JP2011087573A - METHOD FOR EFFICIENTLY SYNTHESIZING cDNA/mRNA-PROTEIN CONJUGATE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve efficiency of protein synthesis in an IVV (in vitro virus) method (mRNA display method, cDNA display method) which is one display technique in biotechnology, and plan further to shorten a working process. <P>SOLUTION: Shortening of the whole process in a display method is planned by optimizing conditions of a link reaction process to eliminate purification and concentration steps which have been essential in conventional techniques (selected figure). By the method, reduction in cost and time of the display technique is actualized, whereby high throughput in a molecular search by a molecular evolution engineering method can be attained. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for efficiently synthesizing mRNA (messenger RNA) -linker-protein conjugates in evolutionary engineering display technology.

With the development of genetic engineering, the theme of development research has shifted from the era of “genome analysis” to clarify gene sequences to “functional analysis” using gene expression products. This is because a genetic trait that depends on a gene appears as a function of a tissue, an organ, a human body, or the like via a gene expression product. Among these gene expression products, proteins and peptides are objects of basic and important functional analysis.
In the post-genome era, rational molecular target biopharmaceuticals (antibody drugs, peptide drugs) by clarifying gene targets based on elucidation of genes identified by genome analysis and their functions, and clarifying their mechanism of action Development is possible. In recent years, an evolutionary molecular engineering technique that has been noted for its effectiveness in the synthesis of nucleic acid drugs has attracted attention.
Peptide drugs are drugs that exhibit the same function with smaller protein molecules than antibody drugs while performing the same functions as antibody drugs that are now widely used. In the future, it is considered that antibody drugs will be replaced due to its high productivity.

Evolutionary engineering for the purpose of creating new high-performance molecules is to select peptides that specifically bind to specific molecules, not limited to proteins, from a huge library of peptides or proteins with random sequences. With the goal.
In particular, it can be said that the in vitro virus (IVV) method that molecularly associates or displays a gene that is a source of sequence randomization and a protein that is an expression product thereof is one of the core technologies.
IVV is a molecule capable of associating 1: 1 the function and phenotype of a gene expression product such as a protein with the mRNA encoded by the corresponding gene. In the IVV method, the most functional protein is selected from several IVVs each having a random protein, and then the mRNA linked to the selected protein is reverse transcribed and subjected to PCR amplification, etc. Can be detected as high-gene information.

The cDNA display method, which is one of the evolutionary engineering methods, starts with a DNA library, and includes transcription, linker linking, translation and protein linking, solid phase immobilization, reverse transcription, separation from the solid phase, purification, screening, cDNA amplification. It is standard to introduce mutations (library) and promoter ligation in one cycle and repeat multiple cycles. On the other hand, in the mRNA display method, the reverse transcription step is performed after the screening step.
By repeating this display (association) and screening cycle, the number of “screening” and “mutation introduction” to the molecule can be increased, so that a better molecule can be created and selected.
Details of the evolutionary engineering method and the IVV method are described in Non-Patent Documents 1, 2, 3, 7, and 8 and Patent Documents 1, 4, 5, and 6.
In the above literature, a method called mRNA display method has been adopted, but in recent progress of IVV method, cDNA display method has been developed to improve the stability of molecules after in vitro virus synthesis. This is described in detail in Non-Patent Documents 4, 5, 6, and 9 and Patent Documents 2, 3, and 7.

Further, in relation to IVV, the functional analysis of the protein is performed through a biochemical functional analysis by a test such as a protein-protein interaction or a protein-nucleic acid interaction.
As an analysis method of such protein-protein interaction, the yeast two-hybrid method, the phage display method, the GST-fusion protein pull-down method, the co-immunoprecipitation method and the like have been known for a long time. On the other hand, as methods for analyzing protein-nucleic acid interactions, an electrophoretic mobility shift assay method, a DNase I footprint method, a methylation buffer method, and the like are known.
The synthesized IVV must meet the same quality as the protein required for these tests.

In the IVV method, a linker having a protein binding site (a molecule for intermolecular linkage) is linked to mRNA encoding a gene, so that a protein synthesized from the mRNA is bound to the linker and captured. The For this reason, it is known that a gene and a gene expression product can be made to correspond for every molecule.
In order to trap a protein in a linker, a technique of incorporating a linker having an aminoacyl tRNA analog into an extended protein chain is used in a translation synthesis system. Such analogs include puromycin and the like, and the IVV method can be carried out by using a nucleic acid construct modified by these.

Conventionally, in the step of linking mRNA and puromycin linker, from the viewpoint of reaction efficiency, an excessive amount of puromycin linker is added to a very small amount of mRNA, and this is removed during purification. It was. These are because it was thought that it became an inhibitor of protein synthesis and the formation of a conjugate in subsequent translation reactions (see Non-Patent Documents 1 to 10 and Patent Documents 1 to 8). These removals include phenol / chloroform extraction, gel filtration, ion exchange, ODS reverse phase column, HPLC and other column purification, silica membrane and ultrafiltration filtration, acrylamide gel excision extraction, dialysis and other Various methods have been used.
In addition, a method has been proposed in which a linker and mRNA are linked by ultraviolet light irradiation cross-linking to avoid a ligation reaction and to omit the subsequent purification step (Non-patent Document 3).
On the other hand, a method has been developed in which a linker is fixed to a solid phase in advance and the reaction solution is removed by buffer exchange to avoid purification (Patent Document 3, Non-Patent Document 9).

International publication WO01 / 016600 International Publication WO06 / 041194 International Publication WO07 / 046520 JP2002-291491 JP2003-299489 JP2004-097213 JP2008-253176 JP2005-245223

Nemoto et al., FEBS Lett. 414, 405 (1997) Roberts et al., Proc. Natl. Acad. Sci. USA 94, 12297 (1997) Kurz et al., Nucleic Acids Res. 28, e83 (2000) Kurz et al., CHEMBIOCHEM 2, 666 (2001)

Tabuchi et al., FEBS Lett. 508, 309 (2001) Tabuchi et al., Biol. Proced. Online 4, 49 (2002) Miyamoto-Sato et al., Nucleic Acids Reserch 31, e78 (2003) Ja & Roberts, Biochemistry 43, 9265 (2004) Biyani et al., Nucleic Acids Res 34, e140 (2006) Retic Lysate IVT Kit (Part Number AM1200) Protocol (Ambion Inc.) Abdella et al., Biochem. Biophys. Res. Commun., 87, 734 (1979)

The conventional method is excellent in that a gene and a gene expression product can correspond to each molecule. However, there were the following problems.
That is, when the linker concentration to be added to the system is increased, the yield of the conjugate of m-RNA and linker increases, but the concentration at which the linker can be added is limited because it causes aggregation between the linkers and the linker and mRNA. Considering the ligation efficiency, the input amount of mRNA and linker must be at most about mRNA: linker ratio = 1: 200, so that a sufficient concentration of mRNA can be input to obtain an unconcentrated conjugate. There wasn't.
Furthermore, it is essential to concentrate the mRNA-linker conjugate that is a reaction product, and extraction, ethanol precipitation, ultrafiltration, evaporation and the like after the above column purification have been performed.
Such purification is not only complicated and time-consuming, but also not suitable for automating the screening of a wide variety of peptides obtained by the IVV method.

On the other hand, it is desirable to remove unreacted mRNA that may affect the yield of protein as well as the linker. In the conventional method in which the linker is fixed to the solid phase in advance, unreacted mRNA can be removed, but it has a problem that the yield is remarkably reduced, and can always meet the demand for various peptide screenings. It can not be said. Thus, an effective means for removing only unreacted mRNA in the liquid phase has not been found at present.
On the other hand, a method has been proposed that uses a cross-link by ultraviolet light irradiation instead of a ligation reaction for the linker and mRNA. This method is particularly excellent in that the purification step can be omitted. However, this method has a problem that the structure of the linker is further complicated, resulting in an increase in manufacturing cost and that mRNA may be damaged by ultraviolet light irradiation.
Further, as described above, since the evolutionary engineering method can obtain better molecular species as the number of displays and screenings is increased, the series of cycles described above needs to be as many as possible. And the fewer the number of work steps in the cycle, the better, and the less work that people are involved in.
However, the evolutionary engineering method includes many chemical reaction steps that cannot be omitted, and it has been difficult to shorten the work steps and save labor in the entire cycle.

From the above viewpoints, the method for producing the ligated body that facilitates development of practical high-throughput screening by greatly reducing the labor, time and cost required for purification and concentration after ligation of mRNA and linker There was a social request for
Furthermore, the development and popularization of peptide screening techniques are required in order to meet the development promotion needs of the above-mentioned peptide drugs and the like. For this reason, in the search method (peptide screening) of the lead peptide as a next generation medicine, there was a strong demand for automation and labor saving.

  That is, according to the first aspect of the present invention, in the reaction of individual molecules in the liquid phase, a linker having a protein binding site and comprising a main chain containing RNA and / or DNA analog and DNA, and a predetermined 3 ′ end A step of mixing mRNA having a neighboring sequence at a predetermined ratio, and then binding by a ligation reaction to obtain a ligation solution containing an mRNA-linker conjugate; and adding the ligation solution as it is to a cell-free translation system; Translating the mRNA having the predetermined sequence in the mRNA-linker conjugate to synthesize a protein; and binding the protein to the protein binding site in the mRNA-linker conjugate. , A method for producing an mRNA-linker-protein conjugate.

The predetermined ratio is preferably the linker: mRNA = 3: 1 to 1: 6, and particularly preferably the linker: mRNA = 1: 1 to 1: 1.5. The chain length of the main chain is preferably 16 to 60 bases, particularly preferably 21 to 38 bases.
The linker main chain is located between the 5 ′ end phosphorylated at the 5 ′ position, the first and second cleavage sites, and the first and second cleavage sites, and a solid phase. A 5 ′ end of the main chain having a non-complementary sequence selected from the group consisting of the main chain, side chain and the mRNA of the linker. It is preferable to comprise a vicinity portion and a main chain 3 ′ end side region portion having a sequence complementary to the 3 ′ end vicinity sequence of the mRNA.
The linker further comprises a side chain containing a nucleic acid analog and DNA, the main chain further comprises a side chain linking site in the main chain 3 ′ end side region, and up to the 3 ′ end of the main chain. It is particularly preferred that the sequence further comprises a sequence complementary to a sequence near the end; the side chain has the main chain linking site located at the 5 ′ end and the protein binding site located at the 3 ′ end.
The cleavage site preferably has a restriction enzyme recognition sequence. The method for producing the mRNA-linker-protein conjugate further comprises a step of binding to a solid phase via the solid phase binding site and a step of cleaving the first and second cleavage sites with a restriction enzyme. Is preferred. The restriction enzyme is preferably PvuII.
It is preferred that the cleavage site also has guanosine. The method for producing the mRNA-linker-protein conjugate further comprises a step of binding to a solid phase via the solid phase binding site, and a step of cleaving the first and second cleavage sites with an RNA cleaving enzyme. It is preferable. The RNA cleaving enzyme is particularly preferably ribonuclease T1.

  In a second aspect of the present invention, in the reaction of individual molecules in a liquid phase, a linker having a protein binding site and comprising a main chain containing RNA and / or a DNA analog and DNA, and a vicinity of a predetermined 3 ′ end A step of mixing mRNA having a sequence at a predetermined ratio and then binding by a ligation reaction to obtain a ligation solution containing an mRNA-linker conjugate; and adding the ligation solution as it is to a cell-free translation system, translating mRNA having the predetermined sequence in the mRNA-linker conjugate to synthesize a protein; and binding the protein to the protein binding site in the mRNA-linker conjugate, and mRNA-linker-protein Generating a conjugate; and binding the mRNA-linker-protein conjugate to a solid phase via a solid phase binding site comprising said linker. A step of performing a reverse transcription reaction on a solid phase using the 3 ′ end of the main chain as a reaction start point and the mRNA as a template to form a cDNA chain; a first and a second provided in the main chain; And cleaving the two cleavage sites with a restriction enzyme or an RNA cleavage enzyme; and a method for producing an mRNA-linker-protein-cDNA conjugate.

  The linker further comprises a side chain containing a nucleic acid analog and DNA, the main chain of the linker is a main chain 5 ′ end phosphorylated at the 5 ′ position, the first and second cleavage sites, The solid phase binding site located between the first and second cleavage sites and used to form a bond with the solid phase, the group consisting of the main chain of the linker, the side chain and the mRNA A non-complementary sequence selected from any of the above, comprising a portion near the 5 ′ end of the main chain and a side chain linking site, and a sequence complementary to the sequence near the 3 ′ end of the mRNA up to the 3 ′ end of the main chain It is preferable that the main chain 3 ′ terminal region region having The side chain preferably has the main chain linking site located at the 5 ′ end and the protein binding site located at the 3 ′ end.

Until the present invention was made, it was common technical knowledge in the cDNA / mRNA display technology that the purification and concentration step after the ligation reaction cannot be omitted. According to the method of the present invention, this can be omitted.
The effect of the present invention is to enable efficient generation of IVV, that is, mRNA-linker-protein conjugate, and further simplifies the process for the entire mRNA display method, cDNA display method, and molecular evolutionary method. Is to make it possible.

FIG. 1 is a structural diagram of a linker main chain. FIG. 2 is a schematic diagram of a linker. FIG. 3 is a schematic diagram showing a conventional IVV synthesis method. FIG. 4 is a schematic diagram showing the IVV synthesis method of the present invention. FIG. 5 is an electrophoresis image (FITC fluorescence) of the ligation reaction product. FIG. 6 is an electrophoresis image (SYBR (registered trademark) Gold) of the ligation reaction product. FIG. 7 is an electrophoresis image (FITC fluorescence) of the translation ligation reaction product.

The present invention is described in further detail below.
1. Linker for producing a conjugate In the present invention, “linker” refers to an mRNA-linker conjugate (conjugate A), mRNA-linker-protein conjugate (conjugate B), and mRNA-linker-protein used in the IVV method. -A linker used in producing any one of the linkages selected from the group consisting of cDNA linkages.
The linker used in the present invention is formed of at least one substance selected from the group consisting of DNA (ie, deoxyribonucleic acid monomer), RNA (ie, ribonucleic acid monomer, such as rG: guanosine), and DNA analog. The main chain has a solid phase binding site for binding the conjugated body to the solid phase site, and a pair of cleavage sites provided at positions sandwiching the solid phase binding site. And Here, the DNA analog includes Biotin-dT, Fluorescein-dT, and the like.
The linker used in the present invention is preferably designed so as to have a sequence that is flexible, hydrophilic, and less likely to form a specific structure as a whole.

In the detailed description of the present invention, “liquid phase reaction” refers to a reaction in a uniform reaction solution in a reaction vessel such as a normal test tube, a minute sample tube, or a microwell plate. The “solid phase reaction” is a reaction in the same reaction vessel as described above, but the molecules to be reacted are bonded to the solid phase surface such as the solid phase beads or the bottom of the vessel, and the reaction is concentrated on the solid phase surface. Refers to doing.
Here, the “predetermined sequence possessed by mRNA” includes a sequence encoding a gene, formation of a ligated body, a sequence necessary for promoting a translation reaction and other sequences, and is not particularly limited.
In the detailed description of the present invention, the term “aminoacyl tRNA 3 ′ terminal analog” means that the 3 ′ terminal is similar to aminoacyl tRNA in the chemical structure skeleton and is being extended when protein synthesis is performed in the translation system. A compound having the ability to bind to the C-terminus of a peptide chain.

Further, in the detailed description of the present invention, the term “Y portion having X” is defined as follows. Here, X represents a functional molecule such as FITC or other fluorescent dye, RnaseT1 or other enzyme substrate, and Y represents a function exhibited by the functional molecule X. The “Y site having X” refers to a specific sequence in a linker consisting of one base to several tens of bases that exhibits the function Y when X is bound or contains X. The specific sequence may be composed only of a functional molecule X, a modifying group that binds to X, and the base included in the specific sequence includes a nucleic acid analog or the like.
As an example, the “cleavage site having rG” refers to a site where ribonuclease T1 can specifically recognize rG and hydrolyze a phosphodiester bond by including guanosine (rG).

In the present invention, the linker main chain includes an RNA and / or DNA analog and a main chain containing DNA. In the method of the present invention, it is preferable that at least 60% or more of the main chain of the linker is composed of single-stranded DNA and RNA and / or DNA analog. More preferably, it is composed of 70% or more, more preferably 80% or more, most preferably 90% or more of the linker main chain, and single-stranded DNA and RNA and / or DNA analog.
In the linker used in the present invention, the length of the main chain is determined in consideration of the reactivity in various reaction steps, the production efficiency of the above-mentioned linked body, etc., preferably 16 to 60 mer, More preferably, it is 21-38mer. The linker used in the present invention can be synthesized using a known chemical synthesis method as described later.
The structure of the main chain of the linker used in the present invention will be described with reference to FIG. The main chain 200 has a main chain 5 ′ end 201. The main chain 5 ′ terminal vicinity 202 is a region not including the main chain 5 ′ terminal 201, and the main chain 3 ′ terminal region 203 is a region not including the main chain 3 ′ terminal. In addition, FIG. 1 does not limit the base length of each said part.

2. MRNA having a predetermined sequence
The mRNA used in the present invention includes both those with unknown sequences and those with known sequences. The sequence may be either a sequence existing in the natural world as genome information or an artificially designed sequence, and its structure is not particularly limited.
In the case of searching for or quantifying a substance that binds to a protein with a known sequence using the above-mentioned conjugate, mRNA encoding a gene of the protein can be used. On the other hand, when the sequence of a protein with an unknown sequence is analyzed in association with a specific function using the above ligated product, mRNA encoding a gene with an unknown sequence can be used.

Examples of such mRNA include mRNA encoding various receptor proteins with known sequences, mRNA encoding various antibodies or fragments thereof, mRNA encoding various enzymes, and mRNA of unknown sequence transcribed from DNA in various gene libraries. Transcribed from DNA having random sequence transcribed by organic synthesis, mRNA having random sequence transcribed from DNA encoding random protein by insertion of random mutation using PCR method mRNA and other mRNAs can be mentioned and selected from these.
The mRNA used in the present invention is not limited to mRNA existing in nature or a structure peculiar to mRNA. However, for the efficiency of protein synthesis, a 7-methylated guanosine cap structure at the 5 ′ end or a polymorph at the 3 ′ end. It is preferable to have at least one of the A tail structure. More preferably, it has a Kozak sequence that promotes the initiation of translation or a Shine-Dlgarno sequence.
mRNA can be obtained using a given method such as in vitro transcription reaction, chemical synthesis, extraction from living organisms, cells or microorganisms. From the viewpoint of the reaction efficiency of linker linking and cell-free translation, it is preferable to produce the linker using an in vitro transcription reaction.
The length of mRNA used in the present invention is preferably 50 to 1,000 bases from the viewpoint of reaction efficiency, and more preferably 200 to 500 bases because reaction efficiency is the highest.

3. Hybridization and Linkage Method The linkage between mRNA and linker can be performed directly or indirectly, chemically or physically, using a known technique. For example, if a sequence complementary to the end of the linker is provided at the 3 ′ end of mRNA, both can be linked by hybridization. Moreover, it can also link | link by a chemical bond by the ligation reaction by a terminal, or the photocrosslinking reaction using the psoralen in each part containing a terminal.
When the present invention is carried out in the mRNA display method, the linker and the mRNA are preferably linked by a ligation reaction. In order to promote the chemical binding reaction between the mRNA and the linker, the sites to be linked are preferably close to each other due to hybridization (annealing).
In annealing, the third nucleic acid molecule may form complementary strands both near the 3 ′ end of the mRNA and near the 5 ′ end of the linker (Splint DNA method). It is more preferable that the vicinity of the end forms a complementary strand (Y-ligation method) (Non-patent Document 6). More specifically, it is preferable that the vicinity of the 3 ′ end of mRNA and the vicinity of the 5 ′ end of the linker form a complementary strand over several bases to several tens of bases. In addition, it is more preferable that the 5 ′ end of the linker and the mRNA intermediate sequence form a complementary strand in order to stabilize the ligation by ligation. However, the above requirement does not require that several bases connected to the terminal base always form a complementary strand, or that the region forming the complementary strand is always continuous. The linker conditions that can be suitably used in the cDNA display method will be described later.

In the present invention, “ligation” or “ligation reaction” refers to a reaction promoted by an enzyme. These enzymes are ligases corresponding to the 6 groups of enzyme numbers. For DNA and RNA ligation reactions, EC6.5.1.1 (ATP-dependent DNA ligase), EC6.5.1.2 (NAD ⁺ -dependent DNA ligase), EC6.5.1.3 (RNA ligase) can be used. In addition to RNA ligase that performs RNA / DNA ligation, it is also possible to use DNA ligase that performs ligation between RNA and RNA / DNA.
Specifically, T4 RNA ligase or TS2126 thermostable phage-derived DNA ligase can be preferably used, and it is more preferable to use T4 RNA ligase for reasons of ligation efficiency.

For annealing before the ligation reaction, heat at 60 to 100 ° C for 2 to 60 minutes, warm with a heat block, aluminum block, water bath or other heating equipment, then leave it at room temperature for 2 to 60 minutes to increase the liquid temperature. It is preferable that the temperature is lowered gently and further cooled to -5 to 10 ° C. For example, it is preferable to warm on an aluminum block at 90 ° C. for 5 minutes, then warm on the aluminum block at 70 ° C. for 5 minutes, and finally leave at room temperature for 10 minutes before placing on ice.
The composition of the ligation reaction was 0.1-2.5 U / μl T4 polynucleotide kinase; 0.4-5 U / μl T4 RNA ligase; 10-250 mM Tris-HCl (pH 7.0-8.0); 2.0-50 mM Magnesium chloride; 2.0 to 50 mM dithiothreitol (DTT); preferably 0.2 to 5.0 mM ATP. In terms of reaction efficiency, 0.5 U / μl T4 polynucleotide kinase; 2.0 U / μl T4 RNA ligase; 50 mM Tris-HCl (pH 7.5); 10 mM magnesium chloride; 10 mM DTT; 1.0 mM It is preferable that it contains ATP. If necessary, an RNA-degrading enzyme inhibitor such as SUPERase RNase inhibitor (Ambion) may be added to this reaction solution.

The volume of the reaction system for performing the ligation reaction is preferably 4.0 to 100 μl. The reaction efficiency is preferably 20 to 40 μl, and the reaction efficiency is highest when 20 μl.
The amount ratio of RNA and linker in this reaction system is preferably 5.0 to 100 pmol of mRNA with respect to 5.0 to 100 pmol of linker. In order to increase the reaction efficiency and minimize the residue, it is preferable to react 1/3 to 6 times the amount of mRNA to 10 to 20 pmol of the linker, and 10 to 15 pmol of mRNA to react with 10 pmol of the linker. Is most preferred.
The annealing reaction can be performed in a ligation reaction solution excluding T4 polynucleotide kinase and T4 RNA ligase. Then, the ligation reaction can be started by introducing a necessary amount of these enzymes into the reaction solution after the annealing.
The ligation reaction is preferably performed at 10 to 40 ° C. for 1 minute to 16 hours. From the viewpoint of work efficiency and reaction efficiency, it is preferably 5 to 180 minutes at 20 to 30 ° C, and most preferably 15 minutes at 25 ° C.

4). Cell-free protein synthesis and formation of a conjugate A protein is synthesized by bringing the conjugate A (conjugate of mRNA and linker) into contact with a cell-free translation system. Here, the cell-free translation system for synthesizing the protein may be obtained from a group selected from the group consisting of animal-derived cells, plant-derived cells, fungi and bacteria, and partially or wholly artificially. It may be synthesized. Specifically, Escherichia coli, rabbit reticulocyte, wheat germ extract and the like can be used (Lamfrom H., Grunberg-Manago M., Biochem. Biophys. Res. Commun., 27, 1 (1967)).
Since the type of codon used for translation differs depending on the biological species, it is preferable to select a cell-free translation system in accordance with the target gene and the origin of the gene. Alternatively, it is preferable to redesign the sequence and structure of mRNA according to the translation system to be used.

  In an embodiment of the present invention, it is preferable to use a lysate of mammalian reticulocyte cells as the cell-free translation system, and it is more preferable to use a lysate of reticulocyte cells obtained from rabbit blood. In order to increase the ratio of reticulocytes in the blood, it is preferable to administer acetylphenylhydrazine to the mammal in advance to induce hemolytic anemia and the like, and then raise and collect blood for several days. The lysate is obtained by degrading cell-derived mRNA with micro-coccal nuclease, adding glycol ether diamine tetraacetic acid (EGTA) to chelate calcium, and inactivating the nuclease (hereinafter referred to as “micro-coccal nuclease treatment”). It is more preferable to say “finished”).

The composition of the translation reaction solution was 3.4 to 85 μl of rabbit reticulocyte lysate (micrococcal nuclease treated) and a reaction buffer (final concentration: 16 to 400 mM potassium acetate; 0.24 to 10 pmol) 0.1 to 2.5 mM magnesium acetate; 0.2 to 50 mM creatine phosphate; each containing 0.00 to 0.25 mM amino acids) is preferably used in 10 to 100 μl. From the viewpoint of reaction efficiency, a reaction buffer (final concentration: 80 mM potassium acetate; 0.5 mM magnesium acetate) containing 17 μl of rabbit reticulocyte lysate (treated with micrococcal nuclease) and 1.2 to 2.0 pmol of the above-mentioned conjugate. More preferably, 25 μl of 10 mM creatine phosphate; each 0.05 mM amino acid (0.025 mM each for methionine and leucine).
If necessary, an RNase inhibitor such as SUPERase RNase inhibitor (Ambion) may be added to the reaction solution.

The translation reaction is preferably performed at 20 to 40 ° C. for 10 to 90 minutes, and particularly preferably performed at 30 ° C. for 20 minutes from the viewpoint of production efficiency and work efficiency.
After the translation reaction, ligation between the ligation A and the translation product (protein) (formation of the ligation B) can be promoted under high salt concentration conditions. It is preferable to promote the formation of Conjugate B in the presence of a final concentration of 0.3 to 1.6 M potassium chloride and a final concentration of 40 to 170 mM magnesium chloride. From the viewpoint of production efficiency, 7 to 10 μl of 3M potassium chloride and 3 μl of 1M magnesium chloride (final concentration: 0.60 to 0.79 mM potassium chloride; 79 to 86 mM magnesium chloride) are added to 25 μl of the above reaction solution. More preferably.
It is preferable that the formation of the linked body B is promoted under reaction conditions of 27 to 47 ° C. and 30 minutes to 3 hours. From the viewpoint of production efficiency and work efficiency, it is particularly preferable to react at 37 ° C. for 90 to 120 minutes.

5). Conjugate A and protein (IVV formation)
When the conjugate A is introduced into a translation system to synthesize a protein or polypeptide chain in a liquid phase, the mRNA and the translated protein are individually linked to the C-terminal via a linker and a protein binding site of the linker. Linked to each molecule. Here, the protein binding site preferably includes an aminoacyl tRNA 3 ′ terminal analog, and the aminoacyl tRNA 3 ′ terminal analog is preferably puromycin.
That is, when the ligated body A in which a linker having puromycin or the like is bound to mRNA is brought into contact with the translation system, IVV in which the mRNA is bound to a protein translated through puromycin is generated. (Refer nonpatent literature 1).

The translated protein further includes removal of the N-terminal peptide, addition of non-amino acid components such as sugar chains and lipids, post-translational modification of amino acids, or affinity binding to the mRNA based on the three-dimensional structure or sequence structure, Chemical modification and other post-translational modification of mRNA by the enzymatic activity of may be made.
Such post-translational modification may be performed based on genetic information encoded in the mRNA, or may be performed based on external factors instead of the genetic information. Moreover, it may be based on a combination of these. Reactions based on genetic information include dimer and multimer formation, self-folding, intramolecular degradation reaction, complex formation with the original mRNA, and the like. Reactions due to external factors include addition of modifying groups, lipids, sugar chains, etc., folding or complex formation with molecular chaperones, and affinity binding with other macrostructures (viruses / cells). Examples of the reaction based on a combination of genetic information and external factors include chemical modification and degradation at a specific sequence site of the protein.
However, the said reaction is not limited to these. In addition, these reactions can be carried out for the aid of analysis and screening, and the reaction itself can be used for analysis and screening.

  Puromycin, one of the aminoacyl tRNA 3 ′ terminal analogs, is a compound represented by the following formula I, and has the ability to bind to the C terminus of the synthesized protein when the protein is synthesized in a translation system. Have.

Puromycin has the ability to bind to the C-terminus of the synthesized protein when the protein is synthesized in the translation system. Examples of other aminoacyl tRNA 3 ′ terminal analogs include 3′-N-aminoacylpuromycin aminonucleoside (PANS-amino acid). Examples of PANS-amino acids include PANS-Gly whose amino acid part is glycine, PANS-Val of valine, PANS-Ala of alanine, or a PANS-amino acid mixture whose amino acid part corresponds to all amino acids.
In addition, 3'-Aminoacyladenosine aminonucleoside, AANS-amino acid, formed by dehydration condensation of the amino group of 3'-aminoadenosine and the carboxyl group of amino acid, linked by an amide bond ). Examples of the AANS-amino acid include AANS-Gly whose amino acid part is glycine, AANS-Val of valine, AANS-Ala of alanine, or an AANS-amino acid mixture in which the amino acid part corresponds to each amino acid of all amino acids.
Further, nucleosides or those obtained by ester bonding of nucleosides and amino acids can also be used.
Examples of aminoacyl-tRNA 3 ′ terminal analogs that can be suitably used in addition to puromycin include ribocytidylpuromycin (rCpPur), deoxydylpuromycin (dCpPur), and deoxyuridylpuromycin (dUpPur). II) to (VI).

In addition, the aminoacyl tRNA analog can be added to the linker by a known technique. In the mRNA display method, a protein linking site having an aminoacyl tRNA analog may be formed at the 5 ′ end of the linker main chain. On the other hand, in the cDNA display method, when the 5 ′ end of the main chain of the linker is used to form a complementary chain with mRNA in order to form cDNA type IVV, the above protein binding site is placed in the side chain of the linker. It is preferable to form.
When the side chain of the linker is short, the translation complex undergoes steric hindrance when the aminoacyl tRNA analog is incorporated, so that the side chain of the linker is preferably of a sufficient length. The side chain of the linker preferably contains, for example, about 1 to 8 spacer molecules such as Spacer 18 Phosphoramidite of the following formula (VII). From the balance of linker synthesis efficiency and conjugate formation efficiency, such a spacer molecule It is more preferable that it contains four.

  The main chain and side chain of the linker can be linked according to a known method. For example, when the main chain of the linker contains Amino-Modifier C6 dT of the following formula (VIII) and the side chain contains a 5 ′ end synthesized from 5′-Thiol-Modifier C6 of the following formula (IX): EMCS represented by the following formula (X) can be used as a crosslinking agent.

The linker used in the present invention can be provided with a cleavage site (hereinafter sometimes referred to as “protein release site”) in order to release the protein from the conjugate B, if necessary. Examples of such a protein release site include a photocleavage site.
Here, the “photocleavable site” refers to a site having a compound having a property of being decomposed when irradiated with light such as ultraviolet rays, and a commercially available compound can also be used. For example, PC Linker Phosphoramidite (Glen research) represented by the following formula (XI), a compound related to a composition for photocleavage of nucleic acid containing fullerene (Patent Document 8), a chain by a photolysis (SBIP) technique A compound related to cleavage is preferred. From the viewpoint of cutting efficiency, it is preferable to use PC Linker Phosphoramidite.

6). Detection of IVV The linked body A or the linked body B can be easily detected by binding a labeling substance to a linker in advance. Examples of such labeling substances include fluorescent compounds, epitope peptides of antigens, radioisotopes and the like. As the radioisotope, it is preferable to use a radioisotope such as ³ H, ¹⁴ C, ³² P, ¹²⁵ I or ¹³¹ I.
When it is difficult to use a radioisotope, it is preferable to use a fluorescent compound. Fluorescent compounds have various functional groups that have free functional groups (for example, a carboxyl group that can be converted into an active ester, a hydroxyl group that can be converted into a phosphoramidide, or an amino group) and can be linked to a linker as a labeled base. It is preferable to use a dye. Examples of such fluorescent dyes include fluorescein isothiocyanate (FITC), phycobiliprotein, rare earth metal chelate, dansyl chloride or tetramethylrhodamine isothiocyanate. As the fluorescent labeling molecule, it is more preferable to use FITC-dT or Fluorescein-dT represented by the following formula (XII).

7). Solid phase immobilization of linking body B The linker used in the present invention preferably has a solid phase binding site for binding the linking body B to the solid phase site in the main chain.
The solid phase to which the linked body B is fixed is not particularly limited, and is appropriately selected according to the purpose for which the linked body is used. As such a solid phase, those having various shapes serving as a carrier for immobilizing a biomolecule can be used. For example, beads such as styrene beads, glass beads, agarose beads, sepharose beads, magnetic beads; substrates such as glass substrates, silicon (quartz) substrates, plastic substrates, metal substrates (for example, gold foil substrates); glass containers, plastic containers Examples of such containers include membranes made of materials such as nitrocellulose and polyvinylidene fluoride (PVDF).

In the above-mentioned conjugate B, by providing a solid phase binding site in the linker, the conjugate B is brought into the solid phase via the solid phase binding site and the “solid phase binding site recognition site” bound to the solid phase. Can be fixed. The “solid phase binding site recognition site” is a site provided on the solid phase, and is a site that binds to the solid phase binding site according to the substance possessed by the solid phase binding site. The solid phase binding site is not particularly limited as long as it can bind the conjugate B to a desired solid phase. For example, when a structural unit that specifically binds to a ligand, epitope peptide or other specific polypeptide is used, a specific polypeptide or the like that specifically binds to the solid phase surface should be bound. Become.
Examples of combinations of substances possessed by each of the solid phase binding site recognition site / solid phase binding site include, for example, avidin and streptavidin and other biotin binding proteins / biotin, maltose binding protein / maltose, G protein / guanine nucleotide, Polyhistidine peptide / metal ion such as nickel or cobalt, glutathione-S-transferase / glutathione, sequence-specific DNA or RNA-binding protein / DNA having specific sequence, antibody or aptamer / antibody or epitope peptide, calmodulin / calmodulin Examples include binding peptides, various receptor proteins such as ATP binding protein / ATP, estradiol receptor protein / estradiol, and ligands thereof.
Of these, the biotin-binding protein / biotin, maltose-binding protein / maltose, polyhistidine peptide / the metal ion, glutathione-S-transferase / glutathione, antibody / antigen molecule (epitope peptide), etc. are preferably used. From the viewpoint of ease of synthesis, it is more preferable to use a combination of streptavidin / biotin.

The solid phase binding site is, for example, a site for binding the conjugate B to the solid phase via a linker. Specifically, the solid phase binding site is a base capable of binding a compound, a binding group, or the like that enables such binding. Composed. More specifically, a base capable of binding biotin (for example, deoxythymine (dT)), a base bound to biotin (for example, biotin-deoxythymine (Biotin-dT)), a base modified with an amino group (for example, , Amino-modified deoxythymine (eg, Amino-Modifier C6-dT: manufactured by Glen Research Search), base modified by carboxy group (eg, carboxy-modified deoxythymine (Carboxy-dT)), base modified by thiol group (For example, thiol-modified deoxythymine (4-Thio-dT)) and the like.
In the linker, biotin-deoxythymine represented by the following formula (XIII) can be preferably used as the solid phase binding site. Then, via such a solid-phase binding site, utilizing the affinity between biotin and avidin, linked body B to which biotin is linked can be bound to the solid phase to which avidin is fixed. In addition, when a base to which a carboxy group or an amino group is bonded is selected as the solid phase binding site, the solid phase and the conjugate B can be bonded by an ester bond or an amide bond.

A reverse transcription reaction is performed by a known method after washing using the ligated body B immobilized on the solid phase by the above method. After the reverse transcription reaction, it is washed again and released from the solid phase in a newly added predetermined buffer.
In the linker used in the present invention, first and second cleavage sites (hereinafter referred to as “solid phase release sites”) are provided as needed to release the conjugated B from the solid phase. The solid phase free site may be cleaved or decomposed under the same conditions as the protein free site. In order to smoothly proceed the screening process and the like, it is preferable that the solid phase free site is configured not to be cleaved or decomposed under the same conditions as the protein free site.
In the above linker, for example, the solid-phase release site is configured as a sequence containing a base that can be recognized by a specific enzyme such as riboG (guanosine; rG; guanosine), and is bound to the solid phase using such an enzyme. It is preferable to release the conjugate B from the solid phase.

  The enzyme used here can be determined according to the design of the linker. For example, if there is a margin in the length of the main chain, a restriction enzyme (former EC number 3.1.23, 3.1.24, 3.1.25) can be selected. Can be used. When the linker is mainly composed of DNA, RNA cleaving enzymes (EC numbers 3.1.26 and 3.1.27) can be used with RNA as a cleavage site. In addition, the enzyme cleavage site can be appropriately selected and designed according to the type of enzyme used.

As the RNA cleaving enzyme, RNase T1, RNase A, RNase I, pancreatic RNase, S1 nuclease, snake venom nuclease, spleen phosphodiesterase, staphylococcal nuclease, mung bean nuclease, red bread fungus nuclease and the like can be used. Among these enzymes, the cleavage site is preferably a site that is cleaved by RNaseT1, RNaseA, RNaseI, or pancreatic RNase, and particularly includes riboG (rG; guanosine) that is recognized and cleaved by RNaseT1. A sequence is preferred.
Since RNaseT1 recognizes rG with one base and cleaves the linker, using rG can shorten the linker main chain length. As a result, there is an advantage that the above-described ligation hardly causes aggregation even when the linker concentration is increased.

  In a linker that is scheduled to be cleaved by a single-strand-specific RNA cleaving enzyme such as ribonuclease T1 (RNaseT1), the solid-phase binding site 2b and the first cleavage site 2c1 in the linker 2 as shown in FIG. Located between the two cleavage sites 2c2 and these must be located in a single stranded loop near the 3 'end of the linker backbone. FIG. 2 also shows mRNA 1 that forms a complementary chain with the linker, the main chain 200 of the linker, the side chain 300 of the linker, and the like.

The binding between the above-mentioned specific polypeptide or the like and the solid phase binding site is preferably performed using only the affinity between molecules in order to avoid denaturation of IVV, but other known methods can also be used. . Examples of such a known method include a method using tannic acid, formalin, glutaraldehyde, pyruvic aldehyde, bis-diazotized benzidone, toluene-2,4-diisocyanate and the like. Moreover, as a molecule | numerator which said specific polypeptide etc. and a solid-phase binding site have, the molecule | numerator which comprises functional groups, such as an amino group, a carboxyl group, or a hydroxyl group, is preferable.
The above-mentioned specific polypeptide or the like can be bound to an element constituting the solid phase by a known technique, and the above-described commercially available beads, the substrate, or the like may be used. The solid phase free site can also be provided on the solid phase side. The above-mentioned immobilization means is an immobilization method using two substances having an affinity for each other. However, if the solid phase is composed of a plastic material such as styrene beads or a styrene substrate, it is necessary if necessary. In addition, a part of the linker can be covalently bonded directly to the solid phase using a known technique (see Qiagen, LiquidChip Applications Handbook, etc.). In the present invention, the immobilization means is not limited to the above method, and any means can be used as long as it is known to those skilled in the art.

8). Comparison of IVV Synthesis Method and Conventional Method in the Present Invention Here, with reference to FIG. 3 and FIG. 4, the conventional method in the IVV method using the above-mentioned connector A or connector B is compared with the method according to the present invention. I will explain. In the figure, the same elements are denoted by the same reference numerals and description thereof is omitted.
In FIG. 3 showing the conventional method, mRNA 1 having genetic information and linker 2 are linked by a predetermined method. Here, the first cleavage site 2c1 and the second cleavage site 2c2 are collectively referred to as a solid phase release site 2c. In FIG. 3, mRNA 1 and linker 2 are linked by a ligation reaction following the hybridization reaction to form linked body A (Step 1). In order to increase the synthesis efficiency of the conjugate, mRNA1 and linker 2 are mixed in the range of 1: 1.5 to 1: 3, and as a result, excess linker 2 is left unreacted.

Column purification is performed to remove the unreacted linker 2 (Step 2), and then ethanol precipitation is performed to concentrate the purified conjugate 3 (conjugate A) (Step 3). Next, by subjecting the conjugate A to, for example, a cell-free translation system (Step 4), the protein 5 corresponding to the mRNA 1 is synthesized on the conjugate A in a state of being bound to the protein binding site 2a ( Step 5). As a result, a linked body 4 (linked body B) composed of mRNA1, linker 2, and protein 5 is generated. Conjugate B is so-called In Vitro Virus (IVV).
The obtained conjugate B is fixed to the solid-phase bead 6 having the solid-phase binding site recognition site 6a (Step 5), and this is subjected to a reverse transcription reaction, thereby generating cDNA 7 corresponding to mRNA (Step 5). 6). Here, when the linker is cleaved at the solid phase release site 2c using an enzyme such as RNase T1, it can be released from the solid phase beads 6 (Step 7). Furthermore, by using the label 2d, the synthesis efficiency of the protein 5, the linked body 3 or the linked body 4 in each step can be quantified.

Although omitted in FIG. 3, when mRNA is degraded with RNase H, a DNA-protein conjugate (IVV having a gene only in DNA) in which the protein and the DNA encoding it are linked is generated. Protein 5 can be released by screening based on the function and structure of the protein and then cleaving the linker at the protein release site 2e by a technique such as photocleavage.
In FIG. 4 showing the method according to the present invention, the same reference numerals as those in FIG. 3 denote the same elements. The conjugate A synthesized in Step 1 is subjected to Step 4 without undergoing purification and concentration steps, and a protein is synthesized. The subsequent solid phase step (Step 5) and subsequent steps are the same as in the conventional method.
By using the method of the present invention, two-step shortening and column purification can be omitted in IVV formation or mRNA / cDNA display methods. Furthermore, since there is no purification step, peptide screening can be performed efficiently by the mRNA / cDNA display method.

9. Method for screening gene encoding target protein from random gene library According to another aspect of the present invention, an efficient method for clarifying the correspondence between a protein and a gene using IVV is used. Peptide screening methods are provided. In this analysis method, (a) a step of introducing a library of mRNA containing one or more genes into an IVV synthesis system to form a ligation B; (b) one or more proteins synthesized in step (a) And (c) measuring whether or not the protein and the target substance interact with each other.
This analysis method is used, for example, when (i) screening a substance that acts on a protein with a known sequence, (ii) screening a protein with an unknown sequence to which a specific substance (for example, a ligand) binds. Can do. For example, in the case of (i), a plurality of conjugates of mRNA having a nucleic acid sequence encoding an orphan receptor protein and puromycin are prepared (that is, mRNAs corresponding to a plurality of orphan receptor proteins are respectively prepared. Prepare a plurality of connected bodies A or connected bodies B.), and put them into the translation system. Then, a plurality of orphan receptor proteins are synthesized from the mRNA of each ligation body A or ligation body B.

Each orphan receptor protein is immobilized by binding the C-terminal to puromycin of Conjugate A or Conjugate B immobilized on a solid phase. If necessary, unnecessary components are washed and removed, and a target substance and a buffer are added thereto to perform a test for binding the target substance to the orphan receptor protein. In the case of (ii), for example, a plurality of mRNAs are obtained from a certain gene library, and a conjugate of a plurality of mRNAs and puromycin is prepared and immobilized on a solid phase. Thereafter, the protein is synthesized in the same manner, and a binding test is performed by bringing the target substance into contact with the protein.
In the step (b), the protein synthesized in the step (a) is brought into contact with one or more target substances. As used herein, the “target substance” means a substance for examining whether or not it interacts with the protein synthesized in the present invention, and specifically includes proteins, nucleic acids, sugar chains, low molecular weight compounds and the like. Can be mentioned.

The protein as the target substance may be a protein whose amino acid sequence and function are known or an unknown protein, and may be a full-length protein or a partial peptide including a binding active site. These may be a synthesized peptide chain, a protein purified from a living body, a protein that has been translated from a cDNA library or the like using an appropriate translation system, and purified. A sugar chain may be bound to the synthesized peptide chain. Among these, it is possible to suitably target a purified protein having a known amino acid sequence or a protein translated and purified from a cDNA library or the like using an appropriate method.
The nucleic acid as the target substance is not particularly limited, and both DNA and RNA can be targeted. Further, not only nucleic acids with unknown base sequences or functions, but also known nucleic acids can be used. The method of the present invention is preferably used for a nucleic acid having a binding ability to a protein and a nucleotide sequence that is known or that has been cleaved and isolated from a genomic library using a restriction enzyme or the like. be able to.

The sugar chain as the target substance is not particularly limited, and the sugar sequence or function thereof can target not only unknown sugar chains but also known sugar chains. Preferably, a sugar chain that has already been separated and analyzed and whose sugar sequence or function is known is used.
There are no particular limitations on the low molecular weight compound as the target substance, and those having an unknown function or those already known for the ability to bind to proteins can be used.
The “interaction” between the target substance and the protein linked to the linker usually means a covalent bond, hydrophobic bond, hydrogen bond, van der Waals bond, and electrostatic force between the protein and the target molecule. The effect | action by the force which acts on the molecule | numerator resulting from at least 1 of the coupling | bonding by is shown. The covalent bond here includes a coordination bond and a dipole bond. Moreover, the coupling | bonding by an electrostatic force includes the coupling | bonding resulting from the three-dimensional structure by an electric repulsion other than an electrostatic coupling | bonding. In addition, a binding reaction, a synthesis reaction, and a decomposition reaction resulting from the above action are also included in the interaction. Specific examples of the interaction include binding and dissociation between antigen and antibody, binding and dissociation between protein receptor and ligand, binding and dissociation between adhesion molecule and partner molecule, binding and dissociation between enzyme and substrate, Binding and dissociation between nucleic acids and proteins that bind to them, binding and dissociation between proteins in information transmission systems, binding and dissociation between glycoproteins and proteins, or binding and dissociation between sugar chains and proteins Is mentioned.

The target substance used here can be used after being labeled with the above-described labeling substance, if necessary. These labeling substances should just select the thing suitable for the measurement or analysis method of the change of the signal which generate | occur | produces based on interaction between a target substance and immobilized protein. The labeling substance can be bound to the target substance based on a known method.
Next, according to the present analysis method, in the step (c), it is measured whether or not the protein and the target substance interact. Whether or not the protein and the target substance are interacting is measured by measuring and detecting a change in signal generated based on the interaction between the two molecules.
Examples of such measurement techniques include surface plasmon resonance (Cullen DC, et al., Biosensors, 3 (4), 211-225 (1987-88)), evanescent field molecular imaging (Funatsu T., et al. al., Nature, 374, 555-559 (1995)), fluorescence imaging analysis, Enzyme Linked Immunosorbent Assay (ELISA): Crowther JR, Methods in Molecular Biology, 42 (1995)), fluorescence Depolarization (Perran J., et al., J. Phys. Rad., 1, 390-401 (1926)), and Fluorescence Correlation Spectroscopy (FCS): Eigen M., et al., Proc. Natl. Acad. Sci. USA, 91, 5740-5747 (1994)).

In the analysis method of the present invention, if necessary, the protein and / or target substance in the protein-target substance conjugate determined to interact in step (c) is further identified. Protein identification can also be performed with a normal amino acid sequencer, or by reverse transcription of DNA from RNA bound to such protein and analysis of the base sequence of the obtained DNA. Identification of the target substance can be performed using NMR, IR, various mass spectrometry and the like.
When analyzing the protein-protein interaction using the RNA chip and protein chip of the present invention, the time-of-flight mass spectrometer (MALDI-TOF MS) is used in the same manner as the sample analysis on a normal protein chip. Can be used.

(IVV synthesis using a linker with rG as a cleavage site)
Hereinafter, the present invention will be described more specifically based on examples. The present invention is not limited to these examples.
1. Synthesis of Linker Short-Biotin-Puromycin linker (SBP linker) used in the present invention was synthesized. First, the following special DNA synthesis was entrusted to GeneWorld (Tokyo) and BEX (Tokyo).

(A) Puro-FS [sequence: 5 '-(S)-(PL) C (F)-(Spacer18)-(Spacer18)-(Spacer18)-(Spacer18) -CC- (Puro) -3']
Where (S) is 5'-Thiol-Modifier C6 and the compound name is S-Trityl-6-mercaptohexyl-1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite; (PL) is PC Linker Phosphoramidite with 3- (4,4'-Dimethoxytrityl) -1- (2-nitrophenyl) -1-propanyl-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite; (F) is Fluorescein -dT (5'-Dimethoxytrityloxy-5- [N-((3 ', 6'-dipivaloylfluoresceinyl) -aminohexyl) -3-acryimido] -2'-deoxyUridine-3'-succinoyl-long chain alkylamino); (Puro ) Is puromycin; (Spacer18) is Spacer Phosphoramidite 18 (18-0-Dimethoxytritylhexaethyleneglycol, 1-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite) (all manufactured by Glen Research).
(B) Hybri [(26mer) SEQ ID NO: 1 5'-CC (rG) C (TB) C (rG) CCC CGCCG CCCCC CG (T) CC T-3 ']
Where (rG) is ribo-G; (T) is Amino-Modifier C6 dT (5'-Dimethoxytrityl-5- [N- (trifluoroacetylaminohexyl) -3-acrylimido] -2'-deoxyUridine, 3 '-[(2 -cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite); (TB) is Biotin-dT (5'-Dimethoxytrityloxy-5- [N-((4-t-butylbenzoyl) -biotinyl) -aminohexyl) -3 -acrylimido] -2'-deoxyUridine-3 '-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite) (all made by Glen Research Search).

The SBP linker was obtained by cross-linking (A) Puro-FS and (B) Hybri according to the following method.
Dissolve Puro-FS 10 nmol in 100 μl of 50 mM phosphate buffer (pH 7.0) and add 1 μl of 100 mM Tris [2-carboxyethyl] phosphine (TCEP, manufactured by Pierce) (final concentration 1 mM) for 6 hours at room temperature. The tritylated mercapto group of Puro-FS was reduced to a thiol group. Immediately before the cross-linking reaction, TCEP was removed and desalted using NAP-5 Columns (manufactured by GE Healthcare Japan) equilibrated with 50 mM phosphate buffer (pH 7.0).
0.2 μM phosphate buffer (pH 7.0) 100 μl, 500 μmol / μl Hybri 20 μl, 100 mM cross-linking agent EMCS (6-Maleimidohexanoic acid N-hydroxysuccinide ester), manufactured by Dojin Chemical Laboratory 20 μl was added, stirred well, and reacted at 37 ° C. for 30 minutes. Thereafter, ethanol precipitation was performed at 4 ° C. to precipitate the reaction product, and unreacted EMCS was removed. The precipitate was washed with 500 μl of cold 70% ethanol, dried under reduced pressure, and then dissolved in 10 μl of 0.2 M phosphate buffer (pH 7.0).
To this reaction product, the reduced Puro-FS (˜10 nmol) was quickly added and left at 4 ° C. overnight. TCEP was added to the sample to a final concentration of 4 mM, and the mixture was allowed to stand at 37 ° C. for 15 minutes to stop the thiol group crosslinking reaction. Precipitate the synthesized linker at room temperature, remove unreacted Puro-FS, and then perform HPLC purification under the following conditions in order to remove unreacted Hybri and their EMCS cross-linked products .

(HPLC conditions)
Column: COSMOSIL (registered trademark) 10 x 250 mm C18-AR-300 (manufactured by Nacalai Tesque)
Buffer A: 0.1 M TEAA (triethylammonium acetate)
Buffer B: 80% acetonitrile (diluted with ultrapure water)
Flow rate: 0.5 ml / min Concentration gradient: The mixing buffer concentration of A and B is changed from 85:15 to 65:15 (A: B) (33 minutes).
The product fractionated by HPLC was detected by electrophoresis on 18% acrylamide gel (8M urea, 62 ° C.), and the target fraction was dried under reduced pressure. Then, it was dissolved in DEPC (Diethylpyrocarbonate) -treated water to make 10 pmol / μl.

2. Synthesis of mRNA BDA (B domain of Protein A) was used as a model mRNA. The BDA gene (BDA gene; SEQ ID NO: 2; 192 bases long) is complemented with a T7 promoter sequence and translation promoting sequence upstream of the 5 'side, a spacer region, a histidine tag (His tag), and a puromycin linker downstream of the 3' side. After synthesizing and purifying DNA added with a sequence having a chain region (BDA whole; SEQ ID NO: 3; 367 bases in length) by T7 RiboMAX Express Large Scale RNA Production System (Promega Corp.)), According to the attached protocol, 5 to 30 pmol / μl of mRNA (BDA mRNA; SEQ ID NO: 4; 337 base length) was synthesized.

3. Ligation enzyme reaction using T4 RNA ligase In the ligation reaction, 1 to 6 times as much mRNA as linker 1 was added, and 20 μl of T4 RNA ligase buffer (50 mM Tris-HCl, pH 7.5) was used. 10 mM magnesium chloride; 10 mM DTT; 1 mM ATP). To anneal before adding enzyme, warmed on aluminum block at 90 ° C for 5 minutes, then warmed on aluminum block at 70 ° C for 5 minutes, finally left at room temperature for 10 minutes, then placed on ice . To this, 1 μl of T4 polynucleotide kinase (10 U / μl) and 1 μl of T4 RNA ligase (40 U / μl) (both manufactured by Takara Bio Inc.) were added and held at 25 ° C. for 15 minutes.
In order to divide the generated reaction solution in half and compare it in the subsequent experimental step, for the sample in which 10 pmol of mRNA was reacted with 10 pmol of linker, all the substances were doubled to make a 40 μl system. (Reaction system R1).

3.2 Results of ligation reaction The ligation efficiency of mRNA and SBP linker was compared at the mixing ratio shown below. The comparison was performed by electrophoresis at 65 ° C., 8M urea, 5% denatured acrylamide gel at 200V for 25 minutes. Each sample was dispensed in 1 μl, mixed with 3 μl loading buffer and 2 μl DEPC water and loaded onto the gel. The results are shown in FIG. The band was detected by the fluorescence of FITC bound to the linker, and the unreacted linker or the above-mentioned conjugate A was detected.
FIG. 5 shows 10 pmol mRNA added with 10 pmol linker (reacted in the 40 μl reaction system) (lane 1, reaction system R1), 6.6 pmol (lane 2, reaction system R2). ), 3.3 pmol (lane 3, reaction system R3), 1.66 pmol (lane 4, reaction system R4), and electrophoresis images of those subjected to the ligation reaction as described above. In FIG. 5, an arrow indicates an unreacted linker, and * indicates a reaction product (linker A) to which the linker is linked.
As can be seen from FIG. 5, a linker residue was seen in lane 1, but almost no other lane was seen. In addition, except for the case of a mixing ratio of 1: 1, there was no difference in the amount of synthesis of ligated A, and it was observed that almost the same amount of ligation reaction occurred in all cases.

FIG. 6 shows an electrophoresis image of the above sample stained with SYBR Gold. The electrophoresis conditions were the same as described above. In FIG. 6, an arrow indicates an unreacted linker, * indicates a synthesized product (linker A) in which the linker is linked, and ** indicates mRNA that remains without forming a linkage. Lane M represents a molecular weight marker, and the number on the left side represents the molecular weight of each band.
As can be seen from FIG. 6, a linker residue was seen in lane 1, but almost no other lane was seen. Further, except for lane 1, there was no difference in the amount of conjugate A synthesized, and it was observed that the remaining amount of mRNA increased as the input amount of mRNA increased.
From the above, it was shown that the presence of 1.0 to 1.5 times the amount of mRNA relative to the linker is sufficient for the formation of conjugate A.

4). Purification and Concentration of Conjugate A In order to confirm the necessity of purification and concentration of the reaction product after ligation, experimental results on purification and concentration are shown.
20 μl was separated from R1 and subjected to the following purification and concentration step (reaction system R1 ′). The rest of R1 and R2 to R4 were cooled and held as they were after the ligation reaction.
In order to remove the unlinked linker and the reaction solution, purification was performed using RNeasy (registered trademark) Kit (Qiagen Co., Ltd.) according to the attached protocol. Conjugate A (30-50 μl) eluted with DEPC water was ethanol precipitated using a coprecipitation agent Quick-Precip Plus solution (EdgeBio) according to the attached protocol. This was dissolved in 12 μl of DEPC water to obtain a template for translation.

5). IVV formation (linkage of conjugate A and protein in parallel with translation)
5.1 IVV (conjugate B) synthesis Retic Lysate IVT Kit (Ambion) was used for the cell-free translation system. The mixing method was performed with reference to the protocol of the kit. All reagents used in the cell-free translation reaction were gently agitated and centrifuged, then placed on ice. A 25 μl scale reaction solution was prepared by mixing and reacting in the following order.
0.625 μl of 20X Translation Mix minus-Met, 0.625 μl of 20X Translation Mix minus-Leu, and 17 μ of Retic lysate were carefully pipetted and mixed without bubbling.
This mixture was added to 2.4 μl R1 ′ and 4.0 μl R1-4. Further, DEPC water was added so that the reaction solution became 25 μl. And it was mixed carefully so that there was no foam again. After mixing, the translation reaction was allowed to proceed at 30 ° C. for 20 minutes.
After the translation reaction, 10 μl of 3M potassium chloride and 3 μl of 1M magnesium chloride were added to the reaction solution and reacted at 37 ° C. for 90 minutes in order to promote the connection between the conjugate A and the synthetic protein.

5.2 Comparison of IVV (Conjugate B) Synthesis Efficiency For Linkage B synthesized as described above, the coupling efficiency of mRNA and SBP linker was compared at the same mixing ratio. For comparison, electrophoresis was carried out using 6% SDS acrylamide gel of 8M urea at 0.01 A for 60 minutes, and then at 0.02 A for 60 minutes. The result is shown in FIG. Band detection was performed by fluorescence of FITC bound to the linker. Thereby, the coupling body A or B can be observed.
FIG. 7 represents Lane 1: R1 ′, Lane 2: R1, Lane 3: R2, Lane 4: R3, Lane 5: R4, Lane 6: Conjugate A. In FIG. 7, * indicates the molecular weight of the linking body A, and the arrow indicates the molecular weight of the linking body B.
As can be seen from FIG. 7, in R1 and R2, the formation of a ligation B that is inferior to R1 ′ in lane 1 was observed. On the other hand, in R3 and R4, the production amount was small, and it was observed that the formation efficiency of the conjugate decreased as the mRNA ratio increased.

From the above, it was shown that IVV can be formed without performing silica gel membrane column purification after ligation reaction and concentration by ethanol precipitation. In addition, it was shown that IVV formation efficiency comparable to that obtained by the purification and concentration described above can be obtained by setting the mixing ratio of linker and mRNA to 1.0: 1.0 to 1.5.
According to the present invention, it is possible to provide an IVV forming method that can be performed at a low cost. Moreover, since the use in the preferred embodiment of the present invention has IVV synthesis efficiency comparable to that of the conventional method, the process can be simplified without reducing the operating efficiency of the mRNA / cDNA display method. Thus, the present invention is applied to a high-throughput evolutionary molecular engineering method aiming at functional analysis of DNA and protein and acquisition of functional protein, thereby meeting the above-mentioned demand for creation of a new drug.

  The present invention is useful in the fields of medical diagnosis, environmental analysis, food analysis, and research bioimaging, including the above drug discovery.

1 mRNA
2 Linker 2a Protein binding site 2b Solid phase binding site 2c Solid phase release site 2c1 First cleavage site 2c2 Second cleavage site 2d Label 2e Protein release site 3 Conjugate A
4 Conjugate B
5 Protein 6 Solid phase beads 6a Solid phase binding site recognition site 7 cDNA
200 Main chain 201 Main chain 5 ′ end 202 Main chain 5 ′ end vicinity 203 Main chain 3 ′ end side region 300 Side chain

Hybri
Guanosine
Biotin-dT
Amino-Modifier C6 dT
BDA whole
BDA mRNA

Claims (14)

In the reaction of individual molecules in the liquid phase,
A linker having a protein binding site, RNA and / or DNA analog and a main chain containing DNA, and mRNA having a predetermined sequence near the 3 ′ end are mixed at a predetermined ratio, and then bound by a ligation reaction. Obtaining a ligation solution comprising an mRNA-linker conjugate;
Adding the ligation solution as it is to a cell-free translation system, translating the mRNA having the predetermined sequence in the mRNA-linker conjugate, and synthesizing a protein;
Binding the protein to the protein binding site in the mRNA-linker conjugate. A method for producing an mRNA-linker-protein conjugate.   The method for producing an mRNA-linker-protein conjugate according to claim 1, wherein the predetermined ratio is the linker: mRNA = 3: 1 to 1: 6.   The method for producing an mRNA-linker-protein conjugate according to claim 1 or 2, wherein the predetermined ratio is the linker: mRNA = 1: 1 to 1: 1.5.   The method for producing an mRNA-linker-protein conjugate according to any one of claims 1 to 3, wherein the main chain has a chain length of 16 to 60 bases.   The method for producing an mRNA-linker-protein conjugate according to any one of claims 1 to 4, wherein the main chain has a chain length of 21 to 38 bases. The linker main chain is:
A 5′-end phosphorylated main chain 5 ′ end,
A main chain of the linker, comprising: a first and a second cleavage site; and a solid phase binding site which is located between the first and second cleavage sites and used for forming a bond with the solid phase. Each having a non-complementary sequence selected from the group consisting of a side chain and the mRNA, near the main chain 5 ′ end,
A main chain 3 ′ end side region having a sequence complementary to the 3 ′ end vicinity sequence of the mRNA,
The method for producing an mRNA-linker-protein conjugate according to any one of claims 1 to 5, wherein The linker further comprises a side chain comprising a nucleic acid analog and DNA;
The main chain further comprises a side chain linking site in the main chain 3 ′ terminal region, and further has a sequence complementary to the 3 ′ terminal vicinity sequence of the mRNA up to the 3 ′ end of the main chain;
The mRNA-linker-protein conjugate according to claim 6, wherein the side chain has the main chain linking site located at the 5 'end and the protein binding site located at the 3' end. Generation method.   The method for producing an mRNA-linker-protein conjugate according to claim 6 or 7, wherein the cleavage site has a restriction enzyme recognition sequence. Binding to a solid phase via the solid phase binding site;
The method for producing an mRNA-linker-protein conjugate according to claim 8, further comprising the step of cleaving the first and second cleavage sites with a restriction enzyme.   The method for producing an mRNA-linker-protein conjugate according to claim 9, wherein the restriction enzyme is PvuII.   The method for producing an mRNA-linker-protein conjugate according to claim 7, wherein the cleavage site has guanosine. Binding to a solid phase via the solid phase binding site;
The method for producing an mRNA-linker-protein conjugate according to claim 7, further comprising a step of cleaving the first and second cleavage sites with an RNA cleaving enzyme.   The method for producing an mRNA-linker-protein conjugate according to claim 12, wherein the RNA cleaving enzyme is ribonuclease T1. In the reaction of individual molecules in the liquid phase,
A linker having a protein binding site, RNA and / or DNA analog and a main chain containing DNA, and mRNA having a predetermined sequence near the 3 ′ end are mixed at a predetermined ratio, and then bound by a ligation reaction. Obtaining a ligation solution comprising an mRNA-linker conjugate;
Adding the ligation solution as it is to a cell-free translation system, translating the mRNA having the predetermined sequence in the mRNA-linker conjugate, and synthesizing a protein;
Binding the protein to the protein binding site in the mRNA-linker conjugate to produce an mRNA-linker-protein conjugate;
binding an mRNA-linker-protein conjugate to a solid phase via a solid phase binding site of the linker;
A step of performing a reverse transcription reaction on a solid phase using the 3 ′ end of the main strand as a reaction initiation point and the mRNA as a template to form a cDNA strand;
Cleaving the first and second cleavage sites of the main chain with a restriction enzyme or RNA cleaving enzyme, and a method for producing an mRNA-linker-protein-cDNA conjugate comprising:
The linker further comprises a side chain comprising a nucleic acid analog and DNA;
The linker main chain is:
A 5′-end phosphorylated main chain 5 ′ end,
The first and second cleavage sites, and the solid phase binding site that is located between the first and second cleavage sites and used to form a bond with the solid phase, Near the main chain 5 ′ end, having a non-complementary sequence selected from the group consisting of the main chain, side chain and the mRNA,
A main chain 3 ′ terminal region region comprising a side chain linking site and having a sequence complementary to the 3 ′ terminal sequence of the mRNA up to the 3 ′ terminal of the main chain;
Characterized by comprising:
The side chain has the main chain linking site located at the 5 ′ end and the protein binding site located at the 3 ′ end.