pretreated with succinic anhydride and MTX-agarose beads (Sigma, St Louis, MO) 20 μl of the MTX-agarose bead suspension in which the positive charge was neutralized was added to the cell lysate and allowed to bind for 30 minutes at 4 ° C. 200 μl of supernatant containing unbound mRNA and protein was removed from the mixture and MTX-beads were washed twice with 180 μl of binding buffer. Bound mRNA and protein were isolated from MTX-beads by eluting at elevated temperature (95 ° C.) with a solution containing SDS. Thereafter, each sample was subjected to SDS-PAGE on a 15% gel.

(A-6) In vitro selection of mRNA encoding streptavidin from a pool of a mixture of streptavidin mRNA and DHFR mRNA
The construction of pDHFR- (CQ) _3- (Apt) ₃ was performed according to the construction of pDHFR- (RE) _3- (Apt) ₃ . Briefly, a sense and antisense oligonucleotide encoding a CQ peptide (36 amino acids; CFTTK ALGIS YGRKK RRQRR RPPQG SQTBQ VSLSK Q) (SEQ ID NO: 10) was synthesized, and the resulting fragment containing the CQ peptide was DHFR sequence. And introduced between aptamer sequences. Template DNA encoding streptavidin has been reported previously. The DNA fragment containing the streptavidin gene was inserted upstream of the CQ peptide sequence by replacing the DHFR gene. Each ³² P-labeled RNA [Streptavidin- (CQ) _3- (Apt) ₃ and DHFR- (CQ) _3- (Apt) ₃ ] was prepared as described above. A combination of three mRNAs (mRNA mixed with a 1: 1 ratio of streptavidin or DHFR-encoding mRNA, or any mRNA as a control) (total amount of 1.25 μg in each combination) at 30 ° C. Translation was performed in cell lysate (25 μl) for 20 minutes. After translation, the cells were treated with 180 μl of binding buffer [20 mM sodium phosphate buffer (pH 8.0), 70 mM NaCl, 10 μl tRHA] and 20 μl of biotin-agarose beads (Sigma, St Louis, MO) suspension. Added to the lysate and allowed to bind on ice for 10 minutes. The supernatant (200 μl) containing unbound mRNA and protein was removed from the mixture and the biotin-beads were washed twice with 180 μl of binding buffer. Bound mRNA was isolated from biotin-beads by elution with a solution containing urea at high temperature (95 ° C.). Each sample was then subjected to 4% gel denaturing PAGE.

(A-7) Combination of strong interaction between ribosome display system and (RE) ₃ and (Apt) ₃ in selection of functional protein pET30a and pDHFR- (RE) ₃ containing DHFR gene (pDHFR) The-(Apt) ₃ plasmid was used as a template for preparing each ³² P-labeled mRNA [DHFR- (RE) _3- (Apt) _3, DHFR + Stop, and DHFR-Stop mRNA]. ^The mRNA labeled with ³² P was translated and subjected to selection with MTX-beads as described above. Bound mRNA was eluted at high temperature and isolated from MTX-beads. The relative amount of selected mRNA was detected by counting of ³² P-labeled mRNA and RT-PCR.

p (Apt) _3- (RE) ₃ -DHFR was constructed in the same manner as pDHFR- (RE) _3- (Apt) ₃ . Linear DNA used for transcription in vitro was prepared as a PCR product from each template plasmid containing the T7 promoter sequence [p (Apt) _3- (RE) ₃ -DHFR, and pDHFR]. mRNA was prepared by the method described above. Polyclonal IgG antibodies were prepared separately for each E. coli termination factor (E. coli RE1-RE3). The total amount of mRNA in the first pool was 2 μg. Then, in vitro translation was carried out at 37 ° C. for 10 minutes using an E. coli extract system for linear template (Promega, Madison, Wis.). In order to stop the translation reaction, the translation product was allowed to stand on ice for 1 minute and subjected to in vitro selection using MTX-beads as described above. The recovered mRNA was purified with RNeasy mini kit (QIAGEN, Hilden, Germany) and reverse-transcribed with MMLV reverse transcriptase (Promega). This reverse-transcribed sample, ie cDNA, was amplified by PCR (ExTaq; Takara) and analyzed by 2.0% agarose electrophoresis. To perform semi-quantitative analysis, agarose gels were stained with SYBR Green I (Molecular Probes) and analyzed with Fluoro Imager 595 (Molecular Dynamics). The strength of interaction between (RE) ₃ and (Apt) ₃ was estimated based on the relative amount of mRNA recovered as RT-PCR product.

B. Results and discussion
(B-1) Dihydrofolate reductase (DHFR) was selected as a functional protein model for enhancing the interaction between the tandem-linked aptamer and the Tat-derived peptide . The reason is that the properties are well analyzed (Blakley, RL et al. New York, 1985, pp.191-253. Taira, K. et al. J. Trends Biochem. Sci. 1987, 12, 275-278. Taira, K. et al. J. Med. Chem. 1988, 31, 129-137. Iwakura, M. et al. J. Biochem. 1995, 117, 480-488. Fujita, S. et al. Proc. Natl. Acad. Sci. USA 1997, 94, 391-396. Hamada, M. et al. J. Biochem. 1998, 123, 684-692). To examine the effect of several aptamers and Tat-derived peptide (RE) on binding affinity, three proteins [DHFR- (RE) _n ] (Fig. 1) and three tandem RNA aptamers [(Apt) _n ] (FIG. 2A) was prepared. “N” in this case is in the range of 1 to 3, and corresponds to the number of copies of each motif. The band after SDS-PAGE of the purified protein is shown in FIG. Each protein was confirmed to have DHFR activity as expected, demonstrating that binding of the RE peptide motif does not interfere with the correct folding of the DHFR domain. In order to maintain the proper secondary structure of each aptamer, Tat aptamers were tandemly linked to each other via six adenosine nucleotides to yield (Apt) ₂ and (Apt) ₃ . Purified RNA after PAGE using a non-denaturing gel is shown in the control lane (C) of FIG. 2A. The gel in FIG. 2A shows that the Tat aptamer does not interact with DHFR. This gel was mixed with wild-type DHFR at increasing concentrations (in each case, the concentration of DHFR was increased from lane C to the right, as indicated by the triangle above the gel). The mobility has not changed.

Aptamer (Apt) _1-3 gel mobility was altered when mixed with the fusion protein DHFR- (RE) _1-3 (FIGS. 2B-2D). In these experiments, ³² P-labeled aptamer (final concentration 30 pM) was mixed with an excess amount of each fusion protein, and the amount of complex formed was quantified with an image analyzer. Apparent K _d values were calculated to be less than 24 nM, 200 pM, and 16 pM with n = 1, n = 2, and n = 3, respectively. Among the combinations tested, the interaction between (Apt) ₃ and DHFR- (RE) ₃ was the strongest [K _{d (apt)} <16 pM]. This complex appeared to take multiple conformations, but stronger interactions were obtained when the number of both aptamers and RE peptides increased. Maintaining the stability of the RNA-protein complex, i.e. the extremely strong interaction between RNA and protein, is an important factor in the linkage between genotype and phenotype, so (Apt) ₃ and DHFR- (RE) _Three
The following experiment was carried out using the motif.

(B-2) Linkage between genotype and phenotype due to interaction between (Apt) ₃ and (RE) ₃
To see if (Apt) ₃ and DHFR- (RE) ₃ are available, two plasmids were prepared. The plasmid encodes wild-type and mutant DHFR- (RE) ₃ fusion proteins. The encoded mRNA and the protein to be translated are shown in FIG. 3A [wild-type, pDHFR- (RE) ₃ − (Apt) ₃ ; Mutant, pMuDHFR- (RE) _3- (Apt) ₃ ].

Each plasmid contains a sequence encoding DHFR with _three RE peptides [(RE) ₃ ] linked to tandem, and a sequence of three Tat aptamers (Apt) ₃ linked to tandem. The mutant protein derived from pMuDHFR- (RE) _3- (Apt) ₃ [MuDHFR- (RE) ₃ ] has two amino acid substitutions from Asp27 to Ala27 and Phe31 to Ala31 in the active site of DHFR. In that it differs from the wild-type protein [DHFR- (RE) ₃ ] derived from pDHFR- (RE) _3- (Apt) ₃ . These mutations result in loss of ability to bind to methotrexate (MTX) (Blakley, RL et al. New York, 1985, pp.191-253. Taira, K. et al. J. Trends Biochem. Sci. 1987, 12, 275- 278. Taira, K. et al. J. Med. Chem. 1988, 31, 129-137. Iwakura, M. et al. J. Biochem. 1995, 117, 480-488. Fujita, S. et al. Proc. Natl. Acad. Sci USA 1997, 94, 391-396. Hamada, M. et al. J. Biochem. 1998, 123, 684-692.). As a second control, a plasmid (pDHFR- (RE) ₃ ) encoding a wild type DHFR- (RE) ₃ fusion protein and not encoding an aptamer sequence was prepared. This translation product, DHFR- (RE) ₃ , cannot interact with the mRNA (FIG. 3A, right).

If the interaction between (Apt) _3- (RE) ₃ can be used to link the genotype and phenotype, the reaction solution is post-translationally only in the wild type system (FIG. 3A, left). When mixed with MTX-agarose beads, both protein (DHFR) and its mRNA should be bound to MTX-agarose beads and recovered. In contrast, in the mutant system (FIG. 3A, middle), the mutant DHFR is designed so that it cannot bind to MTX, so even when bound to MTX-agarose beads, the protein (DHFR) and its mRNA. None of them should be recovered. In addition, in the second control system (FIG. 3A, right), since there is no (Apt) ₃ aptamer, DHFR- (RE) ₃ cannot interact with the mRNA, and therefore when bound to MTX agarose beads. Only protein (DHFR) should be recovered and mRNA should not be recovered.

Three kinds of capped mRNAs labeled with ³² P were prepared from each plasmid using T7 polymerase, and these were used as templates and translated into each protein in a rabbit reticulocyte lysate. During translation, each protein was labeled with ³⁵ S-methionine. After translation, an appropriate binding buffer and MTX-agarose beads were added to each cell lysate, and the binding reaction was allowed to proceed for 30 minutes at 4 ° C. (In this case, RNase in the cell lysate is performed at low temperature. To reduce the degradation rate of mRNA). The supernatant of the binding solution containing unbound mRNA and protein was removed and the MTX-agarose beads were washed twice with binding buffer. Products eluted from the beads by heating (95 ° C.) in elution buffer were analyzed by SDS-PAGE. By examining each cell lysate after translation, it was confirmed that the translated protein and the amount of the input mRNA were consistent with each other (FIG. 3B, lanes 1 to 3).

  Analysis of the eluted product, as expected, showed that DHFR was detected in the wild type and control systems (FIG. 3B, lanes 4 and 6) and few mutant proteins were detected (FIG. 3B, lane 5). The recovery rates of wild-type DHFR and control DHFR were 9 and 16 times higher than that of mutant DHFR, respectively (FIG. 3C, left). Furthermore, since MTX beads are somewhat positively charged after binding to MTX, nonspecific binding of mRNA to MTX beads could not be completely prevented in this experiment (FIG. 3B). Lanes 5 and 6), mRNA was detected only in the wild type system (FIG. 3B, lane 4). The yield of wild-type mRNA was only 2.5 times higher than the amount of background (ie, nonspecifically bound mutant mRNA and control mRNA) (FIG. 3C, right panel). Quantification of the bands revealed that the amount of protein and mRNA was well above background levels. These results demonstrate the effectiveness of the interaction schematically shown in FIG. 3A.

(B-3) Selection of mRNA bound to streptavidin in vitro In selection of target protein, if (Apt) ₃ and DHFR- (RE) ₃ function properly, two distinct mRNAs can be identified. When translated simultaneously, each protein should bind specifically to its mRNA. Since MTX agarose beads are positively charged and non-specific binding of irrelevant mRNA cannot be completely eliminated (FIGS. 3B and 3C), biotin-agarose beads were tried instead. As a positive control, a new plasmid pStreptavidin- (CQ) _3- (Apt) ₃ was constructed by replacing the DHFR gene in the pDHFR- (CQ) _3- (Apt) ₃ plasmid with the streptavidin gene (RE and Both CQ are Tat-derived peptides and have the same function) (Yamamoto, R. et al. Genes Cells 2000, 5, 371-388.). In this system (Fig. _{4), pStreptavidin- (CQ) 3} - (Apt) 3 and _{pDHFR- (CQ) 3 - (Apt} ) when performed with the first a mixture of mRNA derived from _3, rather than DHFR, Translated streptavidin should bind to biotin-agarose beads (Green, NM et al. Adv. Protein Chem. 1975, 29, 85-133).

From pStreptavidin- (CQ) _3- (Apt) ₃ and pDHFR- (CQ) _3- (Apt) ₃ , two types of capped mRNAs labeled with ³² P were prepared using T7 polymerase. The transcribed mRNA is translated separately in the cell lysate (FIG. 4B, lanes 1 and 2), or mixed at a 1: 1 ratio (FIG. 4B, lane 3). Selected with biotin-agarose beads. This step is basically the same as the step used in the experiment shown in FIG. 3. After translation, an appropriate binding buffer and biotin-agarose beads are added to each cell lysate and bound at 4 ° C. for 10 minutes. The reaction was allowed to proceed. Thereafter, the supernatant of the binding solution containing unbound mRNA and protein was removed, and the biotin-agarose beads were washed twice with a binding buffer. The bound product was eluted from the beads with elution buffer at elevated temperature (95 ° C.) and analyzed by 4% denaturing PAGE to identify selected mRNA (FIG. 4B).

When each mRNA bound to the biotin beads was quantified, the yield of the mRNA encoding the bound streptavidin (Streptavidin- (CQ) _3- (Apt) ₃ mRNA) was determined as the mRNA encoding DHFR (DHFR- (CQ) _3- (Apt) ₃ mRNA; three times higher than the yield of lanes 1 and 2). When mRNA bound to biotin beads was isolated from the group in which each mRNA was mixed at 1: 1, pStreptavidin- (CQ) _3- (Apt) ₃ mRNA was selected (lane 3). This result demonstrates that the strong RNA-protein interaction is effective in selecting functional proteins.

(B-4) Combination of strong interaction between ribosome display system and (RE) ₃ and (Apt) ₃ in selection of functional protein
Since functional streptavidin could be selected using strong interaction between RNA and protein (FIG. 4), it was examined whether it could be further improved by combining with a ribosome display system. In the ribosome display system, protein release from the complex is delayed due to deletion of the stop codon (Hanes, J. et al. Proc. Natl. Acad. Sci. USA 1997, 94, 4937-4942. He, M. et al. Nuclelic Acids Res. 1997, 25, 5132-5134.), Ribosomes form relatively stable complexes with translated proteins and their respective mRNAs. In order to examine the effect of deletion of the stop codon in the system of the present invention, DHFR mRNA lacking the stop codon (DHFR-Stop mRNA, right figure in FIG. 5A) was prepared. As a control, DHFR mRNA having a stop codon (DHFR + Stop mRNA, middle diagram in FIG. 5A) was used.

After translation and selection in the cell lysate, the amount of mRNA bound to the MTX-beads is determined by 3 types of mRNA (ie, DHFR- (RE) _3- (Apt) ₃ , DHFR + Stop, and DHFR-Stop). Compared. During translation, DHFR- (RE) _3- (Apt) ₃ mRNA having a stop codon binds to the translated DHFR protein by the interaction between (RE) ₃ and (Apt) ₃ . In addition, since there is a stop codon, the ribosome is released from the mRNA, so that the ribosome is not involved in this protein-mRNA complex. These three mRNAs were translated in rabbit reticulocyte cell lysate and exposed to MTX-agarose beads under the same conditions as described in FIG. The relative amount of mRNA selected was detected from the RT-PCR product labeled with ³² P and the count of the original bound mRNA (FIGS. 5B and 5C). Since PCR product analysis (FIG. 5B) was performed only once, the quantification of mRNA levels shown in FIG. 5C is more reliable. The results shown in FIG. 5C are an average of 4 independent experiments. On average, the selected DHFR- (RE) _3- (Apt) ₃ mRNA (FIG. 5C, lane 1) is 2.5 times higher than the background level of DHFR + Stop mRNA (FIG. 5C, lane 2). On the other hand, the concentration of DHFR-Stop mRNA was 1.4 times (FIG. 5C, lane 3). Therefore, in this system, the (RE) _3- (Apt) ₃ interaction was more effective than the deletion of the stop codon in the stability of the complex between each mRNA and its product.

  The ribosome display method works quite effectively when functional antibodies are selected for each antigen (Hanes, J. et al. Nat. Biotechnol. 2000, 18, 1287-1292.). However, the present results (FIG. 5C) suggest that the stability of the ribosome-mRNA-protein complex formed by deletion of the stop codon is not sufficient for the selection of common proteins such as DHFR. Selecting a suitable antibody in a ribosome display system in a short period of time may require a fairly strong interaction between the antibody and its antigen.

Combining the (RE) _3- (Apt) ₃ interaction with the ribosome display system is advantageous for improving the stability of the complex of each mRNA and its product. For this purpose, another plasmid was prepared in which the (Apt) _3- (RE) ₃ region is located upstream of the DHFR gene (FIG. 6). Furthermore, in order to further stabilize the complex by deleting the stop codon, polyclonal IgG antibodies against E. coli RF1, RF2 and RF3 were prepared (Moffat, JG et al. Biochimie 1991, 73, 1113-1120. ). 'In order to avoid coming to the _{end, (Apt) 3 - (RE} ) ₃ region 5' (RE) stop codon in the ₃ regions 3 - (Apt) ₃ since the set terminal, in the present invention, each Translation of mRNA was performed in E. coli S30 extract by placing an SD sequence between (Apt) ₃ and (RE) ₃ .

The amount of mRNA bound to MTX-beads in the presence of a polyclonal IgG antibody against the termination factor (lanes 1 and 3 in FIG. 6) and in the absence (lanes 2 and 4 in FIG. 6) That is, DHFR-Stop mRNA (lanes 1 and 2 in FIG. 6) and (Apt) _3- (RE) ₃ DHFR-Stop mRNA (lanes 3 and 4 in FIG. 6) were compared. The translated mRNA was subjected to MTX-agarose-bead selection under the same conditions as described in FIG. The relative amount of selected mRNA was detected by RT-PCR (FIGS. 6B and 6C). The results shown in FIG. 6C are the average of two experiments performed independently. These results indicate that the combination of the (RE) _3- (Apt) ₃ interaction and the ribosome display system is effective in stabilizing the mRNA-ribosome complex, especially in the presence of polyclonal IgG antibodies against the termination factor. Demonstrate.

(B-5) Possibility of existence of RNA-protein network Based on the above experimental results, strong interaction between aptamer / peptide stabilizes ribosome display complex, and this kind of interaction contributes to selection of functional protein. It proved useful. However, from the results of the in vitro binding assay (FIG. 2), it is expected that the efficiency of protein selection is not very high. That is, the formation of an RNA-protein network as shown in the upper diagram of FIG. 7A may make it difficult to select proteins from the library. This problem can be overcome by using different combinations of aptamers and peptides as shown in the lower diagram of FIG. 7A.

[Example 2]
A. Materials and methods
(A-1) Construction of dihydrofolate reductase (DHFR), glutathione-5-transferase (GST), and streptavidin (StAv) expression vectors fused with Ricin-A chain
A DHFR gene derived from PTZDHFR20 was inserted into the multi-cloning site of pET30a to construct pDHFR. In addition, geneIII derived from pCANTABE5 (Pharmacia) (for geneIII sequences, in vitro selection and evolution of functional proteins by using ribosome display. Proc Natl Acad Sci US A. 1997 May 13; 94 (10): 4937-42) Two sequences encoding amino acids 1 to 404 (long spacer) and amino acids (short spacer) including positions 195 to 315 are amplified with the following primers, cleaved with EcoT22I and PstI, and DHFR gene It was inserted into the Pst I site downstream of. The primer used was long spacer up primer: 5'-GTG GCG ATG CAT CGA CTG TTG AAA GTT GTT TAG CAA AAC -3 '(SEQ ID NO: 11), long spacer down primer: 5'-TTC TTA CTG CAG AGA CTC CTT ATT ACG CAG TAT GTTAG-3 '(SEQ ID NO: 12), short spacer up primer: 5'-GTG GCG ATG CAT CGG ATC CAT TCG TTT GTG AAT ATCAAG-3' (SEQ ID NO: 13), short spacer down primer: 5'- TTC TTA CTG CAG ACC AGT AGC ACC ATT ACC ATT AGC AA-3 ′ (SEQ ID NO: 14). Next, a sequence encoding glycine and serine at positions 212-258 of the geneIII sequence as a linker was amplified with the following primers, cleaved with ScaI and EcoRV, and inserted into the EcoRV site between the DHFR gene and spacer. A new NheI site was inserted upstream of this linker, and an NcoI site was inserted downstream. Up primer: 5'-ACT CGG AGT ACT TGC TAG CCC TGT CAA TGC TGG CGG CG-3 '(SEQ ID NO: 15), down primer: 5'-GCT ACG GAT ATC CCG CTT GTA ACA GGA GTG CTC CAT GGA TAA TCA AAA TCA CCG GAA CCG-3 ′ (SEQ ID NO: 16). Next, a sequence encoding ricin A chain derived from pUTA (Texas Univ. Prof. Rpbertus J.) was amplified with the following primers, cleaved with NcoI and EcoRV, and inserted into NcoI and EcoRV between DHFR gene and spacer. . Up primer: 5'-CGC GCG CCA TGG ATG TTT CCC AAA CAA TAC CCA AT-3 '(SEQ ID NO: 17), down primer: 5'-GGG GCG GAT ATC CCA AAC TGT GAG CTC GGT GGA GGC GCG -3 ′ (SEQ ID NO: 18). Mutations that inactivate lysine (Glu-177, Arg-180, Glu-208 added to Gln-177, His-180, Asp-208) were also inserted in the same manner (pDLRS, pDLRL). Finally, in order to replace DHFR with GST and StAv, the GST gene derived from pGEX-4t-3 (Pharmacia) and the Streptavidin gene derived from pGSH were amplified, cleaved at the XbaI and NheI sites, and transferred to the XbaI and NheI sites of the plasmid. Inserted (pGLRS, pGLmRS, pSLRS, pSLRL, pSLmRS). The primer used was for GST, up primer: 5'-GGC CGG TCT AGA AAT AAT TTT GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG TCC CCT ATA CTA GGT TAT TG (SEQ ID NO: 19), down primer: 5'- CGG TGG GCT AGC CAG ATC CGA TTT TGG AGG ATG GTC GC (SEQ ID NO: 20), for StAv, up primer: 5'-GGC CGG TCT AGA AAT AAT TTT GTT TAA CTT TAA GAA GGA GAT ATA CAT ATG CAC CAT CAT CAT CAT CAT TC-3 ′ (SEQ ID NO: 21), down primer: 5′-CGG TGG GCT AGC CCG CCG CTC CAG AAT CTC AAA GCA-3 ′ (SEQ ID NO: 22).

  The DNA sequence of pSLRL (Streptavidin-Linker-Ricin-GeneIII (long)) is shown in SEQ ID NO: 1 in the sequence listing, and the DNA sequence of pSLRS (Streptavidin-Linker-Ricin-GeneIII (Short)) is shown in SEQ ID NO: 2 in the sequence listing. Show.

The structure of the base sequence of SEQ ID NO: 1 is as follows.
Translation Position; 5076-8155
T7 promoter; 4987-5004
lac operator ； 5007-5030
SD sequence; 5061-5067
Steptacidin sequence; 5121-5600
Linker; 5601-5768
Ricin A chain; 5769-6657
Spacer (containing GeneIII (L) sequences); 6573-7856

The structure of the base sequence of SEQ ID NO: 2 is as follows.
Translation Position; 5076-7007
T7 promoter; 4987-5004
lac operator ； 5007-5030
SD sequence; 5061-5067
Steptacidin sequence; 5121-5600
Linker; 5601-5768
Ricin A chain ； 5769-6657
Spacer (containing GeneIII (S) sequences); 6573-7007

(A-2) mRNA binding and selection with Biotin affinity column and Glutathione affinity column Each plasmid (pDLRS, pDLRL, pSLRS, pSLRL, pSLmRS, pGLRS, pGLmRS) was cleaved with XhoI and then AmpliScribe T7 in the presence of Cap Analog. Transcription Kit (Epicentre Technologies) was used for transcription from T7 promoter. In some cases it was isotope labeled with α ³² P-CTP. These mRNAs were translated using the Rabit Reticulocyte Lysate System (Promega). The reaction translated 1 μg of RNA on a 25 μl scale. The composition of the solution followed the attached protocol. Incubate at 30 ° C for 10 minutes, add 20 μl of reaction solution to Sample Buffer (if streptavidin is selected, 50 mM Potassium phoshate buffer (pH 7.9), 300 mM NaCl, 0.05% Tween 20, 2% Block Ace (Yukizirushi); GST In this case, 80 μl of pH 7.4, 10 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ , 2.7 mM KCl, 140 mM NaCl and 1% Triton X-100) is added, and Biotin- 10 μl of Agarose (Sigma) was added. In some cases, the amount of RNA bound after the binding and washing steps was quantified by isotopic counting. After 10 minutes of incubation at room temperature, the supernatant was removed and washed 3 times with 200 μl of Sample Buffer. Then, 100 μl of Elution Buffer (50 mM Potassium phoshate buffer (pH 7.9), 300 mM NaCl, 0.05% Tween20, 2% Block Ace, 3M Guanisine, 50 mM) is used to recover the bound RNA. EDTA; for GST selection, add pH 7.4, 10 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ , 2.7 mM KCl, 140 mM NaCl and 1% Triton X-100, 3 M guanidine and 250 mM EDTA) The mixture was stirred vigorously for 10 minutes and centrifuged to collect the supernatant. This operation was repeated twice. The recovered RNA was purified by RNeasy minin kit (QIAGEN). Purified RNA was reverse transcribed with 5 μM down primer according to the protocol of Reverse Transcription System (Promega), and PCR was performed with Ex Taq polymerase (TAKARA). Up and down primers were added at 1 μM. The primers used were up primer: 5′-CTT TAA GAA GGA GAT ATA CAT ATG-3 ′ (SEQ ID NO: 23), down primer: 5′-CTC CTC CGG TCG ACG ATA TC-3 ′ (SEQ ID NO: 24).

B. Results and discussion
(B-1) Construction of dihydrofolate reductase (DHFR), glutathione-5-transferase (GST) and streptavidin (StAv) expression vectors fused with ricin A chain
DHFR, GST, and StAv proteins are well-studied proteins. DHFR was inserted for use as a negative control for the binding of StAv and Biotin. Linker was introduced to prevent folding of DHFR, GST, StAv and lysine due to steric hindrance. This sequence is encoded on the GeneIII gene and encodes a large amount of glycine and serine. Lysine introduced only the A chain, a domain that inactivates ribosomes. Mutant lysine completely loses the activity of hydrolyzing the N-glucoside bond at the α-salicine site present in 28S rRNA. A sequence inserted by the GeneIII gene was inserted as Spacer downstream of lysine. This is so that lysine can attack the ribosome with cis before the translation of the protein by the ribosome is completed. A plasmid was constructed in which two types of spacers, long and short, were inserted (FIG. 9A).

(B-2) Translation inhibition by ricin A chain In order to confirm whether ricin A chain has activity, inhibits ribosome translation activity, and can stop ribosome, the plasmids constructed above (pSLmRS, pSLRS , pSLmRL, pSLRL) were used as templates for transcription and translation. First, it was cleaved at the XhoI site downstream of the Spacer sequence of the plasmid to form a linear dsDNA, and the mRNA was transcribed using T7 polymerase. A cap was introduced at the 5 'end for translation in Lysate. Each of the four mRNAs was translated under appropriate conditions, and the resulting protein was detected by SDS-PAGE (B and C in FIG. 9). As a result, the expression level of the protein fused with inactive lysine with mutations in the ricin sequence was higher than the expression level of the protein fused with wild-type lysine (SLmRS, SLmRL). This is thought to be due to the fact that lysine inhibited the ribosome, resulting in a reduced amount of protein translation. Therefore, it was indirectly confirmed that the ricin fusion protein constructed by the present inventor has an activity of stopping the ribosome. It was also found that the expression of the protein decreased as the Spacer sequence lengthened.

(B-3) mRNA binding by Biotin affinity column
pDLRS, pSLRS, and pSLmRS were cleaved at the XhoI site downstream of the Spacer sequence in the same manner as described above to form linear dsDNA, and mRNA was transcribed using T7 polymerase. A cap was introduced at the 5 ′ end so that it could be translated in lysate, and body-labeled with α-CTP for RNA quantification. Each of the three types of mRNA was translated under appropriate conditions and bound to Biotin-Agarose. Biotin-Agarose was washed several times to remove unbound mRNA. In the binding and washing steps, mRNA bound to Biotin-Agarose was quantified. Biotin has a high affinity with StAv.

  When DLRS mRNA is translated, ricin stops the ribosome and a protein-mRNA complex is formed, but DHFR does not bind to Biotin, so it is thought that the mRNA hardly binds to Biotin-Agarose (FIG. 10B). left). On the other hand, in SLRS mRNA, StAv is expected to bind to Biotin-Agarose and mRNA is recovered (middle of FIG. 10B). Since SLmRS mRNA has a non-active lysine inserted, StAv binds to Biotin-Agarose, but the mRNA and protein are complex because a stop codon is inserted at the end of the Open Reading Frame (ORF) on the mRNA. It is thought that it becomes difficult to form a body (right of FIG. 10B). If only SLRS mRNA is selectively recovered as described above, the system of the present invention is considered very effective for protein selection.

  When washing was repeated and mRNA that nonspecifically bound to agarose was removed, SLRS mRNA was selectively bound (FIG. 10C). 30% of SLRS mRNA was bound to biotin agarose and recovered. This is 2000 times the efficiency of 0.015% of the recovery rate of mRNA by ribosome display, which is conventionally performed, and it is considered that there is a great advantage for ensuring the diversity of the random pool. On the other hand, DLRS mRNA and SLmRS mRNA bound only 0.3% or less. Furthermore, when lysine is inactivated, the mRNA recovery efficiency falls to the same level as in the case of DLRS mRNA. Therefore, it is considered that ricin stops the ribosome and stabilizes the protein-mRNA complex. In addition, since Lysate is used, there may be less RNA degradation compared to E. coli. Moreover, SLRS mRNA is concentrated 100 times or more with respect to DLRS mRNA and SLmRS mRNA by three washings (FIG. 10C). This is equivalent to the concentration efficiency of ribosome display. That is, it is suggested that the system of the present invention is a system capable of selecting proteins with an efficiency equal to or higher than that of the ribosome display method.

  Furthermore, different selection models were also verified. Using the GLRS mRNA, SLRS mRNA, and SLmRS mRNA obtained by transcription from the plasmids constructed above (pGLRS, pSLRS, pSLmRS), the GLRS mRNA, SLRS mRNA, and SLmRS mRNA were translated according to the same procedure as described above, and the products were respectively bound to Biotin-Agarose. (FIG. 11). If the protein selection system is working correctly, only SLRS mRNA is thought to bind to Biotin-Agarose (middle of FIG. 11A). Further, SLRS mRNA, GLRS mRNA, and GLmRS mRNA obtained by transcription from (pSLRS, pGLRS, pGLmRS) were translated according to the same method as described above, and the products were respectively bound to Glutathione-Sepharose (FIG. 12). ). In this case, only GLRS mRNA is considered to bind to Glutathione-Sepharose (middle of FIG. 12A). When washing is repeated and non-specifically bound mRNA is removed, it can be seen that SLRS mRNA and GLRS mRNA are selectively bound to each other (FIGS. 11B and 12B). In each case, mRNA recovery was 5.1% (Biotin-Agarose) and 1.3% (Glutathione-Sepharose). Concentration efficiency was> 25 times and> 6 times, respectively. Therefore, these results also suggest that the system of the present invention can select proteins with an efficiency equal to or higher than that of the ribosome display method, and the possibility that it can be applied to various proteins.

(B-4) Protein selection by Biotin affinity column
As described above, four types of mRNA (SLRL mRNA, DLRL mRNA, SLRS mRNA, DLRS mRNA) were transcribed from four types of plasmids (pSLRL, pDLRL, pSLRS, pDLRS). In SLRL mRNA and DLRL mRNA, a longer spacer was inserted downstream of lysine. Each of these mRNAs was translated under appropriate conditions and bound to Biotin-bound agarose (A in FIG. 13). In addition, SLRL mRNA and DLRL mRNA, and SLRS mRNA and DLRS mRNA were mixed in an amount of 1: 1 (0.5 μg each), and these were similarly translated under appropriate conditions and bound to agarose bound to Biotin. After washing the agarose three times, mRNA was separated with Elution Buffer. This mRNA was purified and amplified by reverse transcription and PCR. As a result, cDNA encoding StAv was selectively amplified by 15 cycles of PCR (B in FIG. 13). The cDNA encoding DHFR was not amplified even after 25 cycles of PCR. In addition, even when mixed at 1: 1, the cDNA encoding StAv was selectively amplified. That is, it is suggested that the StAv protein is normally selected and the gene is recovered in the system of the present invention. It also indicates that the genotype mRNA corresponding to the protein and phenotype are correctly bound. Interestingly, SLRS mRNA binds to agarose more efficiently than SLRL mRNA.

  As a control, a certain amount (0.025 μg) of each mRNA was reverse transcribed and PCR was performed. In addition, SLRL mRNA and DLRL mRNA, SLRS mRNA and DLRS mRNA were mixed in an amount of 1: 1 (each 0.0125 μg), and reverse transcription and PCR were performed in the same manner. This confirmed that the amplification of the cDNA was concentration dependent. In addition, when PCR was performed without reverse transcription of mRNA, cDNA was not amplified, and it was confirmed that there was no DNA contamination.

  In addition, according to the same selection method, a pool in which GLRS mRNA and SLRS mRNA were mixed in an amount of 1: 1 was translated under appropriate conditions and combined with Biotin-Agarose or Glutathione-Sepharose. SLRS mRNA is selected when bound to Biotin-Agarose (FIG. 14A), and GLRS mRNA is considered to be selected when bound to Glutathione-Sepharose (FIG. 14B). As controls, SLRS mRNA, SLmRS mRNA, and GLRS mRNA were each transcribed and bound to Biotin-Agarose (FIG. 14A), and GLRS mRNA, GLmRS mRNA, and SLRS mRNA were each transcribed and bound to Glutathione-Sepharose (FIG. 14A). 14B). After washing the agarose three times, the mRNA was separated with Elution Buffer. This mRNA was purified and amplified by reverse transcription and PCR. As a result, when the translation was performed using a pool of GLRS mRNA and SLRS mRNA mixed at 1: 1 and bound to Biotin-Agarose, the cDNA encoding StAv was selectively amplified by PCR and bound to Glutathione-Sepharose. The cDNA encoding GST was selectively amplified by PCR. Therefore, this result also showed that the genotype mRNA corresponding to the protein and phenotype was correctly bound.

[Example 3]
In order to assess the general applicability of the ribosome display system, we will examine the stability of the ribosome-mRNA complex in the presence and absence of stop codons and even within the ribosome-mRNA complex. It was investigated in the presence and absence of additional interactions between the translated peptide and its mRNA.

The additional interaction utilized by the inventor was the MS2 coat protein (MSp) dimer fused in tandem with the corresponding RNA sequence of the specific binding motif (named C variant (or Cv)) Is the interaction between.
Specifically, the construct shown in FIG. 16 was transcribed and translated in vitro, and the complex formed thereby was selected using Ni-NTA agarose.
As a result, the efficiency of the system was improved by introducing additional mMSp-Cv interaction (FIG. 18), the recovery of mRNA after selection was 0.46% after 3 repeated washes, There was a clear advantage over the recovery rate of 0.24% without -Cv interaction. The inventor defined selectivity as the ratio of the amount of mRNA encoding a histidine tag to the amount of mRNA not encoding a histidine tag, as assessed by quantitative RT-PCR, but the calculated selectivity was 3 Increased from 6 (without Cv) to 6 (with Cv). Thus, the introduction of the mmSp-Cv interaction improved not only the yield of the target mRNA but also the selectivity of the polysome display system.

  Selection of linkers for Tat aptamers and Tat-derived peptides (RE and CQ) demonstrates the possibility that extremely strong interactions between RNA and proteins can be used for genotype-phenotype binding by avoiding network structures It was done. By using such interactions, strong RNA-protein interactions can be selected and manipulated. In addition, protein-mRNA complexes with longer half-lives can be generated, making selection much easier. Other approaches, such as the ribosome display system, are difficult to change the natural half-life of the ribosome-mRNA complex.

Furthermore, the techniques of the present invention can be combined with a ribosome display system simply by deleting the stop codon from the fusion gene. As a result, as shown in lane 3 of FIG. 6, a significantly more stable protein-mRNA complex is obtained as a result of binding at two sites.
Also, the selection method of the present invention has the advantage that the operation is simple and straightforward.

  By connecting the ricin gene downstream of the StAv gene, it was demonstrated by the present invention that the ribosome is stopped by lysine and genotype and phenotype are efficiently bound. As a result, the target mRNA can be selected with extremely high efficiency. Two types of spacer sequences, long and short, were constructed downstream of the ricin gene. It was found that mRNA can be recovered more efficiently by inserting a short spacer sequence. In the case of SLRS mRNA, the recovery efficiency was 30%, which was as high as before. This selection system also showed that SLRS mRNA was concentrated more than 100 times with respect to DLRS mRNA.

  By using the above-described system of the present invention, it is possible to efficiently screen a novel functional protein from a random DNA pool.

FIG. 1 is a schematic diagram showing purified DHFR- (RE) _1-3 fusion protein and wild type (wt) DHFR and a photograph of the gel after SDS-PAGE. The purified recombinant protein (about 100 μg) was subjected to SDS-PAGE and stained with Coomassie blue for visualization. Lane 1, marker; Lane 2, wild-type DHFR; Lane 3, DHFR- (RE) ₁ ; Lane 4, DHFR- (RE) ₂ ; Lane 5, DHFR- (RE) ₃ The molecular weight of the marker protein is shown on the left side. A polyglycine linker was inserted between DHFR and the first RE peptide and between the RE peptide adjacent to RE. Polyhistidine (His) was ligated to the N-terminus of DHFR for purification. FIG. 2 is a diagram and a photograph showing the results of measurement of the dissociation constant (K _d ). Each lane C was loaded with aptamer only. Black arrows indicate protein-RNA complexes, and white arrows indicate free RNA. The relative amount of complex and free RNA was calculated from the radioactivity of ³² P. The equilibrium dissociation constant was determined from a theoretical curve obtained by the method of least squares from the concentration of free RNA or complex RNA with respect to the peptide concentration. Diamonds indicate the relative amount of free RNA and squares indicate the relative amount of complex. In each panel, elongated black triangles indicate an increase in protein concentration. (A) A photograph showing the lack of interaction between (Apt) _1-3 and wild-type DHFR. Each lane C was loaded with 1 nM (Apt) _1-3 and added with 20, 100, 500 nM wild-type DHFR as indicated by the elongated triangles. (B) A diagram and a photograph showing the binding of (Apt) ₁ and DHFR- (RE) ₁ . 30 pM (Apt) ₁ (lane C) was mixed with 0.25, 0.5, ₁ , 2, 4, 8, 16, 32 nM DHFR- (RE) ₁ as indicated by elongated triangles. (C and D) Figures and photographs showing the binding of (Apt) _2-3 and DHFR- (RE) _2-3 protein. Mix 30 pM (Apt) ₂ or (Apt) ₃ RNA (lane C) with 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16 nM protein as shown by the elongated triangles did. FIG. 3 is a diagram and a photograph showing a model of functional protein selection using the interaction between (Apt) ₃ and (RE) ₃ in vitro. (A) Schematic diagram of the interaction between transcribed RNA, translated protein, and methotrexate-bound agarose beads (MTX ligand). (B) A photograph of the results of SDS-PAGE showing the concentration of DHFR- (RE) ₃ protein and mRNA (wild type) by MTX-beads. Each sample was subjected to SDS-PAGE of 15% gel. Lanes 1 to 3, after each translation reaction, 1 μl of cell lysate was loaded. Lanes 4-6, 10 μl of eluate containing mRNA and protein bound to MTX-beads was loaded. Lanes 1 and 4 show wild type protein; lanes 2 and 5 show mutant protein; and lanes 3 and 6 were loaded with control samples. (C) It is a figure which shows the quantification result of the protein and mRNA which were shown to (B). FIG. 4 is a diagram and photographs showing in vitro selection of mRNA encoding streptavidin from a pool of mixtures of streptavidin and DHFR mRNA. (A) Schematic diagram of the interaction between transcribed RNA, translated protein, and biotin-conjugated agarose beads (biotin ligand). (B) A photograph of the result of denaturing PAGE showing the concentration of streptavidin- (RE) _3- (Apt) ₃ mRNA by biotin agarose beads. After translation and subsequent selection, each sample was subjected to denaturing PAGE on a 4% gel. Lane 1; isolated streptavidin- (CQ) _3- (Apt) ₃ mRNA. MRNA (1.25 μg) encoding streptavidin was translated in cell lysate and selected with biotin beads. Lane 2; isolated DHFR- (CQ) _3- (Apt) ₃ mRNA. MRNA encoding DHFR (1.25 μg) was translated in cell lysate and selected with biotin beads. Since DHFR is not expected to interact with biotin beads, the level of bound DHFR- (CQ) _3- (Apt) ₃ mRNA indicates a level of non-specific (background) interaction. Lane 3; isolated streptavidin- (CQ) _3- (Apt) ₃ and DHFR mRNA (upper band; DHFR mRNA, lower band; streptavidin). A mixture of mRNA encoding each of streptavidin or DHFR at a ratio of 1: 1 was translated in a lysate and selected with biotin beads. FIG. 5 is a diagram and a photograph showing a comparison of the strength of interaction between (RE) _3- (Apt) ₃ and a ribosome display system in the selection of functional DHFR in a rabbit reticulocyte lysate. (A) Schematic diagram of the interaction between transcribed RNA, translated protein, and methotrexate-bound agarose beads (MTX ligand). (B) Shows the results of electrophoresis of RT-PCR products with 2% agarose to show concentration of DHFR- (RE) _3- (Apt) ₃ , DHFR-Stop or DHFR + Stop mRNA on MTX-beads It is a photograph. The PCR reaction was performed for 20 cycles. Each sample was subjected to a 2% Sea Kem GTG agarose gel (FMC, Riceland, Maine USA). Lane 1, PCR product from DHFR- (RE) _3- (Apt) ₃ mRNA; Lane 2, PCR product from DHFR + Stop mRNA; Lane 3, PCR product from DHFR-Stop mRNA. The PCR product was analyzed once, but the mRNA level quantification shown in FIG. 5C is more reliable. (C) shows the results of quantification of concentrated mRNA. The relative amount of selected mRNA was detected by counting ³² P-labeled mRNA. Each lane number corresponds to the lane number in FIG. 5B. Results are the average of 4 experiments performed independently. FIG. 6 is a graph showing an increase in stability of a complex of each mRNA and its product by combining the interaction of (RE) _3- (Apt) ₃ and a ribosome display system in an E. coli S30 strain extract. And photos. (A) Schematic diagram of the interaction between transcribed RNA, translated protein, and methotrexate-bound agarose beads (MTX ligand). (B) DHFR-Stop mRNA (lane 1) in the presence (lane 1 and lane 3) or absence (lane 2 and lane 4) of polyclonal IgG antibodies against termination factors (E. coli RF1, RF2, and RF3). And lanes 2) and (Apt) _3- (RE) ₃ -DHFR-Stop mRNA (lanes 3 and 4) were electrophoresed with 2% agarose to show enrichment with MTX beads. It is a photograph which shows the result. The selected mRNA was subjected to MTX-agarose beads under the same conditions as described in FIG. (C) It is a figure which shows the result of quantification of RT-PCR product shown to (B). Results are the average of two experiments performed independently. FIG. 7A shows the possibility of networking of RNA-protein bonds. In principle, Tat (RE) peptide and aptamer bound to tandem bind not only within a molecule but also between molecules. When binding between molecules, RNA-protein binding is networked. Rather than linking the same peptide and aptamer in tandem, this intermolecular interaction can be minimized by linking different peptides and aptamers. FIG. 7B is a schematic diagram of the first embodiment of the method for selecting a functional protein in vitro. Binding proteins are produced and enriched by repeating the steps of transcription, translation, affinity selection, and PCR. In the translation process, tandem aptamer (Apt) ₃ binds with tandem RE peptide (RE) ₃ with very high affinity. This binding binds the protein and its mRNA. In Example 1, DHFR and streptavidin were used as targets for the selection step, but any protein can be used in combination with tandem aptamer (Apt) ₃ and tandem RE peptide (RE) ₃ . FIG. 8 is a diagram showing a model of an in vitro protein selection system using lysine. Prepare a random DNA library or cDNA library. A lysine sequence and a spacer sequence are inserted on the 3 'end side of this sequence. When transcription / translation is performed, lysine is translated following the library protein, and this ricin inactivates the ribosome by an intramolecular reaction and stops the translation of the ribosome. As a result, an RNA-ribosome-protein complex is formed. Since the RNA of this complex encodes the genetic information of a protein, when a specific functional protein is selected, the RNA having the genetic information of that protein can be recovered together. This RNA can be amplified by reverse transcription and PCR, and the genetic sequence can be determined by reading the genetic information. Also, the above cycle can be repeated to apply more selective pressure. FIG. 9A is a diagram showing a DNA sequence used in a model system of a protein selection system. 5'-side T7 promoter, streptavidin or GST sequence, linker sequence, ricin A chain sequence or inactive ricin A chain sequence with a part of ricin A chain sequence, spacer derived from M13 phage Gene III gene Genes are inserted in the order of the sequence (360 bp or 1212 bp). Streptavidin and GST sequences are proteins to be selected. The linker sequence encoded a peptide linker and was inserted to eliminate steric hindrance of streptavidin and GST and ricin A chain. The spacer sequence was inserted to prevent the ribosome from inhibiting the folding and activity of other proteins. FIG. 9B is a schematic diagram showing translation of SLmRS, SLRS, SLmRL, and SLRL genes. Since lysine does not function in the SLmRS and SLmRL genes, an RNA-ribosome-protein complex is not formed. Therefore, the protein is dissociated from the ribosome, and the protein is efficiently created. FIG. 9C is a photograph showing proteins translated from SLmRS, SLRS, SLmRL, and SLRL genes. The protein was labeled with S35-methionine and detected with 10% SDS-PAGE. A shorter linker produced more protein. More proteins were created when lysine was inactivated. Almost no protein was detected from the SLRL gene. FIG. 10A is a schematic diagram showing selection of mRNA (SLRS) encoding streptavidin protein by biotin agarose. On the other hand, mRNA encoding DHFR does not bind to biotin agarose (DLRS). Even when lysine is inactivated, mRNA encoding streptavidin protein does not bind to biotin agarose (SLmRS). FIG. 10B is a diagram showing the recovery amount of mRNA encoding streptavidin protein by biotin agarose. After translation, after binding to biotin agarose, unbound mRNA was washed and the amount of remaining mRNA was measured. After SLRS mRNA was washed three times, 30% of the amount of mRNA used for translation was bound to biotin agarose. On the other hand, DLRS and SLmRS mRNA showed almost no binding after 3 washes. SLRS mRNA was concentrated 100 times or more after three washes. FIG. 11A is a schematic diagram showing selection of mRNA (SLRS) encoding a streptavidin protein by biotin agarose. On the other hand, mRNA encoding GST does not bind to biotin agarose (GLRS). Even when lysine is inactivated, mRNA encoding streptavidin protein does not bind to biotin agarose (SLmRS). FIG. 11B is a diagram showing the recovered amount of mRNA encoding streptavidin protein by biotin agarose. After translation, after binding to biotin agarose, unbound mRNA was washed and the amount of remaining mRNA was measured. After SLRS mRNA was washed 4 times, 5% of the amount of mRNA used for translation bound to biotin agarose. On the other hand, after GLRS and SLmRS mRNA were washed 4 times, almost no binding was observed. SLRS mRNA was concentrated 25 times or more after 4 washes. FIG. 12A is a schematic diagram showing selection of mRNA (GLRS) encoding GST protein by glutathione sepharose. On the other hand, mRNA encoding streptavidin does not bind to glutathione sepharose (SLRS). Even when lysine is inactivated, mRNA encoding GST protein does not bind to glutathione sepharose (GLmRS). FIG. 12B is a diagram showing the recovery amount of mRNA encoding GST protein by glutathione sepharose. After translation, after binding to glutathione sepharose, unbound mRNA was washed and the amount of remaining mRNA was measured. GLRS mRNA was washed 4 times, and 1.2% of the amount of mRNA used for translation was bound to glutathione sepharose. On the other hand, SLRS and GLmRS mRNA showed almost no binding (less than 0.2%) after 4 washes. GLRS mRNA was concentrated 6 times or more after 4 washes. FIG. 13A is a schematic diagram showing selection of mRNA (SLRS, SLRL) encoding a streptavidin protein by biotin agarose. On the other hand, mRNA encoding DHFR does not bind to biotin agarose (DLRS, SLRL). The spacer length of SLRS and DLRS is shorter than that of SLRL and DLRL. FIG. 13B is a photograph showing selection of mRNA encoding streptavidin protein (SLRS and SLRL) by biotin agarose from a mixed pool of mRNA encoding streptavidin and DHFR protein (SLRS + DLRS and SLRL + DLRL). After the mRNA pool was translated and bound to biotin agarose, the bound mRNA was amplified by RT-PCR. As a result, SLRS mRNA and SLRL mRNA were amplified. On the other hand, SLmRS (L) mRNA and DLRS (L) mRNA were not amplified by RT-PCR. SLRS (L) mRNA was mixed with a 1: 1 mixture of DLRS (L) mRNA, and after binding to biotin agarose, RT-PCR was performed. SLRS (L) mRNA was selectively amplified. . The shorter the spacer, the better the selection. FIG. 14A is a photograph showing selection of mRNA encoding streptavidin protein (SLRS) using biotin agarose from a mixed pool of mRNA encoding SLTP + and GST protein (SLRS + GLRS). After SLRS mRNA was translated and bound to biotin agarose, the bound mRNA was amplified by RT-PCR. As a result, SLRS mRNA was amplified. On the other hand, SLmRS mRNA and GLRS mRNA were not amplified by RT-PCR. A pool in which SLRS mRNA and GLRS mRNA were mixed at a ratio of 1: 1 was translated, bound to biotin agarose, and subjected to RT-PCR. As a result, SLRS mRNA was selectively amplified. FIG. 14B is a photograph showing selection of mRNA encoding GST protein (GLRS) by glutathione agarose from a mixed pool of streptavidin and mRNA encoding GST protein (SLRS + GLRS). After GLRS mRNA was translated and bound to glutathione agarose, the bound mRNA was amplified by RT-PCR. As a result, GLRS mRNA was amplified. On the other hand, GLmRS mRNA and SLRS mRNA were not amplified by RT-PCR. A pool in which SLRS mRNA and GLRS mRNA were mixed at a ratio of 1: 1 was translated, bound to biotin agarose, and subjected to RT-PCR. As a result, GLRS mRNA was selectively amplified. FIG. 15 is a predicted view of a possible cyclized polysome display system. FIG. 16A shows a construct prepared as a template for in vitro transcription for our novel selection system. The following template DNAs were designed: dCvH (+) mM2D (+), (I); dCvH (-) mM2D (+), (II); dCvH (+) mM2D (-), (III); dCvH (- ) mM2D (-), (IV). dCvH (+) mM2D (+) and dCvH (−) mM2D (+) both have stop codons, but not dCvH (+) mM2D (−) and dCvH (−) mM2D (−). The expected lengths were 1786 bp (I), 1723 bp (II), 1716 bp (III) and 1653 bp (IV), respectively. FIG. 16B is a photograph showing the confirmation result of in vitro translation. Lane 1, translation product of rCvH (+) mM2D (+); lane 2, translation product of rCvH (−) mM2D (+); lane 3, translation product of rCvH (+) mM2D (−); lane 4, rCvH ( -) Translation product of mM2D (-). The expected molecular weights were 55.7 kDa (lanes 1 and 3) and 53.5 kDa (lanes 2 and 4), respectively. FIG. 17 is a photograph showing the results of in vitro selection performed to evaluate the efficiency of the inventors' new system. The pattern after RT-PCR product agarose gel electrophoresis is shown. Lanes 1-3 and lanes 5-7 show RT-PCR products amplified from each mRNA pool after in vitro selection. Lane 1, initial mRNA pool containing only rCvH (+) mM2D (+); Lane 5, initial mRNA pool containing only rCvH (+) mM2D (-). These lanes serve as positive controls for selection. Lane 2, initial mRNA pool containing only rCvH (-) mM2D (+); Lane 6, initial mRNA pool containing only rCvH (-) mM2D (-). These two lanes serve as negative controls for selection. Lane 3, initial mRNA pool was a 1: 1 mixture of rCvH (+) mM2D (+) and rCvH (-) mM2D (+); Lane 7, initial mRNA pool was rCvH (+) mM2D (-) What was a 1: 1 mixture of rCvH (−) mM2D (−). These two lanes show the selection efficiency. Lane 4, RT-PCR product amplified from a 1: 1 mixture of rCvH (+) mM2D (+) and rCvH (-) mM2D (+) before in vitro selection; Lane 8, rCvH (+ before in vitro selection) ) RT-PCR product amplified from a 1: 1 mixture of mM2D (−) and rCvH (−) mM2D (−). FIG. 18 shows the correlation between including mMS2p-Cv interaction in the system and in vitro selection. The horizontal axis shows the yield defined as the ratio between the radioactivity associated with pelleted Ni-NTA agarose and the radioactivity of the input radiolabeled mRNA prior to the elution step of choice. The yield of each mRNA is the average of the results from two independent experiments. (I) Positive control with histidine tag. Has Cv but no MSp (rCvH (+) D (+)). (II) Positive control with histidine tag. It has one Cv and one MSp (rCvH (+) mM1D (+)). (III) Positive control with histidine tag. It has one Cv and two MSp (rCvH (+) mM2D (+)). (IV) Negative control without histidine tag. Has two Cv and MSp (rCvH (-) mM2D (+)).

Claims (30)

A DNA construct comprising the DNA of (i) and (ii) below, and wherein the DNAs of (i) and (ii) below are joined to express a fused transcript and translation product.
(I) DNA encoding any polypeptide
(Ii) DNA encoding a polypeptide having a function of stabilizing a complex comprising a transcription product and a translation product of the DNA of (i)   The DNA construct according to claim 1, wherein the DNA of (ii) is a DNA encoding a polypeptide having a function of inactivating a ribosome.   The DNA construct according to claim 2, wherein a DNA encoding a spacer peptide is bound downstream of a DNA encoding a polypeptide having a function of inactivating a ribosome.   The DNA construct according to claim 2, wherein a DNA encoding a linker peptide is inserted between a DNA encoding an arbitrary polypeptide and a DNA encoding a polypeptide having a function of inactivating a ribosome.   The DNA construct according to claim 1, wherein the DNA of (ii) is a combination of a DNA encoding an RNA binding protein and a DNA encoding an RNA to which the RNA binding protein binds.   6. The DNA construct according to claim 5, wherein the fused transcript of the DNA of (i) and (ii) has been modified to lack a stop codon.   6. The DNA construct according to claim 5, wherein a plurality of DNAs encoding RNA binding proteins are juxtaposed, and a plurality of DNAs encoding RNAs to which the RNA binding protein binds are juxtaposed.   The DNAs encoding a plurality of juxtaposed RNA binding proteins are DNAs encoding a plurality of different RNA binding proteins, and the DNAs encoding the RNAs to which a plurality of juxtaposed RNA binding proteins bind are different from each other. The nucleic acid construct according to claim 7, wherein the nucleic acid construct is DNA encoding RNA to which a plurality of RNA binding proteins bind. A nucleic acid construct which is a complex of the mRNA of the following (i) and the DNA of the following (ii).
(I) Fusion mRNA of RNA encoding a DNA binding protein and RNA encoding an arbitrary polypeptide
(Ii) DNA comprising a DNA region to which the translation product of the fusion mRNA of (i) binds   The nucleic acid construct according to claim 9, wherein a complex is formed by hybridization of the mRNA of (i) and the DNA of (ii). The nucleic acid construct according to claim 9, wherein the 3 'region of the mRNA of (i) and the 3' region of the DNA of (ii) are hybridized. The DNA construct of the following (i) or (ii) for use in the preparation of the DNA construct according to claim 1.
(I) a DNA construct comprising DNA having a function of stabilizing a complex of a transcription product and a translation product of a DNA encoding an arbitrary polypeptide; and (ii) a transcription product and a translation product of a DNA encoding an arbitrary polypeptide. DNA construct comprising DNA having a function of stabilizing the complex and a cloning site of DNA encoding the arbitrary polypeptide A method for producing a complex comprising a transcription product and a translation product of the DNA of (i) and (ii), wherein the DNA of (i) and (ii) is expressed in the DNA construct according to claim 1 .   The translation product of claim 9 wherein the mRNA of (i) in the nucleic acid construct of claim 9 is translated and the translation product is bound to the DNA of (ii) in the nucleic acid construct of claim 9. 10. A method for producing a complex with the nucleic acid construct according to 9.   The method according to claim 14, wherein a nucleic acid construct which forms a complex by hybridizing the mRNA of (i) and the DNA of (ii).   The method according to claim 14, wherein a nucleic acid construct in which the 3 'region of the mRNA of (i) and the 3' region of the DNA of (ii) are hybridized is used.   A method of extending the 3 ′ region of the DNA of (ii) in the complex produced by the method according to claim 15, comprising contacting the complex with a reverse transcriptase, and converting the mRNA of (i) Performing a reverse transcription reaction on the template.   A method of extending the DNA of (ii) in the complex produced by the method according to claim 16, wherein the complex is contacted with a reverse transcriptase, and the mRNA of (i) is used as a template (ii) A method comprising performing a reverse transcription reaction using the DNA of as a primer.   The method according to any one of claims 13 to 18, which is performed in a cell-free system. A composite produced by the method of claim 13.   A composite produced by the method according to claim 14. A method for screening a polypeptide that binds to a specific target substance or mRNA encoding the polypeptide, comprising the following steps (a) to (c):
(A) a step of expressing a DNA of (i) and (ii) in the DNA construct according to claim 1 to form a complex containing a transcription product and a translation product of the DNA of (i) and (ii) (B) a step of bringing the specific target substance into contact with the complex formed in the step (a) (c) a step of recovering the complex bound to the target substance A method for screening a polypeptide that binds to a specific target substance or mRNA encoding the polypeptide, comprising the following steps (a) to (c):
(A) by translating the mRNA of (i) in the nucleic acid construct of claim 9, and binding the translation product to the DNA of (ii) in the nucleic acid construct of claim 9, A step of forming a complex containing the nucleic acid construct according to claim 9 (b) a step of bringing the specific target substance into contact with the complex formed in the step (a) (c) binding to the target substance Process for recovering the complex A method for screening a polypeptide that binds to a specific target substance or mRNA or DNA encoding the polypeptide, comprising the following steps (a) to (d):
(A) by translating the mRNA of (i) in the nucleic acid construct of claim 10 and binding the translation product to the DNA of (ii) in the nucleic acid construct of claim 10, A step comprising forming a complex comprising the nucleic acid construct according to claim 10 (b) contacting the complex formed in step (a) with a reverse transcriptase, and using the mRNA of (i) in the complex as a template (C) a step of extending the DNA of (ii) in the complex (c) by contacting the specific target substance with the complex formed in the step (b) (d) Recovering the complex bound to the target substance A method for screening a polypeptide that binds to a specific target substance or mRNA or DNA encoding the polypeptide, comprising the following steps (a) to (d):
(A) by translating the mRNA of (i) in the nucleic acid construct of claim 11 and binding the translation product to the DNA of (ii) in the nucleic acid construct of claim 11, A step of forming a complex comprising the nucleic acid construct according to claim 11 (b) contacting the complex formed in step (a) with a reverse transcriptase, and using the mRNA of (i) in the complex as a template And (c) extending the 3 ′ region of the DNA of (ii) by performing a reverse transcription reaction using the DNA of (ii) in the complex as a primer, and (c) the specific target substance, A step of contacting the complex formed in the step (b) (d) a step of recovering the complex bound to the target substance A method for screening a polypeptide that binds to a specific target substance or mRNA or DNA encoding the polypeptide, comprising the following steps (a) to (d):
(A) by translating the mRNA of (i) in the nucleic acid construct of claim 10 and binding the translation product to the DNA of (ii) in the nucleic acid construct of claim 10, A step of forming a complex comprising the nucleic acid construct according to claim 10 (b) a step of bringing the specific target substance into contact with the complex formed in the step (a) (c) binding to the target substance (D) extending the DNA of (ii) in the complex by bringing a reverse transcriptase into contact with the complex and performing a reverse transcription reaction using the mRNA of (i) in the complex as a template Recovering the complex bound to the substance A method for screening a polypeptide that binds to a specific target substance or mRNA or DNA encoding the polypeptide, comprising the following steps (a) to (d):
(A) by translating the mRNA of (i) in the nucleic acid construct of claim 11 and binding the translation product to the DNA of (ii) in the nucleic acid construct of claim 11, A step of forming a complex comprising the nucleic acid construct according to claim 11 (b) a step of bringing the specific target substance into contact with the complex formed in the step (a) (c) binding to the target substance A reverse transcriptase is brought into contact with the complex, and a reverse transcription reaction is performed using the mRNA of (i) in the complex as a template and the DNA of (ii) in the complex as a primer. ii) a step of extending the 3′-side region of the DNA of (ii) a step of recovering a complex bound to the target substance   The method according to any one of claims 22 to 27, wherein the target substance is immobilized on a carrier.   A kit for use in the screening method according to claim 21, comprising the DNA construct according to claim 1 or 12, or the complex according to claim 20.   A kit for use in the screening method according to any one of claims 23 to 27, comprising the nucleic acid construct according to claim 9 or the complex according to claim 21.

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