JP2016123343A - Nucleic acid linker - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel nucleic acid linker for a rapidly, simple, and accurate cDNA display method that is applicable to an automation high throughput system.SOLUTION: A novel nucleic acid linker consists of a branched single stranded DNA having a side chain in which puromycin is bound to the 3' end and not having a biotinylated base and a site for cleaving the base, wherein the side chain is bound to a principal chain through an oligo dA provided in a root region thereof. Using this nucleic acid linker enables the execution of a series of all steps of a purification of a mRNA-protein cointegrate produced by a cell-free translation system, a production of a mRNA/cDNA-protein cointegrate by a reverse transcription, a production of a cDNA-protein cointegrate by RNase, and a purification of the mRNA/cDNA-protein cointegrate or the cDNA-protein cointegrate, on a Ni magnetic bead surface, as well as enables acquisition of a high affinity protein to a target substance in a short time.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a novel puromycin linker oligonucleotide for cDNA display, and further relates to a simple and efficient cDNA display method using the puromycin linker and high-throughput in vitro selection.

In recent years, an evolutionary molecular engineering display technique has attracted attention as a technique for screening proteins and peptides having desired functions and properties in a wide range of fields including diagnostic reagents and drug discovery fields. As such display technology, in addition to conventional methods using cells such as phage display and cell surface display, display technology using cell-free translation systems such as ribosome display, mRNA display, and cDNA display has recently been developed. Remarkably, there is an expectation as a powerful research tool that contributes to the development of new functional peptide molecules such as peptide drugs (Non-Patent Documents 1 to 3).
Currently, the most widely used display method is the phage display method (Non-Patent Document 4), and there are many successful examples (Non-Patent Documents 5 to 7). In the case of the phage display method, it is about 10 ^{8 at the} maximum, but in the cell-free translation system display technology, it is 10 ¹² or more, so higher molecular diversity is possible. In addition, the cell-free translation system has the flexibility that non-natural residues can be incorporated into proteins / peptides, and is also cell-dependent in that post-translational modifications such as disulfide shuffling can be performed. (Non-Patent Documents 8 to 10).
However, despite such beneficial characteristics and subsequent technology development (Non-Patent Document 11), cell-free translation system display technology is not as general as the phage display method (Non-Patent Document 1). . The major factor is that, in the phage display method as compared with the cell-free translation system, an accurate correspondence between the genotype and the expressed peptide can be secured, and a simple and rapid method has already been established. The entire selection / evolution process using phage display is generally accomplished in about 2 to 3 weeks (Non-Patent Documents 1 and 4), whereas cell-free translation-based display technology allows more than 10 cycles of selection / evolution. When included, the whole process takes 5 to 8 weeks (Non-Patent Documents 12 to 14).

The automation technology in evolutionary engineering is relatively simple in the case of nucleic acids, and both the in vitro selection process and the evolution process for creating new ligands have been successfully automated, improving the accuracy and speed of analysis (non- Patent Document 15). On the other hand, in the case of the displayed protein, there is a problem in the accuracy of the coupling process downstream, and thus it is generally difficult to adopt an automated system. For example, in the case of a cell-dependent method such as phage display, a coupling process via a cell is expected to be a major obstacle. However, there are already reports of semi-automated and automated high-throughput systems for phage display (Non-patent Documents 16 and 17). On the other hand, the cell-free translation system, especially cDNA display, should be a technology that is inherently suitable for automation. However, although there are attempts to automate based on microfluidic analysis technology (Non-patent Document 18), it is quite difficult to achieve complete automation technology. Not proceed. This has led to the creation of new ligands that are excellently optimized in the process of producing mRNA complexes that serve as templates in cDNA displays and in the process of fusion with proteins, but these optimization preparation methods are suitable for automated operations. This is due to the absence (Non-Patent Documents 12 to 14, 18).
However, in order to create new ligands and reagents on a larger scale in the future, it is essential to construct an automated high-throughput system for cDNA display. For this purpose, an accurate and rapid cDNA display technology and the cDNA display The development of nucleic acid linkers is urgent.

JP 2003-299489 A Japanese Patent No. 4318721 JP 2011-87573 A JP 2011-217708 A JP 2012-139197 A WO2013 / 065827 Japanese Patent No. 4959226 WO2010 / 104114

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  An object of the present invention is to provide a rapid, simple and accurate cDNA display method and in vitro selection method applicable to an automated high-throughput system, and to provide a novel nucleic acid linker therefor.

  A typical nucleic acid linker used in cDNA display is a puromycin linker, which is a T-shaped single-stranded DNA molecule consisting of a main chain and a side chain (arm part). In the form of sandwiching a biotin site and a biotin site for binding to a streptavidin-immobilized bead (SA bead) along with a ligation site for linking to mRNA on the 5 'end side of the single-stranded DNA molecule that becomes the main chain And has a primer for reverse transcription of mRNA at the 3 'end. The side chain has puromycin for linking a protein synthesized from mRNA at the end, and is bound to the main chain via a spacer sequence (Patent Documents 1 to 3, Non-Patent Documents 13, 22, and 28). Such). In the selection step by cDNA display using the linker, first, mRNA transcribed from the template DNA library and the linker are linked at the ligation site, and the resulting mRNA-linker conjugate is converted into a cell-free translation system. A protein is produced and linked to a linker via puromycin to produce an mRNA-linker-protein fusion. Thereafter, it is bound to a solid phase carrier (SA beads) on which streptavidin is immobilized, purification of mRNA-linker-protein fusion (hereinafter also referred to as mRNA protein fusion) on the SA beads and cDNA by reverse transcriptase. Synthesis is performed to produce a stable cDNA-linker-protein fusion (hereinafter also referred to as cDNA protein fusion or cDNA display). Next, the cDNA display is separated from the SA beads by cleaving the enzyme cleavage site such as the restriction enzyme site provided in the vicinity of biotin on the 5 'end side of the main chain of the nucleic acid linker with the restriction enzyme, and provided to the protein. The obtained His-tag is purified and subjected to a selection step using the binding property to the target substance as an index (Patent Document 1, Non-Patent Documents 13, 22, and 23). Thereafter, DNA from the selected protein fusion is amplified by PCR to obtain a mutation-introduced DNA library, and the same series of selection steps are repeated several times. As a method for separating the region containing biotin from the cDNA display, a method of cleaving with a RNase using a cleavage site provided in advance in the vicinity of biotin on the 5 ′ end side of the main chain of the nucleic acid linker as riboguanosine (Patent Documents 2 and 3). Non-Patent Document 29), a method of introducing deoxyinosine and cleaving with DNA repair enzyme Endo V (Patent Document 5), and a method of providing a photocleavage site and cleaving with light (Patent Document 6). Proposed. As a purification method for cell-free translation systems of mRNA-linker-protein fusions that do not use biotin, a poly-A sequence provided in a DNA chain connected by a photoreactive cross-linking agent (soralen) separate from the main chain is used. Although a purification method using dT-immobilized beads has been proposed (Patent Document 4, Non-Patent Document 37), cDNA protein fusion synthesis by reverse transcription on dT-immobilized beads has not been performed. Thus, in the conventional puromycin linker, in order to efficiently perform the purification step and reverse transcription step of the mRNA protein fusion from the cell-free translation system on a single solid phase, biotin provided in the linker is used. It was thought that the use of SA beads to bind to was essential.

In contrast, the present inventors have previously designed a template DNA for cDNA display using a His-tag downstream of the gene coding sequence to ensure that the protein has been translated to the end of the stop codon. It is common to provide a sequence, and metal (Ni) affinity purification using His-tag sequences is the usual method for purification of the final cDNA display (cDNA protein fusion) before moving to the selection step. Focused on that. If all steps from purifying mRNA protein fusion from cell-free translation system to reverse transcription and cDNA protein derivative purification can be performed on single metal (Ni) beads, in vitro selection process I thought that I could simplify and shorten the time.
However, affinity purification by His-tag uses affinity between the His-tag sequence and the metal (Ni) on the surface of the bead (carrier resin), so that it is also described in paragraph [0009] of Patent Document 4. In addition, since the amount of mRNA protein fusion synthesized in a cell-free translation system has been small in the past, it has been common technical knowledge that it is difficult to remove contaminating proteins having similar amino acid sequences in purification from cell-free lysates. It was.

Therefore, the present inventors diligently studied the design of a puromycin linker in order to increase translation efficiency in a cell-free translation system. Specifically, by providing an oligo dA in a region between the flexible spacer sequence usually provided in the side chain having puromycin and the base portion, the flexible spacer sequence supported by the rigid oligo dA is interposed. Thus, it was conceived that puromycin could be appropriately arranged in the vicinity of the P site of ribosomal RNA (FIG. 9d) and translation efficiency would be improved. In order to confirm this idea, a puromycin linker with oligo dA with a different length in the base region of the side chain was subjected to a cell-free translation system under the same conditions. As a result, oligo of sufficient length (12dA or more) It was found that high translation efficiency can be achieved even with a cell-free translation system when dA is provided. When the mRNA protein fusion obtained in the cell-free translation system was purified with metal (Ni) magnetic beads, the mRNA protein fusion could be purified with high purity. Furthermore, it was possible to proceed to the reverse transcription process on the metal (Ni) beads as it was, and succeeded in obtaining the cDNA protein fusion with high purity.
In addition, since SA beads are not used for purification from cell-free translation systems, there is no need to provide biotin in the linker or a cleavage site for biotin removal in the vicinity of the ligation site and the region that hybridizes with mRNA. This led to the removal of steric obstacles that hindered This may have increased the efficiency of ligation with mRNA and helped to build an accurate and compact conformation between mRNA and nucleic acid linker, which may have resulted in higher translation efficiency in cell-free translation systems. is there. In addition, in the ligation step of the present invention, the reaction between the mRNA and the linker proceeds almost stoichiometrically, so there is no need to have excess mRNA in the cell-free translation system, and purification is simplified. This is a major factor that enables purification by affinity affinity using His-tag sequences.
In any case, in the puromycin linker oligonucleotide, an oligo dA is provided in the base region of the side chain without incorporating biotin, thereby eliminating the step of removing the sequence containing biotin, and at the same time, high fusion with mRNA. As a result of the improvement of efficiency (Fig. 4c) and translation efficiency, surprisingly, it is possible to purify the mRNA protein fusion with high purity from the cell-free translation system using metal (Ni) beads. (Fig. 10). Moreover, an efficient cDNA display method in which a series of steps from purification of mRNA protein fusion, formation of cDNA protein fusion by reverse transcription, and purification of cDNA protein fusion can all be performed on a single carrier. Could be provided.

In a model experiment in which the in vitro selection cycle using the cDNA display method is applied to known targets (immunoglobulin, acetylcholine binding protein and interleukin 6 receptor), one selection cycle can be performed in a short time of less than 8 hours. As a result of in vitro selection using a three-finger library that actually targets VEGF, a VEGF high-affinity peptide comparable to anti-VEGF antibody could be provided (FIG. 11a). Table 4).
In addition, the method for synthesizing the puromycin linker oligonucleotide was replaced with the conventional method using the crosslinking agent EMCS, and a simple production method using branched deoxycytosine (5-Me-dC) could be provided.
By obtaining the above knowledge, the present invention could be completed.

That is, the present invention is as follows.
[1] A branched nucleic acid linker composed of a main chain composed of a single-stranded DNA capable of specifically hybridizing with a target mRNA, and a side chain having puromycin at the 3 ′ end,
The side chain has an oligo dA region at the other end, is covalently bonded to the modified base in the single-stranded DNA polynucleotide chain via the oligo dA, and the oligo dA region and the puromycin A spacer region is included between and
The main chain has a site that can be ligated with the mRNA at the 5 ′ end, and a region that can hybridize with the mRNA adjacent to the site, and the mRNA is reverse transcribed at the 3 ′ end. A nucleic acid linker comprising a primer site serving as a primer.
[2] The nucleic acid linker according to [1], wherein the modified base is 5-methyldeoxycytosine (5-Me-dC).
[3] The nucleic acid linker according to [1] or [2], wherein the oligo dA comprises a sequence in which at least 12 deoxyadenines (dA) are continuous.
[4] The nucleic acid linker according to any one of [1] to [3], wherein the spacer sequence is a sequence containing 2 to 8 Sp.18 (Spacer 18 Phosphoramidite).
[5] The method for producing the nucleic acid linker according to [1],
From the 3 ′ end to the 5 ′ end, a protein ligation site bound with puromycin, a spacer sequence, an oligo dA sequence, 5-methyldeoxycytosine (5-Me-dC), a region capable of hybridizing with the target mRNA, and The 5-Me-dC of single-stranded DNA containing a site that can be ligated with the mRNA reacts with the phosphate group at the 5 ′ end of the DNA sequence that serves as a primer for reverse transcription of the mRNA. A manufacturing method characterized by combining them.
[6] An mRNA-protein fusion in which a target mRNA and a protein encoded by the mRNA are linked via the nucleic acid linker according to any one of [1] to [4].
[7] The mRNA-protein fusion according to [6], wherein the mRNA has a sequence encoding a His-tag downstream of a sequence encoding a protein.
[8] A method for producing the mRNA-protein fusion according to [6] or [7], comprising the following (a) to (c);
(A) annealing the nucleic acid linker according to [1], which targets the mRNA, with the mRNA;
(B) ligating the 3 ′ end of the mRNA and the 5 ′ end of the nucleic acid linker to produce an mRNA-nucleic acid linker;
(C) A step of producing an mRNA-protein fusion by synthesizing a protein from the mRNA using a cell-free protein translation system and binding the C-terminus of the protein to puromycin in the nucleic acid linker.
[9] In the step (c), a step is provided in which the protein is matured in the presence of a high concentration salt for 10 minutes to 2 hours during or after the production of the mRNA-protein fusion. The method according to [8] above.
[10] An mRNA / cDNA-protein fusion comprising the nucleic acid linker according to any one of [1] to [4], wherein the mRNA and the cDNA complementary to the mRNA are passed through the nucleic acid linker. An mRNA / cDNA-protein fusion in which the mRNA / cDNA complex is linked to the protein encoded by the mRNA.
[11] An mRNA / cDNA-protein fusion comprising the nucleic acid linker according to any one of [1] to [4], wherein the cDNA is complementary to the mRNA via the nucleic acid linker, and the mRNA. A cDNA-protein fusion in which the protein encoded by is linked.
[12] An mRNA / cDNA-protein fusion comprising the nucleic acid linker according to any one of [1] to [4], wherein the mRNA and the cDNA complementary to the mRNA are passed through the nucleic acid linker. A method for producing a purified mRNA / cDNA-protein fusion, wherein the mRNA / cDNA complex and the protein encoded by the mRNA are linked, comprising the following (a) to (f):
(A) annealing the nucleic acid linker with the target mRNA;
Wherein the target mRNA is an mRNA having a sequence encoding a His-tag downstream of a sequence encoding a protein;
(B) ligating the 3 ′ end of the mRNA and the 5 ′ end of the nucleic acid linker to produce an mRNA-nucleic acid linker;
(C) preparing a mRNA-protein fusion by synthesizing a protein from the mRNA using a cell-free protein translation system and binding the C-terminus of the protein to puromycin in the nucleic acid linker;
(D) binding the mRNA-protein fusion to a metal matrix having an affinity for the His-tag sequence, and washing;
(E) subjecting the mRNA-protein fusion on the surface of the metal matrix to a reverse transcription reaction to produce an mRNA / cDNA-protein fusion;
(F) washing the mRNA / cDNA-protein fusion on the surface of the metal matrix and eluting the purified mRNA / cDNA-protein fusion.
[13] In the step (c), a step of maturing the protein in the presence of a high concentration salt for 10 minutes to 2 hours is provided during or after the production of the mRNA-protein fusion. The method according to [12] above.
[14] The method according to [12] or [13], wherein the metal matrix is Ni-coated magnetic beads.
[15] An mRNA / cDNA-protein fusion comprising the nucleic acid linker according to any one of [1] to [4], wherein the cDNA is complementary to the mRNA via the nucleic acid linker, and the mRNA. A method for producing a purified cDNA-protein fusion linked to a protein encoded by the method comprising the following (a) to (g);
(A) annealing the nucleic acid linker with the target mRNA;
Wherein the target mRNA is an mRNA having a sequence encoding a His-tag downstream of a sequence encoding a protein;
(B) ligating the 3 ′ end of the mRNA and the 5 ′ end of the nucleic acid linker to produce an mRNA-nucleic acid linker;
(C) preparing a mRNA-protein fusion by synthesizing a protein from the mRNA using a cell-free protein translation system and binding the C-terminus of the protein to puromycin in the nucleic acid linker;
(D) binding the mRNA-protein fusion to a metal matrix having an affinity for the His-tag sequence, and washing;
(E) subjecting the mRNA-protein fusion on the surface of the metal matrix to a reverse transcription reaction to produce an mRNA / cDNA-protein fusion;
(F) a step of producing a cDNA-protein fusion by degrading mRNA by allowing an RNase to act on the mRNA / cDNA-protein fusion;
(G) A step of washing the cDNA-protein fusion on the surface of the metal matrix and eluting the purified cDNA-protein fusion.
[16] In the step (c), a step is provided in which the protein is matured in the presence of a high concentration salt for 10 minutes to 2 hours during or after the production of the mRNA-protein fusion. The method according to [15] above.
[17] The method according to [15] or [16] above, wherein the metal matrix is Ni-coated magnetic beads.
[18] A method for selecting a DNA encoding a protein having high affinity for a target substance using the mRNA / cDNA-protein fusion according to [10] or the cDNA-protein fusion according to [11] A method comprising the following steps (a) to (c) and (j) or steps (a) to (j):
(A) a step of immobilizing the target substance on a solid surface;
(B) An mRNA / cDNA-protein fusion or cDNA-protein fusion comprising a protein that binds to a target substance by contacting the mRNA / cDNA-protein fusion or cDNA-protein fusion with an immobilized target substance. The process of selecting the body,
(C) separating a cDNA encoding a protein from the selected mRNA / cDNA-protein fusion or cDNA-protein fusion, and PCR-amplifying the obtained cDNA fragment;
(D) A template DNA containing a sequence necessary for promoting a translation reaction and a sequence encoding a His-tag is prepared using the amplified cDNA fragment, transcribed into mRNA, and any one of claims 1 to 4. Annealing the nucleic acid linker according to claim 2 and ligating the 3 ′ end of the mRNA and the 5 ′ end of the nucleic acid linker to produce an mRNA-nucleic acid linker;
(E) An mRNA-protein fusion is prepared by synthesizing a protein from the mRNA of step (d) using a cell-free protein translation system and binding the C-terminus of the protein to puromycin in the nucleic acid linker. Process,
(F) binding and washing the mRNA-protein fusion to a metal matrix having an affinity for a His-tag sequence;
(G) subjecting the mRNA-protein fusion on the surface of the metal matrix to a reverse transcription reaction to produce an mRNA / cDNA-protein fusion;
Alternatively, a step of further degrading mRNA by causing an RNase to act on the mRNA / cDNA-protein fusion to produce a cDNA-protein fusion,
(H) washing the mRNA / cDNA-protein fusion or cDNA-protein fusion on the surface of the metal matrix and eluting the purified mRNA / cDNA-protein fusion or purified cDNA-protein fusion;
(I) a step of repeating the steps (b) and thereafter;
(J) The amplified DNA fragment obtained in the step (c) or the DNA fragment obtained in the step (c) after repeating the steps (b) to (i) at least once is used as a target substance. A process of selecting DNA encoding a protein with high affinity.
[19] In the step (a), the target substance is biotinylated in advance prior to immobilization, and a magnetic bead bound with streptavidin is used as the solid phase to be immobilized. 18].
[20] A method for identifying or obtaining a protein having a high affinity for a target substance,
A method comprising a step of recovering DNA encoding a protein having a high affinity for a target substance selected by the method according to [18] or [19] and determining a base sequence thereof.

The puromycin linker oligonucleotide (hereinafter referred to as “linker-N”) of the present invention does not contain a biotin molecule and a sequence for removing the molecule, and oligo dA is added to the base region of the side chain having puromycin. It has the main feature of having it. As a result, even with a cell-free translation system, high translation efficiency can be achieved, and a series of steps from purification of mRNA protein fusion to formation of cDNA protein fusion by reverse transcription and purification of cDNA protein fusion are performed. , All with a single metal (Ni) support surface could be performed quickly and with high yield to high purity. In addition, since the biotin molecule is not provided in the linker, the step of cutting the region containing the biotin molecule can be omitted, and the efficiency of the ligation reaction and the transcription reaction due to the removal of the steric hindrance material near the ligation. It also has the advantage of improved efficiency. In vitro selection by the cDNA display method using the linker-N of the present invention is completed in 6 to 8 hours in one cycle, and the time required for all rounds to obtain a functional peptide, for example, a binding peptide to a target substance, is 2 Within a week (Figure 15).
Furthermore, the present invention has developed an efficient method for producing puromycin linker oligonucleotides using branched deoxycytosine (5-Me-dC). By applying to a compact linker-N having no biotin molecule, it can be produced very simply and inexpensively.
Combining the cDNA display method of the present invention with a high-throughput automated system can be expected to contribute greatly to the rapid discovery of novel ligands and lead molecules. For laboratory-scale throughput, parallel processing of large numbers of samples is possible by using microtubes / microplates, and combinatorial drug discovery by combining with high-throughput systems (Non-patent Documents 35 and 36). It is expected to be an important support tool.

The structure of linker-N of the present invention, and a method for producing the same. (A) Four functional properties of Linker-N; ligation site for ligation by T4 RNA ligase, puromycin site for covalent binding of the synthesized protein, spacer sequence and oligo dA site, and synthetic cDNA Priming site for reverse transcription. (B) A method for producing linker-N. C in the figure indicates 5-Me-dC. Schematic diagram of (1) mRNA protein fusion and (2) cDNA protein fusion (cDNA display) formation. A gene construct introduced into a cDNA display. In the figure, SP6 represents the SP6 promoter and cap site, UTR represents the 5 ′ untranslated region of Xenopus β-globin, Spc represents the spacer sequence, and the Y-tag sequence has a sequence for further hybridization with the linker at the 3 ′ end. Including. Ligation reaction with PDO (3F, 6R14, BDA) mRNA and linker-N (ratio 1: 1). (A) FITC fluorescence detection after ligation of PDO mRNA and linker-N. In the figure, M represents a size marker, and 10 min, 20 min, and 40 min represent ligation reaction times, respectively. (B) Detection of all nucleic acid components by the Sybr Gold. (C) Sybr Green staining of all nucleic acid components after ligation of 3F, 6R14 and BDA mRNA and linker-N for 10 minutes, 3F on the left in the figure is 3F-mRNA used as a size marker. Ligation efficiency with PDO (3F, 6R14, BDA) mRNA and linker-N Examination of the length of oligo dA in linker-N. (A) Linker-N (18 A residues) (b) Linker-N-10A (10 A residues) (c) Linker-N-0A (0 A residues). Comparison of the ability to form protein fusions based on the length of oligo dA in the rabbit reticulocyte translation system. (A) Electrophoretic analysis (Fluorescein fluorescence image). (B) Comparison of fusion rate (%). Comparison of the ability to form protein fusions with different oligo dA lengths in the wheat germ extract translation system. (A) Maturation effect in the presence of a high concentration salt on the ability to form a protein fusion when linker-N is used. In the figure, 0: post-translational sampling for 10 minutes, 1; maturation for 1 hour in the absence of high concentration of salt, 2; maturation for 30 minutes in the presence of high concentration of salt, 3; maturation for 1 hour in the presence of high concentration of salt The fractions obtained are shown. (B) The ability to form a protein fusion when linker-N-10A and linker-N-0A are used. (C) Comparison of fusion rate (%). The conceptual diagram which shows the effect of presence of oligo dA in a linker. (A) Ribosome size. (B) When there is no oligo dA (linker-N-0A). (C) When oligo dA is present (linker-N). (D) A schematic diagram showing the effect of the “stem” structure of the oligo dA in the linker-N increasing the range of motion of the spacer flexible structure in response to a change in the position of the ribosome in the solution after protein synthesis. Purification of cDNA protein fusion using His-tag. In the figure, F.T. represents the flow-through fraction, E1 represents eluate 1 and E2 represents eluate 2, and PDO corresponds to the mRNA protein (PDO) fusion that has not undergone reverse transcription. The mRNA protein fusion is slower to migrate than the cDNA protein fusion. Therefore, the PDO lane is shifted downward for comparison of concentration. Demonstration of high-throughput cDNA display using linker-N and its in vitro selection. (A) Schematic diagram showing essential steps and required time for functioning a cDNA display using wheat germ lysate. Transcription of template DNA into mRNA, purification and quantification (˜1.5 hours). Ligating mRNA to linker-N (0.2 hours). A process of translation into a wheat germ lysate system and maturation under high salt conditions to form a display protein (mRNA protein fusion) (˜1 hour). Step of elution after purification of mRNA protein fusion on Ni-NTA magnetic beads, reverse transcription, and purification of cDNA protein fusion (0.5 hours). A step of selecting a target protein immobilized on a streptavidin (SA) matrix, followed by elution and amplifying DNA corresponding to the target protein (˜2 hours). PCR amplification analysis step (~ 1 hour). The whole process can be completed in 6-8 hours. (B) In vitro selection using PDO and 3F, 6R14, BDA display proteins. Bind 20: 1 mixture of non-binding PDO and binding BDA or 3F or 6R14 to (1) IgG binding to BDA, (2) AChBP binding to 3F, and (3) IL6R binding to 6R14 The selection fraction using the SA beads was subjected to electrophoresis after PCR. (C) The selection efficiency was expressed as “the number of fold enrichments” from the change in the ratio before and after the selection of each selection fraction obtained in (b). FLAG-tag sequence selection from 8-mer peptide library. (A) DNA construct for preparation of 8-mer peptide library. (B) Selection cycle. An 8-mer peptide library complex mixture containing 0.1% FLAG-tag sequence is subjected to a selection cycle process of transcription, ligated to linker-N, translated in WGE to synthesize mRNA fusion protein, and His-tag The cDNA fusion protein is synthesized after purification on a solid phase using reverse transcription, followed by reverse transcription, followed by purification, selection on an anti-FLAG M2 antibody matrix, and PCR amplification. The amplified product is further subjected to two cycles of the same process for cloning and sequencing. (C) Ratio of FLAG-tag sequence in the PCR amplification product after 3 selection cycles. In “puro-Linker” shown as a comparison, the same composite mixture was applied to the selection procedure of Non-Patent Document 13 and selection was performed three times. Three finger (3F) structure and gene construct of 3F library. Binding assay of cDNA protein fusion (R7) after 7 rounds of in vitro selection of VEGF affinity protein using 3F library targeting VEGF. (A) (1) 1/3 fraction of R7 to streptavidin beads (SA), (2) 1/3 fraction of R7 to VEGF immobilized beads, (3) R7 cDNA (R7 not translated) (4) After binding 1/3 fraction of R7 to Fc-immobilized beads, the eluted fraction was PCR amplified and analyzed by gel electrophoresis. . (B) ELISA was applied to each of the elution fractions after the binding steps (1), (2) and (4), and binding to beads was detected at a 450 nm wavelength absorption. Evolutionary engineering process with a new linker that enables shorter time The fully automated high-throughput system for finding lead compounds for drug discovery is about 8 hours per round, and lead compounds in about 2 weeks even if 8-10 rounds are performed That you can get. Estimated 3D structure of VEGF-binding peptide

1. About linker-N (1-1) Structure of linker-N The linker-N of the present invention is constituted as follows. As in the case of a normal T-shaped puromycin linker oligonucleotide, when a horizontal DNA strand is referred to as a main chain and a DNA strand having a vertical puromycin is referred to as a side chain, from the 5 ′ end side of the main chain, It consists of a “ligation site” with mRNA, a “hybridization region” with mRNA, and a “primer site” for reverse transcription, and the side chains from the base part to “oligo dA”, “spacer sequence” and puromycin The biotin molecule is not included in the linker. A typical sequence is shown as FIG. 1a, but the base sequence of each part is not limited.
Here, since there is no biotin molecule in the vicinity of the “ligation site” of the linker-N of the present invention, and no site for removing the biotin molecule from the linker is necessary, it is covalently bonded directly to the “hybridization site” with mRNA. Preferably, it is composed of a recognition sequence that can be ligated by T4 RNA ligase, for example, “CCC”. The “hybridization site” is appropriately designed in consideration of a sufficient length capable of stably hybridizing with mRNA using a GC sequence, ease of synthesis, and the like. For example, 10 to 50 mer, preferably 12 to 30 mer, more preferably 15 to 20 mer.
As described in (1-2) below, the side chain “spacer sequence” has a flexible structure, and together with “oligo dA”, puromycin is arranged near the P site in the ribosome. It can design suitably so that it may have sufficient length. The “Sp.18 (Spacer 18 Phosphoramidite)” molecule represented by the chemical formula “PO ₄ (CH ₂ CH ₂ O) ₅ CH ₂ CH ₂ PO ₄ ” is preferably used because of its flexibility and low steric hindrance. In the present invention, 2 to 8 are connected. Preferably 3-6, more preferably 4-5 are connected. In addition, it is preferable to bind a fluorescent compound for molecular labeling such as FITC or Fluorescein-dT in the middle of the spacer sequence. Fluorescein-dT, which is a typical fluorescent compound for molecular labeling, is also preferable, but Alexa and the like that are not easily affected by the environment containing the buffer are more preferably used. When using FITC as an example, `` (Sp.18 × 1-4) (T-FITC) (Sp.18 × 1-4) '', preferably `` (Sp.18 × 1) (T- FITC) (Sp.18 × 3) ”or the like can be used.
The distance to the puromycin of the side chain that combines the “spacer sequence” and “oligo dA” is 8 nm or more, preferably 10 to 20 nm, more preferably 12 to 14 nm, as will be discussed in (1-2) below. It is.

(1-2) Oligo dA In the present invention, the length of the “oligo dA” in the linker-N is 12 or more, preferably 15 to 25, more preferably 17 to 20 dA (deoxyadenine). The position in the linker is preferably provided near the branch point at a position away from puromycin. As a result, as shown in (1-3), the translation effect in the cell-free translation system becomes extremely large. Fig. 9 shows an image of this point. Protein binding to puromycin is believed to occur at the P site of ribosome A, but as shown in FIG. 9c, the size of eukaryotic ribosomal RNA is 25-30 nm in height (29.4 for wheat germ). nm), which is said to have a width of 27 to 31 nm (Non-patent Documents 32 and 33), the distance between the mRNA sandwiched between the ribosomal RNA and the P site in the ribosome is considered to be about 15 to 20 nm.
In Example 3, the length of the oligo dA was changed, and a translation experiment using RRL (rabbit reticulocyte lysate) and WGE (wheat germ extract) was performed. As a result, the oligo dA was provided in the linker. This is important for increasing the production efficiency of mRNA-protein fusions. Since “Linker-N-0A (0A)” used in Examples (3-1, 2) lacks oligo dA, “puro-linker” described in Non-Patent Document 13 and Non-Patent Document 27, 31 has a side chain length similar to that of the “SBP linker”. “Linker-N-0A” is 20% to 30% of the amount of protein fusion produced in any of the lysates in the case of “Linker-N” of the present invention. It is consistent with the results described.

(1-3) Synthesis method of linker-N As a synthesis method of linker-N of the present invention, a side chain with respect to the main chain is formed by a known method as in the case of conventional general puromycin-linker oligonucleotide. It can also be combined. For example, Amino-Modifier C6 dT is provided at the branching position of the puromycin-linker oligonucleotide main chain, 5′Tiol-Modifier C6 provided at the 5 ′ end of one side chain, and the crosslinking agent EMCS (N- ( 6-maleimidocaproyloxy) succinimide) can be used (for example, Patent Document 3).
However, the branched deoxycytidine 5-Me-dC (5′-dimethoxytrityl-N4- (O-levulinyl-6-oxyhexyl) -5-methyl-2′-deoxycytidine) developed in the present invention is used. It is preferable to bind 5-Me-dC in which a primer sequence is incorporated into a single-stranded puromycin oligonucleotide using the following synthesis method.
Specifically, a single-stranded puromycin oligonucleotide in which a 5′-ligation site / hybridization region and a puromycin-oligo dA-containing spacer sequence are linked by 5-Me-dC, for example, as shown in FIG. The described "5'-CCCCCCCGCCGCCCCCCG (5-Me-dC) A18 (Spec18) (Spec18) (Spec18) (F-dT) (Spec18) CC (Puro) -3 '" is synthesized with a DNA synthesizer or the like. Here, (F-dT) represents (T-FITC). After removing the levulinyl protecting group of 5-Me-dC, a primer sequence, for example, “5′-CCTTG-3 ′”, is linked to 5-Me-dC to easily produce the linker-N of the present invention. it can. The generation of “Linker-N” can be confirmed by TOF-MS and gel electrophoresis.
Since this synthesis method does not use a cross-linking agent, the step of producing a single-stranded puromycin oligonucleotide for linking primer sequences can use a DNA synthesizer, and can be performed at a low cost when commissioned. And the final yield of the linker-N of the present invention was more than 8 times higher than that of the conventional puromycin oligonucleotide linker, in which the step of using a crosslinking agent had to be performed manually (Table 1). The reproducibility of the downstream process can be greatly enhanced by greatly reducing the time and cost.

2. Preparation of mRNA-linker-N conjugate (2-1) mRNA library The mRNA to be linked to the linker-N of the present invention includes the case of an mRNA library. Transcribe and use DNA library.
The template DNA includes a sequence encoding a gene, a sequence necessary for promoting a translation reaction, a His-tag sequence, and other sequences. The main chain of linker-N is located at the 3 ′ end most. Complementary base sequences are included in the primer site and hybridization region corresponding to (Fig. 3).
Examples of sequences that encode genes include various functional proteins whose sequences are known to be molecularly evolved, such as DNAs that encode receptors, ion channels, enzymes, antibodies, bioactive peptides, or fragments thereof, or sequences Various known structural proteins, for example, DNA encoding actin, myosin, collagen, or fragments thereof, or various gene libraries, or various gene libraries containing sequences randomized by organic synthesis, PCR, etc. Can be mentioned. In particular, a DNA that has been developed as a high-throughput screening polypeptide library, has a three-finger-like scaffold (3F), and encodes a 3F polypeptide library in which loops 1 to 3 are randomized (Patent Document 7), Alternatively, it is preferable to use DNA encoding an ICK polypeptide library having an ICK scaffold and having randomized loops 1 to 3 and tail region (Patent Document 8). The polypeptide library of the high-throughput screening system will be described in detail in the following (5-1) using a typical “3F peptide library” as an example.
In addition, when applied to in vitro selection by the cDNA display method, this template DNA is prepared using the PCR amplification product obtained in the selection step.

It is preferable to have a 7-methylated guanosine cap structure at the 5 'end of the mRNA encoding the gene and a poly A tail structure at the 3' end in order to increase the efficiency of protein synthesis and promote the initiation of translation. It is further preferable to have an omega sequence (5 ′ untranslated region sequence of tobacco mosaic virus) that is known to have a Kozak sequence or a translation promoting effect in a wheat germ lysate system. In particular, it is preferable to provide “SP6 (SP6 promoter and cap site), UTR (Xenopus β-globin 5 ′ untranslated region), and Kozak sequence” used in the examples of the present invention at the 5 ′ end (FIG. 3). ).
The total length of mRNA used in the present invention is preferably 50 to 3,000 bases, more preferably 200 to 1,000 bases, from the viewpoint of ligation efficiency with a linker and translation efficiency in a cell-free translation system.

(2-2) Method for linking mRNA and linker
Ligation between mRNA and linker is performed by ligation after annealing by hybridization by a known technique. The ligation reaction can also utilize RNA ligase or DNA ligase that performs RNA / DNA ligation. For example, T4 RNA ligase or TS2126 thermostable phage-derived DNA ligase is preferable, and T4 RNA ligase is particularly preferable.

As the composition of the reaction solution for the annealing reaction and the ligation reaction of the present invention, a known reaction solution composition (for example, Patent Document 3) can be applied. For example, 10-250 mM Tris-HCl (pH 7.0-8.0); 2.0-50 mM magnesium chloride; 2.0-50 mM dithiothreitol (DTT); 0.2-5.0 mM ATP, annealing reaction After completion, 0.1-2.5 U / μl T4 polynucleotide kinase and 0.4-5 U / μl T4 RNA ligase are added to initiate the ligation reaction.
Known methods can also be applied to the annealing reaction conditions and ligation reaction conditions of the present invention, but a biotin molecule that causes steric hindrance during the reaction is also arranged near the ligation site of the linker-N of the present invention to remove the biotin molecule. Since the reaction proceeds very efficiently, the normal reaction time can be greatly shortened.
Specifically, the general annealing reaction is performed at 60 to 100 ° C. for 2 to 60 minutes with a heating device such as an aluminum block, and then switched off and left for 2 to 60 minutes to react with the reaction solution temperature. Gently lower and cool to -5-10 ° C. The ligation reaction is performed at 20 to 30 ° C. for 5 to 180 minutes.
In the present invention, such a general method can be applied with reduced time. In the embodiment of the present invention, in the annealing step, after heating at 85 to 94 ° C. for 30 seconds to 1 minute, it is gradually cooled to 25 ° C. over 10 minutes with a program of −0.1 ° C./sec, and in the ligation step, 25 C. for 10 minutes.
The volume of the reaction system for performing the ligation reaction is preferably 20 to 40 μl from the viewpoint of reaction efficiency.
In addition, the amount ratio of RNA to linker in the reaction system can react 1/3 to 6 times the amount of mRNA with respect to 10 to 20 pmol of linker, preferably 10 to 15 with respect to 10 pmol of linker. React with pmol of mRNA. In the present invention, the ligation efficiency is extremely high and the reaction proceeds almost stoichiometrically, so that it is not necessary to keep mRNA in excess. Therefore, in order to reduce excess mRNA during purification, in the present invention, it is most preferable to react mRNA and linker-N in equimolar amounts.

(2-3) Ligation efficiency with linker-N and mRNA of the present invention The efficiency and ligation time of the ligation of the present invention were verified using mRNA encoding a model protein. Examples of model proteins include PDO, Oct-1 Pou-binding domain; BDA, protein A B domain; 3F, three finger; and 6R14, interleukin-6 receptor A finger type ligand was used. When mRNA constructs from these mRNAs of different sizes (Figure 3) were reacted with linker-N (Figure 1) in an equimolar ratio, they were rapidly ligated within 10 minutes to reach 90-95% efficiency. (Figures 4a-c). Increasing the reaction time to 20 minutes or 40 minutes does not significantly increase the ligation efficiency. Here, formation of mRNA-linker complex was confirmed by digestion of mRNA with RNaseH (lanes “10 min + RNaseH” in FIGS. 4a and b).
Linker-N does not form complex secondary structures such as “biotin loops” such as “puro-linkers (Non-patent Documents 13, 22, etc.)”, and is a fully aligned hybridization region and simplification. Ligation site. Thus, it is considered that the annealing reaction was completed and stabilized quickly, and the ligation time was shortened and high ligation efficiency was achieved.

The following (Table 2) shows data comparing the time and efficiency of ligation between the linker-N (invention) and various puromycin linker-oligonucleotides of the conventional method and the translation efficiency. That is, the ligation strategy of each linker is a method called a sprint ligation method (Non-Patent Documents 10, 12, and 14), a psoralen crosslinking method (Non-Patent Document 24), a Y-ligation method, and the like (Non-Patent Documents 13 and 19). 22). The sprint ligation method used in the mRNA display of Non-Patent Documents 12 and 14 has a slow ligation reaction, low ligation efficiency, and takes time for gel purification from unused mRNA. The psoralen photocrosslinking method used in the mRNA display of Non-Patent Document 24 is extremely efficient (performed at a molar ratio of mRNA: linker of 1: 2) but fast, but UV irradiation can damage the mRNA In addition, an excessive linker removal step is required. Y-ligation by enzymatic action of T4 RNA ligase used in the cDNA display or mRNA display described in Non-Patent Documents 13, 22 and 25 is also very efficient, but the ligation time and the stoichiometry of mRNA and linker Various arguments have been reported.
In Table 2, when comparing only the ligation efficiency and translation efficiency, some puromycin linkers for mRNA display have high efficiency and have the advantage of at least one purification step, but mRNA display. In this case, compared to the case where a cDNA fusion (cDNA display) is formed, the mRNA itself is more easily decomposed and the secondary structure is more easily formed. (Nonpatent literature 12-14, 26).

3. Preparation of mRNA protein fusion By contacting the mRNA-linker-N conjugate of the present invention with a cell-free translation system, protein synthesis is performed in the ribosome A, and at the same time, the protein translated via puromycin and The mRNA-linker-N conjugate is fused to construct an mRNA-linker-N-protein fusion (hereinafter also referred to as mRNA protein fusion).
Examples of cell-free translation systems that can be used in the present invention include known rabbit reticulocytes and wheat germ extracts, as well as Escherichia coli and insect cells. Preferred are rabbit reticulocytes and wheat germ extract, with wheat germ extract being most preferred. In order to increase the translation efficiency, it is also effective to change the template DNA to a preferred codon that can be easily used by the selected species in accordance with the selected species of the translation system.
Hereinafter, the case of using a typical wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL) will be described in detail, but other cell-free translation systems can also be carried out by known methods according to these methods.

(3-1) In the case of wheat germ extract (WGE) When using wheat germ lysate as the cell-free translation system, it is prepared by a known method as the S30 fraction of the germ extract from wheat excluding the endosperm portion. It is already available as a kit from Promega (Madison, WI, USA).
The translation reaction in WGE is carried out at 20-40 ° C. for 10-40 minutes. Since the translation and fusion efficiency using the linker-N of the present invention is extremely high, a reaction time of 25 ° C. for 10 minutes is sufficient, but it is preferable to carry out the reaction at 25-30 ° C. for 10-20 minutes (FIG. 4).
Furthermore, it is effective to provide a maturation step under high salt concentration conditions after the translation reaction. Specifically, after the translation reaction, potassium chloride and magnesium chloride are added to the reaction system to obtain a high salt concentration condition, and the temperature as it is, for example, at 25 to 30 ° C. for 10 minutes to 2 hours, preferably 30 minutes to 1 Leave for hours. Meanwhile, it is considered that the fusion of the mRNA-linker-N conjugate and the protein can be completed and the three-dimensional structure of the protein can be accurately arranged. In that case, it is preferable to carry out potassium chloride in the presence of a final concentration of 500 to 1,000 mM and magnesium chloride in the presence of a final concentration of 50 to 100 mM. For example, 65 mM MgCl ₂ and 750 mM KCl are added and matured at 25 ° C. for 10 minutes to 1 hour (FIG. 8a lane 3).

(3-2) Rabbit reticulocyte lysate (RRL) When using a lysate of reticulocyte cells obtained from rabbit blood as the cell-free translation system, acetylphenylhydrazine is administered to the rabbit in advance for hemolysis. It is preferable that blood is collected after inducing anemia or the like and reared for several days, and it is more preferable to inactivate the nuclease by degrading cell-derived mRNA with micrococcal nuclease and chelating calcium with EGTA. Such a rabbit reticulocyte lysate is commercially available as an “IVT kit (Ambion, Austin, TX, USA)”, and the kit is also used in the examples of the present invention.
In the case of using RRL, it is sufficient to follow the description of the instruction manual such as “IVT kit”. For example, the mRNA-linker-N conjugate of the present invention is added to a reaction buffer containing RRL (for example, final concentration: 80 mM). Of potassium acetate; 0.5 mM magnesium acetate; 10 mM creatine phosphate; 0.05 mM amino acid (0.025 mM each for methionine and leucine) at 20-40 ° C. for 10-40 minutes. An RNA-degrading enzyme inhibitor such as SUPERase RNase inhibitor (Ambion) may be added to this reaction solution.
Moreover, it is preferable to provide a maturation step under high salt concentration conditions after translation as in the case of WGE.
For example, the translation reaction is performed at 30 ° C. for 10 minutes and then matured in the presence of 65 mM MgCl ₂ and 750 mM KCl at 37 ° C. for 2 hours.

(3-3) Production rate of the mRNA-protein fusion of the present invention The production rate of the fusion between the mRNA-linker-N of the present invention and the four types of translated model proteins (PDO, BDA, 3F and 6R14), Calculated by the following formula, the RRL case is shown in (FIG. 7b), and the WGE case is shown in (FIG. 8c) (Note that, as described in the above (1-2), the oligo dA in the linker was respectively shown. It is also measured when the length is changed.)
Fusion formation% = (A / A + B) × 100
(Here, A is the band intensity on SDS-PAGE of the mRNA-protein fusion, and B is the band intensity of the mRNA-linker that did not react.)
When the percentages of protein fusion formation when WGE was selected as the translation system (FIG. 8c) and when RRL was used as the translation system (FIG. 7b) were compared, the results when linker-N was used were compared. The value is 50 to 65% for RRL, while it is 90 to 95% for WGE. The formation% of 95% indicated for the BOD protein fusion is by far the highest value even when compared with the conventional puromycin linker (Table 2).

By the way, as shown in the above (Table 2), when the conventional puromycin linker is used (Non-patent Documents 13, 22, and 25), the case where WGE is used as the cell-free translation system is more RRL. Protein fusion formation efficiency is about 1.5 to 7 times higher.
These results show that the number of ribosomes in the WGE translation system is 2.5 × 10 ¹⁵ per ml (4 μM), whereas the number of ribosomes in the RRL translation system is 1.2 × 10 5 per 1 ml (0.2 μM). ^This is consistent with the conventional explanation (Non-Patent Document 8, 28 to 30) that is caused by showing a difference of one or more orders of ¹⁴ pieces.
Considering the ratio of ribosome to mRNA-linker, RRL is 1: 1-2: 1 (0.1-0.2 μM ribosome and 0.1 μM mRNA-linker), while WGE is This ratio is about 40: 1.
For these reasons, as a translation system applied to a high-throughput screening system using the cDNA display technology of the present invention, it is preferable to use a WGE translation system exhibiting high fusion production efficiency.

  Also, as discussed in (1-2) above, in (FIG. 7b) and (FIG. 8c), when attention is paid to the length of the oligo dA in the linker used for each protein, In all proteins, the maximum value is shown when the length of oligo dA is the longest, and such high fusion-forming ability in mRNA-linker-N of the present invention is attributed to the long oligo dA region in linker-N. I support you to do.

4). Purification of mRNA protein fusion / Preparation and purification of cDNA-linker-N-protein fusion (hereinafter referred to as cDNA protein fusion) (4-1) Metal (Ni) beads Purification of mRNA protein fusion of the present invention, The same metal carrier or metal-coated carrier is used for preparation of cDNA protein fusion by reverse transcription reaction and purification of the obtained cDNA protein fusion. The metal is preferably a metal having an affinity for the His-tag sequence, such as Ni or Co. The carrier can be used in various shapes, but is preferably on a bead from the viewpoint of ease of handling in the selection process, for example, styrene beads, glass beads, agarose beads, sepharose beads, magnetic beads, etc. are preferred, such as Ni. It is preferable to deposit a metal for use. Ni magnetic beads are commercially available as Ni-NTA magnetic beads (Qiagen). The His-tag sequence usually refers to a sequence of 6 consecutive histidines (His) (His × 6), but the number of consecutive histidines may be 5 to 10, preferably 6 to 8.

(4-2) Purification of mRNA protein fusion Purification of mRNA protein fusion using a His-tag sequence from a cell-free translation system, which has been considered difficult in the past, also uses metal beads such as Ni-NTA magnetic beads. And easy to do.
Specifically, Ni-NTA magnetic beads directly in a reaction solution of a cell-free translation system at a temperature of 25 ° C. to 30 ° C. have an adsorption capacity of 600 to 1500 pmol with respect to 10 to 20 pmol of the mRNA protein fusion. And let stand at 25 ° C for 10 minutes to cause binding via the His-tag, then 3 times with Wash Buffer (WB: 50 mM Na phosphate buffer pH 8.0, 300 mM NaCl, 20 mM imidazole) Wash once with PBS buffer.

(4-3) Reverse transcription step The washed Ni-NTA magnetic beads are directly subjected to a reverse transcription reaction.
Specifically, Ni-NTA magnetic beads are suspended in a reverse transcriptase reaction solution such as 50 mM Tris / HCl pH 8.0, 75 mM KCl, 3 mM MgCl ₂ , 0.5 mM dNTP, and reverse transcriptase such as 50-100 U of M-MLV reverse transcriptase (Takara) is added and reacted at 40-50 ° C. for 5-20 minutes to produce mRNA / cDNA protein fusion. The reaction is stopped by quenching to 10-0 ° C.

(4-4) Purification and elution of cDNA protein fusion The obtained mRNA / cDNA protein fusion can be purified as it is and used as a cDNA display. In the examples of the present invention, mRNA is not degraded without degrading mRNA. / CDNA protein fusion is used as it is, but using RNaseH, which has the resolution of mRNA that forms mRNA / cDNA, a cDNA protein fusion with single-stranded cDNA is prepared and selected for subsequent experimental steps Is also possible.
In general, the term “cDNA display” refers to both mRNA / cDNA protein fusions and cDNA protein fusions. Therefore, in the present invention, they are referred to as cDNA protein fusions without distinguishing between the two.

When creating a cDNA protein fusion, specifically, for example, react RNaseH (Ambion, Austin, TX, USA) with Ni-NTA magnetic beads containing mRNA / cDNA protein fusion at 37 ° C for 30 minutes. To degrade the mRNA complementary to the cDNA.
Next, the cDNA protein fusion (mRNA / cDNA protein fusion) on the metal (Ni) surface is washed with a buffer (for example, WB) and then eluted with an imidazole-containing solution. For example, Ni-NTA magnetic beads were washed 3 times with WB at 25-30 ° C. and then eluted with 250 mM imidazole (EB: 50 mM Na phosphate buffer pH 8.0, 300 mM NaCl, 250 mM imidazole) Elute after standing at 25 ° C for 5 minutes. This elution is performed once to several times, preferably twice. In the eluate, the cDNA protein fusion is purified with high purity (yield: about 60%, FIG. 10).
The obtained cDNA protein fusion is applied to a cDNA display method and subjected to evolutionary engineering screening. In this case, the cDNA protein fusion is sometimes referred to as cDNA display.

(4-5) Merits of one-step purification of the present invention
In the case of the cDNA display method, as previously discussed, it is useful for reducing mRNA degradation and the formation of mRNA secondary structure (especially when a library is used for selection) (Non-patent Documents 12 to 12). 14 and 26), in addition to the purification step from the cell-free translation system lysate of the mRNA protein fusion prior to the reverse transcription step, the purification step of the cDNA protein fusion is indispensable. . In the case of the conventional cDNA display method, at least two steps are required for each purification. In addition, even in the case of mRNA display, separation from cell-free translation lysates, purification steps for removal of ribosomes (due to the absence of stop codons), and translations that have been interrupted without being translated in full length In order to isolate a full-length protein, it is common to provide a two-step purification step as a purification step using a C-terminal tag. It may be 0.2 to 6.5% (Non-Patent Documents 13 and 16).
One of the major advantages of the present invention is that the purification of a cDNA protein fusion through reverse transcription and mRNA degradation from a cell-free translation lysate is performed in only one step of gentle affinity purification using a His-tag. The cDNA protein fusion could be isolated from the lysate to about 60-80% in a short time of less than 20 minutes (FIG. 10).
As described above (Table 2), in consideration of all the steps from the formation of mRNA protein fusion to the production of purified cDNA protein fusion for cDNA display, the method of the present invention is described in Non-Patent Document 13, The yield of cDNA protein fusion is about 60-300 times higher than the methods reported in 22 and 27 (Table 2). Thus, the high yield of cDNA protein fusions directly affects the diversity of mutant proteins obtained when applied to various libraries (Non-patent Documents 28 and 34), so the cDNA of the present invention. The display method has proven very useful for in vitro selection of functional proteins / peptides. Furthermore, the omission of the above-described purification process and the shortening of the time, as well as the availability of a magnetic matrix, indicate characteristics suitable for a high-throughput platform.

5). In Vitro Selection Using the cDNA Display Method of the Present Invention (5-1) Method for Preparing a Random Polypeptide Library Another embodiment of the present invention, the cDNA display method, efficiently screens proteins that have a high interaction with a target substance. Screening of cDNA displays prepared using random peptide libraries, especially with high interaction strength to target substances, and high-throughput proteins with enhanced interaction, such as high affinity proteins A method for screening is provided.
Here, as the “random polypeptide library”, a stable scaffold-forming polypeptide library having a randomized region in a part shown in the above (2-1) can be preferably used. In particular, a three-finger-like scaffold (3F) peptide library (Patent Document 7) or an ICK polypeptide library having an ICK scaffold (Patent Document 8) developed as a high-throughput screening polypeptide library is preferable.

Hereinafter, a three-finger-like scaffold (3F) peptide library, which is a typical high-throughput screening polypeptide library, will be described in detail.
Three-finger-like scaffold (3F) is commonly found in some α-neurotoxins derived from snake venom, as described in (Patent Document 7, Non-Patent Document 22). It is a structure having a small size of 60 to 70 amino acids, and includes 4 to 5 disulfide bonds, 3 to 5 antiparallel β-sheets, and 3 protruding loops forming a three-finger structure. Extremely stable. In particular, it has high thermal stability and can withstand 60 to 70 ° C. (Biochemistry. 2000 Aug 1; 39 (30): 8705-10). In addition, since it is small in size, it is unlikely to be recognized as an antigen and prone to protease degradation, so it is predicted that the half-life in serum is long.
In the present invention, when referring to “3F peptide library” or “3F random peptide library”, loop I and loop of three-finger-like scaffold (3F) constructed in (Patent Document 7, Non-Patent Document 22) A random peptide library in which amino acids in specific regions of II and Loop III are simultaneously randomized. Typically, a library in which each peptide constituting a 3F peptide library exists in a form corresponding to a polynucleotide (cDNA) encoding each peptide so that it can be used as a combinatorial library (Also referred to as cDNA display). However, a 3F polynucleotide library composed of mRNA or cDNA encoding the randomized 3F peptide library used to produce the cDNA display is referred to as a “3F peptide library”.
Methods for randomizing specific regions of Loop I, Loop II, and Loop III in the “3F peptide library” are known and described in detail in, for example, (Patent Document 7, Non-Patent Document 22). Yes.

Even though the three-finger-like scaffold (3F) is composed of different amino acids, the three-dimensional structure is highly conserved. For example, the three-finger-like scaffold (3F) is listed in [Table 1] in (Patent Document 7). It may be a library constructed using the 3F skeleton of neurotoxin derived from the origin organisms, but especially Micrurus corallinus (Kubo, T., et al., (2002), Program No. 137.16. ^{, 32 nd Annual Meeting of Society for} Neuroscience, Washington, DC, 3 rd November, 2002) derived from CTx3 peptide library (SEQ ID NO: 15) is preferred.

(3F) The peptide library is expressed as follows using the amino acid sequence.
A protein library comprising a group of proteins having a three-finger-like scaffold, the following regions:
(i) a region from the C-terminal second amino acid of C1-Cys to the N-terminal seventh amino acid of C2-Cys;
(ii) a region from the second amino acid on the C-terminal side of C3-Cys to the fourth amino acid on the N-terminal side of C4-Cys; and
(iii) A protein library characterized in that amino acid residues in the region from the fourth amino acid on the C-terminal side of C5-Cys to the first amino acid on the N-terminal side of C6-Cys are randomized.
Here, conserved cysteines that form disulfide bonds in the three-finger-like scaffold (3F) are referred to as C1-C8 in order from the N-terminal side, respectively, between C1 and C2, between C3 and C4, and C5. The regions between C6 are referred to as Loop I, Loop II and Loop III, respectively.

The most preferable CTx3-3F library is represented by the following amino acid sequence.
(Formula 1)
LVCY (X) ₆ PGTLETCPDDFTCV (X) ₁₀ TQYCSHACAIP (X) ₇ CCQTDKCNG (SEQ ID NO: 15)
(In the formula, X is an arbitrary amino acid residue.)

(5-2) Target substance which is the target of the cDNA display method of the present invention The “target substance” of the present invention is a substance for examining whether or not it interacts with the protein presented by the cDNA display of the present invention. Specifically, proteins, nucleic acids, sugar chains, lipids, steroids, organic or inorganic substances, low molecular compounds and the like can be mentioned. In any case, the sequence or structure may be known or unknown, and the function may be known or unknown. It is also possible to create a discrimination molecule (group) by targeting microorganisms and cells as a whole.
Preferred target substances include a disease-causing molecule that is a drug discovery target, a molecule involved in disease induction, or a biomarker for examination or diagnosis.
Here, the “interaction” between these target substances and the protein displayed in the cDNA display of the present invention is usually a covalent bond, hydrophobic bond, hydrogen bond, van der Waals between the protein and the target molecule. The effect | action by the force which acts on the molecule | numerator which arises from at least 1 among the coupling | bonding and the coupling | bonding by an electrostatic force is shown. Specifically, 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, nucleic acid and Binding and dissociation between binding proteins, binding and dissociation between proteins in the information transmission system, binding and dissociation between glycoprotein and protein, binding and dissociation between sugar chain and protein, etc. Examples include binding and dissociation of lipids and proteins, steroids and proteins, low molecular compounds and proteins, and the like.

(5-3) Preparation and Immobilization of Target Substance In the present invention, typically, the target substance is immobilized on the surface of a solid phase, and the binding reaction with the cDNA display of the present invention is observed in a liquid. It is also possible to apply a known method of fractionating a cDNA display library to form a microarray and concentrating the library fraction with a labeled target substance. However, the former can be overwhelmingly opposed to a library with a large number of parameters, which is extremely preferable for high-throughput selection.
Therefore, the former typical method will be described below.
Here, as the solid phase, known immobilization carriers can be used, for example, various materials such as beads such as styrene beads, glass beads, magnetic beads, glass substrates, plastic substrates, wells, membranes, Shaped ones can be used. Preferably, beads such as magnetic beads are preferred.
A known binding method can be used for binding between the target substance and the solid phase. For example, biotin-binding protein / biotin such as streptavidin, maltose-binding protein / maltose, antibody / antigen molecule (epitope peptide), G protein / Various guanine nucleotides, glutathione-S-transferase / glutathione, sequence-specific DNA or RNA-binding protein / specific sequence DNA or RNA, calmodulin / calmodulin-binding peptide, ATP-binding protein / ATP, estradiol receptor protein / estradiol, etc. Receptor protein / its ligand and the like. Among these, the most suitable combination for automation of in vitro selection is a streptavidin / biotin combination, and it is most effective to use the affinity with a streptavidin in which a biotinylated target substance is bound to a magnetic bead. preferable.
For example, in Example 7 of the present invention, a case where VEGF (vascular endothelial growth factor) and a VEGF binding peptide that inhibits receptor binding thereof are screened is shown. Human VEGF-A shown in SEQ ID NO: 20 (“VEGF 165” (PeproTech, London)) is biotinylated and immobilized on magnetic beads coated with streptavidin.

As a method for biotinylating a target substance, a known method can be applied. For example, when the target substance is a protein, a base capable of binding biotin (for example, deoxythymine (dT)) or a base bound to biotin (for example, biotin- Deoxythymine (Biotin-dT)), a base modified with an amino group (eg, amino-modified deoxythymine (eg, Amino-Modifier C6-dT: manufactured by Glen Research Search)), a base modified with a carboxy group (eg, , Carboxy-modified deoxythymine (Carboxy-dT)), a base modified with a thiol group (for example, thiol-modified deoxythymine (4-Thio-dT)) and the like. Commercially available EZ-Link Sulfo-NHS-SS-biotin (Pierce, Rockford, USA) can also be used.
Magnetic beads bound with streptavidin can be prepared by coating streptavidin on the surface of beads such as Ni-NTA magnetic beads by a known method, but commercially available streptavidin-coated beads (Magnotex-SA (Takara Bio) or Magnosphere ^™ ) MS300 / Streptavidin (Takara Bio) may be used.
A suitable ratio for immobilizing the target protein on the streptavidin-coated beads can be appropriately set depending on the target molecule.

If necessary, the target substance can be used after being labeled with a labeling substance.
Examples of such a labeling substance include a fluorescent compound and an epitope peptide of an antigen. The fluorescent compound is preferably a fluorescent dye such as fluorescein isothiocyanate (FITC), phycobiliprotein, rare earth metal chelate, dansyl chloride or tetramethylrhodamine isothiocyanate. When the target substance is a protein, FITC-dT or Fluorescein- More preferably, dT is used.

(5-4) Method for Measuring Interaction In the cDNA display method of the present invention, it is determined whether or not the protein being displayed interacts with the target substance. 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)).

(5-5) Identification method of cDNA-displayed protein and / or target substance In the cDNA display method of the present invention, if necessary, display in a protein-target substance conjugate determined to be interacting Identify proteins and / or target substances. Identification of the displayed protein is performed by PCR amplification of the cDNA in the cDNA protein fusion and analysis of the base sequence. Identification of a target substance can be performed using NMR, IR, various mass spectrometry, etc., using a label that has been labeled in advance. If the labeling substance is a protein, it can also be performed with a normal amino acid sequencer.

(5-6) All Steps of In Vitro Selection In vitro selection using the cDNA display method using the linker-N of the present invention is not limited to the following procedure, but will be described according to (FIG. 11a).
(A) Transcribing template DNA (for example, random peptide library) to mRNA, purifying it, and quantifying it as appropriate (mRNA library): ~ 1.5 hours.
(B) Ligating mRNA to linker-N (mRNA-linker-N): 0.2 hours.
(C) Step of maturation under high salt condition (mRNA-protein fusion) after translation in wheat germ lysate translation system: ~ 1 hour.
(D) A step of purifying an mRNA protein fusion on Ni-NTA magnetic beads, performing reverse transcription, and purifying the cDNA protein fusion and then eluting it (cDNA protein fusion): 0.5 hours.
(E) A step of binding a cDNA protein fusion to a target protein immobilized on SA beads or the like, washing, and eluting: ~ 2 hours.
(F) PCR amplification of the eluted cDNA protein fusion cDNA and determining the DNA sequence, or preparing the template DNA and subjecting it to step (a) again:
The whole process can be completed in 6-8 hours.

(5-7) Evaluation of the selection method of the present invention An experiment for evaluating the selection ability of only a candidate molecule that specifically binds from a molecule that does not specifically bind in a library molecule, which is most important for display technology, is concretely performed. The following procedure was carried out (Example 6).
A known ligand (FLAG tag) and a random 8-mer peptide library were mixed at a ratio of 1: 1000, and an evaluation experiment was performed (FIG. 12). For comparison, “puro-linker (Non-Patent Document 13)” was used, and the entire process was performed according to the method of Non-Patent Document 13. When the immobilized anti-FLAG M2 antibody was carried out three times at the same time in both methods, when the cDNA display method of the present invention using linker-N was applied, 80% (16 / 20) The FLAG tag was detected, whereas the puro-linker was 20% (4/20). This result shows that the enrichment rate of candidate molecules that specifically bind is improved by using linker-N. That is, during the selection process of the present invention, it was shown that excess mRNA-linker-N conjugate, mRNA protein fusion, and the like that would cause “noise” could be efficiently removed from the system and the S / N ratio could be reduced. .

The advantages of the linker-N cDNA display compared to the puro-linker (Non-patent Document 22) are summarized as shown below (Table 3). In the preparation of the library using the 3F library, puro-linker is 4 × 10 ¹⁰ molecules / ml per 1 ml of WGE lysate, whereas in the case of linker-N, 6 × 10 ¹² molecules are obtained. Can do. The 3F library used was prepared with an NNS codon (N = A, T, C or G; S = C or G) encoding any amino acid including a stop codon. In Non-patent Document 22 using puro-linker, a library of 1.2 × 10 ¹¹ size per 3 ml was used, whereas in Example 5 of the present invention, a library of 3 × 10 ¹² size per 0.5 ml was used. Prepared and used. There is a big difference in the number of selection rounds required to converge to the same selectivity (60-70%), both in the case of linker-N compared to 10 rounds in the case of “puro-linker” It is 7 rounds, and it turns out that efficiency is about 25-30%. The results of Example 6 using the same target (anti-FLAG antibody) also demonstrated that the efficiency of the cDNA display method of the present invention is 4 times that of the conventional method (FIG. 12c). As will be described later, in the VEGF-binding peptide selection experiment using the 3F random peptide library with linker-N in Example 7, the dissociation constant (Kd value) is about 1 order of magnitude smaller than that of the conventional method. A novel peptide with high affinity could be obtained (2.1 nM: 48 nM). When the affinity was compared about the peptide which occupies the main population (maximum ratio) with the candidate peptide selected by each method, the difference of about 2 order had arisen (2.1nM: 152nM). This is consistent with the theory that selection results depend on the diversity of the initial library. These results reveal the advantage that cDNA display with linker-N can be used to obtain good candidates at a lower cost and labor in an accelerated manner.

6). Method for Screening Gene Encoding Functional Protein of Interest from Known Random Peptide Library When applied to a so-called random peptide library having a large library size, the basic steps are (FIG. 11) or (FIG. 12b). This is the same as the selection process diagram shown in). Although depending on the library size, 3 to 20 rounds, preferably 5 to 15 rounds, more preferably 7 to 10 rounds are usually performed in order to screen for high-affinity novel peptides for the target substance.
As the applicable library size, even if the cDNA display molecule is 10 ¹² molecules or more, more than 10 ¹³ or 10 ¹⁴ or more per ml, it can be applied by increasing the number of rounds.
In Example 7 of the present invention, a 6 × 10 ¹² / ml cDNA display was prepared by applying to a 3F library (Non-patent Document 22) which is a known typical random peptide library, and magnetic beads were prepared. By conducting 7 rounds of affinity screening with VEGF immobilized on the surface, a novel VEGF affinity polypeptide (Kd = 2.1 ± 0.5 nM) could be screened at a high rate of 60% (Tables 3 and 4).

7). VEGF-binding peptide (7-1) VEGF-binding peptide having three-finger-like scaffold (3F) About 60% of three types of novel VEGF affinity polypeptides obtained using the cDNA display method of the present invention G-1 group polypeptides occupying a high affinity of Kd = 2.1 ± 0.5 nM (Table 4).
The VEGF-binding peptide has a three-finger-like scaffold (3F) (Patent Document 7, Non-Patent Document 22) as a basic skeleton, and the amino acid sequences of specific portions of Loop I, Loop II and Loop III are “PLTRVV”, respectively. (SEQ ID NO: 6) ”,“ HGDHHTLSEW (SEQ ID NO: 7) ”and“ EEPTAHV (SEQ ID NO: 8) ”.
The three-finger-like scaffold (3F) of the basic skeleton in the Examples is one embodiment of the VEGF-binding peptide of the present invention because it uses a CTx3-3F library derived from CTx3 (Non-patent Document 15). The VEGF binding peptide (peptide G-1) having CTx3-3F is represented by the amino acid sequence of the following (Formula 2).
(Formula 2)
LVCYPLTRVVPGTLETCPDDFTCVHGDHHTLSEWTQYCSHACAIPEEPTAHVCCQTDKCNG (SEQ ID NO: 16)
The VGEF-binding peptide (peptide G-1) showed high VEGF specificity in both PCR analysis and ELISA binding assay. The predicted conformation diagram for peptide G-1 is shown in FIG.

(7-2) Optimization of VEGF-binding peptide size The three-finger-like scaffold (3F) used in the present invention is composed of a total length of 61 amino acid residues in the case of 3F derived from CTx3, for example. There are three loops, loop I, loop II, and loop III, and each loop has a structure that is firmly fixed by an SS bond in the terminal region. Therefore, the peptide “1F scaffold” having a small size of 12 to 25 amino acid residues cleaved leaving Cys for forming the terminal SS bond of each loop structure also retains the original three-dimensional structure. In Non-Patent Document 14, it is demonstrated that the inhibitory activity, agonist activity, and antagonist activity similar to those shown in the original 3F are maintained in the 1F peptide containing loop I.
The VEGF-binding peptide of the present invention does not need to be a full-length peptide that can constitute a 3F structure, but includes any one of loop I, loop II, and loop III, and can constitute at least a “1F scaffold”. If present, it has the same VEGF-binding activity as the full-length VEGF-binding peptide having a 3F structure and can be expected to have VEGF inhibitory activity. In addition to the ease of synthesis, the small size of the full-length peptide provides advantages such as small antigenicity and serum half-life.
From the above, the VEGF-binding peptide of the present invention is preferably optimized to further reduce the size, and other aspects of the present invention are any of the following (Formula 3) to (Formula 5) It can be said that the peptide comprises the amino acid sequence of In particular, it is a peptide containing any one of the amino acid sequences of (Formula 3) to (Formula 5), and more preferably a partial peptide of (Formula 2). At that time, among the amino acids constituting 1F, Cys that is not involved in cyclization is preferably changed to another amino acid. According to Non-Patent Document 22, (Formula 3) and (Formula 4) Cys is replaced with Gly. In addition, amino acids outside Cys at both ends used for cyclization, for example, “Leu-Val” on the N-terminal side and “Val” on the C-terminal side in (Formula 3) can be replaced with other amino acids.
(Formula 3)
LVCYPLTRVVPGTLET G PDDFTCV (SEQ ID NO: 17)
(Formula 4)
ETCPDDFT G VHGDHHTLSEWTQYCS (SEQ ID NO: 18)
(Formula 5)
ACAIPEEPTAHVC (SEQ ID NO: 19)

8). Automated high-throughput system using the present invention As described in (5-5) above, the present invention is designed so that each process is extremely simplified and can be performed in a short time. It can be applied to a fully automated high-throughput system for finding lead compounds for drug discovery (FIG. 15). For example, a lead compound can be obtained in about 2 hours in 8 to 10 rounds for about 8 hours per round.

EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.
Other terms and concepts in the present invention are based on the meanings of terms that are conventionally used in the field, and various techniques used to implement the present invention include those that clearly indicate the source. Except for this, it can be easily and reliably carried out by those skilled in the art based on known documents and the like. In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, kit instruction manuals, catalogs, and the like.
In addition, the description content in prior art documents, patent publications, and patent application specifications cited in the present specification shall be referred to as the description content of the present invention.

(Example 1) Synthesis of nucleic acid linker (linker-N) of the present invention 5'-CCCCCCCGCCGCCCCCCG (5-Me-dC) A18 (Spec18) (Spec18) (Spec18) (F- A puromycin-oligonucleotide consisting of dT) (Spec18) CC (Puro) -3 ′ was synthesized by DNA synthesizer ABI394 (Applied Biosystem, Japan) outsourced to BEX (Japan). The 5 ′ end of the puromycin-oligonucleotide was protected by acetylation. In the formula, the symbol (5-Me-dC) represents 5′-dimethoxytrityl-N4- (O-levulinyl-6-oxyhexyl) -5-methyl-2′-deoxycytidine (Glen Research). (Spec18) indicates C18 spacer phosphoramidite, (F-dT) indicates fluorescein-dT, and (Puro) indicates puromycin CPG.
The levulinyl protecting group of 5-Me-dC was removed with 0.5 M hydrazine hydrate in 1: 1 pyridine / acetic acid and the column was further washed with pyridine / acetic acid (1: 1) and then with acetonitrile. Primer sequence 5′-CCTTG-3 ′ was linked to 5-Me-dC in puromycin-oligonucleotide to branch the oligonucleotide chain. The resulting puromycin-branched oligonucleotide was cleaved from the column with K ₂ CO ₃ in methanol to deprotect the 5 ′ terminal acetyl group. In order to complete this deprotection, the reaction was carried out in 25% ammonium hydroxide at 65 ° C. for 1.5 hours and purified by reverse phase HPLC to purify the puromycin-linker oligonucleotide of the present invention (hereinafter “linker- N ”) was obtained (FIG. 1). The generation of “Linker-N” was confirmed by TOF-MS and gel electrophoresis. All phosphoramidite reagents used for modification were obtained from Glen Research (Sterling, VA, USA).

(Example 2) Ligation efficiency of linker-N (2-1) Preparation of template DNA construct In order to examine the ligation efficiency of linker-N, the template DNA of mRNA linked to linker-N was used as a B-protein of known protein A. Domain (BDA: Non-patent document 20), Oct-1 Pou domain (PDO: Non-patent document 21), Three finger (3F: Non-patent document 22) and IL-6 receptor (6R14: Non-patent document 22) It was prepared using DNA.
“BDA (B-domain of protein A)” is amplified from the pEFZZ18 protein A fusion vector (GE Healthcare) described in Non-patent document 20, and “PDO (Pou domain of Oct-1)” is non-patent document. Synthesized according to 21. According to Non-Patent Document 22, “3F (three finger)” is cloned from the South American coral snake Micrurus coralinus, and “6R14” (3F peptide having binding activity with human IL6 receptor α) is described in Non-Patent Document 22. PBADThio / TOPO vector IL6R10-14.
The 5 ′ terminal side of these DNAs is linked to “an oligonucleotide containing SP6 promoter / cap site / Xenopus β-globin 5 ′ untranslated region (UTR)” and a translation initiation codon (ATG) described in Non-Patent Document 22, and On the 3 ′ end side, “spacer (G3S) ₂ / His-tag sequence (6 × His) / spacer (G3S) / Y-tag sequence” was added and amplified by PCR to obtain a template DNA construct ( FIG. 3). Here, the spacer (G3S) is “GGGS (G = glycine; S = serine)”. PCR amplification was performed with a 20 second denaturation (94 ° C) step, a 15 second annealing (60 ° C) step and a 30 second extension step (72 ° C).

(2-2) Ligation between linker-N and various mRNAs The four types of template DNA constructs obtained in (2-1) were respectively obtained at 37 ° C. in RiboMAX Large Scale Production System (Promega, Madison, WI, USA). Transcripted with SP6 RNA polymerase for 1 hour to produce capped transcripts and purified with RNeasy purification kit (Qiagen) to obtain BDA-mRNA, PDO-mRNA, 3F-mRNA, 6R14-mRNA, with absorbance at 260 nm Quantified.
Next, each mRNA and linker-N were annealed at a ratio of 1: 1 (50 pmol) at 94 ° C. for 15 to 30 minutes, and then gradually cooled to 4 ° C. Ligation was performed at 25 ° C. for 10 minutes, 20 minutes and 40 minutes using 3U T4 kinase (NEB, USA) and 20U T4 RNA ligase (Takara, Japan). The formation of an RNA-linker complex can be confirmed by degrading mRNA hybridized with DNA with RNaseH (Ambion, Austin, TX, USA) and performing electrophoresis on a denaturing polyacrylamide gel.
(Figure 4) shows an example of ligase reaction of PDO-mRNA and linker in the presence or absence of RNaseH for 10 minutes, 20 minutes, and 40 minutes, and ligase reaction for 10 minutes. These were separated by denaturing polyacrylamide gel electrophoresis (6% gel and 8M urea). The results were obtained by using a fluorescence image (FIG. 4a) by FITC incorporated in linker-N and a nucleic acid staining image (FIG. 4b) by Sybr Gold (Molecular Probes, USA) with fluoroimagers (Bio-Rad, Hercules, CA, USA). FIG. 4c shows similarly the ligation results after 10 minutes of 3F-mRNA, 6R14-mRNA and BDA-mRNA other than PDO and linker-N (Sybr Gold staining).
Next, the ligation efficiency of each was calculated by the following formula, and the following results were obtained: PDO 96 ± 4%, 3F 91 ± 5%, 6R14 95 ± 3%, BDA 96 ± 2% (FIG. 5).
Ligation% = (A / A + B) × 100
(A in the formula is the band intensity of the ligated product and B is the intensity of the residual mRNA).
The above results indicate that Linker-N achieves both a low linker: mRNA ratio and high ligation efficiency compared to conventional nucleic acid linkers for cDNA display or mRNA display such as puromycin linker. (Table 2).

(Example 3) Optimization of the length of "oligo dA" in linker-N from the viewpoint of protein translation efficiency in various cell-free translation systems (3-1) Synthesis of mRNA-linkers having different oligo dA lengths The length of oligo dA in N was changed to 18, 10, 0, and linker-N (18A), linker-N-10A (10A) and linker-N-0A ( 0A) was synthesized (FIG. 6). Using each linker, an mRNA (PDO, BDA, 3F, 6R14) -linker fusion was prepared for each linker in the same manner as in Example 2 (2-2).

(3-2) Rabbit Reticulocyte Translation System Each mRNA (PDO, BDA, 3F, 6R14) -linker fusion is used as a rabbit reticulocyte lysate IVT kit (Ambion, Austin, TX, USA) (hereinafter referred to as “Rabbit Reticulocyte Lysate”). , And referred to as RRL), each mRNA was translated, and mRNA (PDO, BDA, 3F, 6R14) -protein fusion was prepared.
Specifically, according to Non-Patent Documents 13 and 22, 100 nM of mRNA-linker fusion was added to 25 μl of RRL reaction system (16.75 μl of lysate, 80 mM potassium acetate, 0.5 mM magnesium acetate, 10 mM creatine phosphate, 0.05 mM amino acid (methionine). And leucine were each 0.025 mM)) and the protein synthesis reaction was carried out at 30 ° C. for 10 minutes. The protein fusion (ie, covalent binding to the puromycin portion of the protein) was then matured in the presence of 65 mM MgCl ₂ and 750 mM KCl for 2 hours at 37 ° C. Sampling for electrophoresis analysis was performed at 10 minutes (FIG. 7a).
The efficiency of fusion formation was calculated according to the following formula, and the values for linker-N (18A), linker-N-10A (10A) and linker-N-0A (0A) for each mRNA were PDO (18A, 60 ± 5; 10A, 30 ± 2; OA, 20 ± 3), BDA (18A, 65 ± 3; 10A, 35 ± 3; 0A, 22 ± 2), 3F (18A, 50 ± 4; 10A, 30 ± 3) 0A, 18 ± 3) and 6R14 (18A, 55 ± 5; 10A, 35 ± 4; 0A, 20 ± 2).
Fusion formation% = (A / A + B) × 100
(Here, A is the band intensity of the protein fusion (mRNA-linker-protein), and B is the band intensity of the remaining mRNA-linker.)
The ability of each mRNA-linker to form a fusion is shown in FIG. 7b.
In any mRNA, it was found that the longer the oligo dA in the linker, the higher the translation efficiency.

(3-3) Wheat germ extract translation system About the fusion of each mRNA (PDO, BDA, 3F, 6R14) and linker-N, wheat germ extract (Wheat Germ Extract (Promega, Madison, WI, USA)) (Hereinafter referred to as WGE), each mRNA was translated in the same manner as in (3-2) above, and an mRNA (PDO, BDA, 3F, 6R14) -protein fusion was prepared.
For translation in WGE, 100 nM of mRNA-linker fusion was performed in 25 μl of WGE reaction system (12.5 μl of lysate, according to other product protocol), followed by protein synthesis reaction at 25 ° C. for 10 minutes, and then at 65 mM for 1 hour at 25 ° C. It matured protein fusions with the addition of KCl MgCl ₂ and 750 mM. In order to verify the effect of high-concentration salt, it was also carried out as a control when it was matured for 1 hour in the absence of high-concentration salt.
Electrophoretic analysis was sampled at 10 minutes (ie before the addition of high-concentration salt), 30 minutes and 1 hour (after addition of high-concentration salt) (FIG. 8a). In FIG. 8a, the 0 fraction shows the fraction sampled after 10 minutes of translation, 1 fraction shows the control fraction cultured for 1 hour without high concentration salt, 2 fractions contain high concentration salt and 30 fractions. Minutes and 3 fractions represent fractions cultured for 1 hour, respectively. As a result, it can be seen that the addition of a high concentration of salt in the translation process is effective, and the one-hour maturation process is also effective.
Subsequently, each mRNA (PDO, BDA, 3F, 6R14) was fused with linker-N10A and linker-NOA in the same manner in WGE at 25 ° C. for 10 minutes, and then in the presence of high-concentration salt. Time maturation (Figure 8b).
The percentage formation of each mRNA protein fusion was calculated according to the above formula, and the values for Linker-N (18A), Linker-N-10A (10A) and Linker-N-0A (0A) for each mRNA were PDO (18A , 90 ± 5; 10A, 65 ± 3; 0A, 30 ± 4), BDA (18A, 95 ± 4; 10A, 60 ± 6; 0A, 35 ± 4), 3F (18A, 85 ± 4; 10A, 60 ± 4; 0A, 30 ± 4) and 6R14 (18A, 90 ± 6; 10A, 60 ± 5; 0A, 35 ± 5) (FIG. 8c).

When the% of protein fusion formation in the case where WGE is selected as the translation system (FIG. 8c) and the case where RRL is used as the translation system (FIG. 7b) are compared, the value at linker-N is RRL. Is 50 to 65%, whereas WGE shows an extremely high value of 90 to 95%.
The difference is as much as one order in terms of the formation amount of 2.5 × 10 ¹⁵ per 1 ml (4 μM) and 1.2 × 10 ¹⁴ per 1 ml (0.2 μM) in the RRL translation system.
From these results, it is demonstrated that the translation efficiency increases as the length of the oligo dA in the linker increases in WGE translation. The number of dA (deoxyadenine) is at least 10 or more, preferably 12 or more. Was found to be necessary.
It is confirmed that “oligo dA” in linker-N contributes to proper placement of puromycin near the P site of ribosome A via a flexible spacer sequence, as in (FIG. 9d). It was.

(Example 4) mRNA (PDO, BDA, 3F, 6R14) each protein fusion purification and reverse transcription invention protein fusions, prior to reverse transcription (RT), previously provided with His- tag protein Was used for purification for 10 minutes at 25 ° C. on Ni-NTA magnetic beads (Qiagen), which is a kind of IMAC (Immobilized metal affinity chromatography) carrier.
The beads were washed with 1X PBS buffer, 20 U of M-MLV reverse transcriptase (Takara, Japan) was added, RT was performed at 42 ° C. for 10 minutes, and placed on ice.
After several washes, the fusion was eluted twice with 250 mM imidazole at 25 ° C. for 5 minutes.
Each of the obtained cDNA protein fusions was reacted with RNaseH (Ambion, Austin, TX, USA) at 37 ° C. for 30 minutes to decompose and remove mRNAs forming mRNA / cDNA duplexes. As an example, FIG. 10 shows a case where a PDO mRNA protein fusion is used. Each sample was separated by 12% SDS-PAGE containing 8M urea, and the band was confirmed with a fluoroimager. It can be seen that the reverse transcript from either the first (E1) or the second (E2) eluate from the magnetic beads is very pure (FIG. 10).
This means that the mRNA protein fusion is purified, reverse transcription, mRNA degradation and removal, and cDNA protein fusion while the mRNA protein fusion is bound to the surface of the magnetic beads by simple IMAC using His-tag. This means that the entire purification process of the body could be performed efficiently and in a short time, and that the cDNA protein fusion could be purified with high purity and high yield.
In the figure, FT is the filtered fraction, E1 and E2 are the eluates 1 and 2 that have been reverse transcribed and mRNA removed by RNaseH treatment. PDO corresponds to the mRNA protein fusion, but the cDNA protein fusion. In order to show the quantity ratio, the position of the PDO band is shifted upward.

(Example 5) In vitro selection In this example, IgG recognized by BDA, AChBP recognized by 3F, and IL6-R recognized by 6R14 were immobilized on magnetic beads as models, and each target protein was BDA of the present invention. The in vitro selection efficiency of the present invention is evaluated using a PDO cDNA protein fusion as a control together with 3F and 6R14 cDNA protein fusions.

(5-1) Preparation of Magnetic Beads Immobilized with Biotin-Labeled Target Protein Among target proteins used for selection, immunoglobulin G (rabbit IgG) is an interleukin-6 receptor (human IL6Rα) from Sigma. And VEGF (human VEGF165) were purchased from Pero Tech (London, UK). Acetylcholine binding protein (AChBP) cDNA was cloned from Aplysia kurodai [manuscript in preparation], expressed in recombinant E. coli and purified.
In order to immobilize these target proteins on magnetic beads, they were biotinylated using EZ-Link Sulfo-NHS-SS-biotin (Pierce, Rockford, USA) at a molar ratio of 1:10. Unreacted biotin was inactivated with 1M Tris-HCl (pH 8) and removed by dialysis at 4 ° C. for 16 hours. Each biotinylated target protein was confirmed by SDS-PAGE and Western blot using streptavidin-HRP (GE Healthcare). Quantitative immobilization was checked by titrating at 25 ° C. for 30 minutes against a fixed amount of magnetic beads coated with streptavidin (Takara, Japan). The supernatant protein was stored and immobilized, and the target protein was eluted with 100 mM dithiothreitol (DTT; Wako, Japan) at 25 ° C. for 10 minutes.

(5-2) In vitro selection Subsequently, each cDNA protein fusion of PDO and BDA, 3F, and 6R14 was immobilized on the magnetic beads immobilized with the target protein (IgG, IL6R, AChBP) prepared in (5-1) above. To react.
Non-binding PDO and binding BDA (or 3F, 6R14) mRNA- (linker-N) complex were mixed at a ratio of 20: 1, and translated and modified by the same steps as in Example 4 to obtain cDNA. Display protein.
Here, in the case of a ligand rich in disulfide such as 3F and 6R14, it is assumed that a fusion of 70% or more of protein disulfide isomerase (PDI) (input template is formed according to Non-Patent Document 23). (1: 1 molar ratio to cDNA display) in the presence of 10 mM oxidized glutathione (GSSG) and 1 mM reduced glutathione (GSH) and subjected to translation, refolding and maturation steps.
As in Example 4, 50 μl of each sample eluted with His-tag was mixed with 150 μl of selection buffer (SB; 1 × PBS, 100 mM NaCl and 0.1% Tween-20), and 30 minutes at 25 ° C. IgG, IL6R and AChBP were incubated with immobilized magnetic beads. All dithiothreitol (DTT) is removed from the buffer used for each selection.
The beads were washed 10 times with 200 μl of the same buffer, then eluted with 100 mM DTT (micro-spin column, GE Healthcare) and desalted (10 mM TrisHCl pH 7).
The “before selection” fraction and the eluted “after selection” fraction were amplified for 25 cycles using the following 0.2 mM PS and Pv3A primers (combined to Greiner), respectively (denaturation (94 ° C.)) 20 seconds; annealing (60 ° C), 15 seconds; extended (72 ° C), 20 seconds).
PS: 5'-ATTTAGGTGACACTATAGAATACAAGCTTGCT-3 '(SEQ ID NO: 1) and
Pv3 A: 5'-TTTCCCCGCCGCCCCCCGTCCTGCTTCCGCCGTGATGAT-3 '(SEQ ID NO: 2)
Denaturing gel electrophoresis (4.5% gel and 8M urea) was performed, and the amount of DNA encoding each model protein was quantitatively analyzed by Sybr Gold staining.
A series of these steps is shown as FIG. 11a. The average time required from the initial template DNA to mRNA transcription step to the final analysis step is also included. Thus, each process is performed in a very short time, and it is also proved that the screening method of the present invention is performed with high throughput.
The model (1) PDO / BDA and IgG combined with BDA, (2) PDO / 3F and AChBP combined with AChBP, and (3) PDO / 6R1 and IL6R combined with IL6R. It was selectively amplified compared to PDO (FIG. 11b). (1)-(3) “Fold Enrichment Number”, which is the ratio of change in quantitative PCR before and after each selection, is (1) 40 ± 5%, (2) 30 ± 3 %, (3) 35 ± 2% (FIG. 11c).

(Example 6) Selection using library of 8-mer peptide mixture corresponding to FLAG-tag sequence
An 8-mer peptide library, which is a mixture of randomized 8-mer peptides corresponding to “DYKDDDDK (SEQ ID NO: 3)” required for binding of the FLAG-tag to the anti-FLAG M2 antibody, is prepared as follows. did.
The 8-mer peptide library was constructed using two oligonucleotides with complementary “overlap sequences”. (1) upstream primer; oligonucleotide having SP6 promoter and cap on the most 5 'side, followed by 5'-UTR, Kozak and ATG followed by "overlap sequence";
(2) Downstream primer; a mixture of oligonucleotide fragments constructed so that an “overlap sequence” is provided on the 3 ′ end side of the random region and has a His-tag and Y-tag sequence at the 5 ′ end ( FIG. 12a).
Here, “overlapping sequence” means that two or more bases on the 3 ′ side of a synthetic oligonucleotide that is an antisense primer containing two sense primers and a random sequence are complementarily overlapped with each other by PCR. This is a method of constructing a library while extending from the 5 'side to the 3' side.
The two types of primers used here are as follows.
Upstream sense primer: (SEQ ID NO: 4)
5'-ATTTAGGTGACACTATAGAATACAAGCTTGCTTGTTCTTTTTGCAGAAGCT
CAGAATAAACGCTCAACTTTGGCAGATCTACCATGG-3 '
Downstream antisense primer: (SEQ ID NO: 5)
5'-TTTCCCCGCCGCCCCCCGTCCTGCTTCCGCCGTGATGATGATGGTGGTGAG
ACCCTCCGCCTGAGCCTCCACC SNNSNNSNNSNNSNNSNNSNNSNN TGAACCTCCCATGGT-3 '
The random region was encoded by the NNS codon (N = A, T, C or G; S = C or G) and included in the downstream primer.
The two primer fragments were ligated in 15 cycles of overlapping PCR with denaturation (94 ° C) for 20 seconds, annealing (60 ° C) for 15 seconds and extension (72 ° C) for 30 seconds.
The tag construct was prepared in the same way except that the random region was replaced with a FLAG-tag sequence.

The 8-mer peptide library mRNA-linker-N was prepared by ligating the 8-mer peptide library mRNA to the linker-N obtained in Example 1 in the same manner as in Example (2-2). 10 pmol of 8-mer peptide library mRNA-linker-N was mixed with 10 fmol of FLAG-tag mRNA-linker prepared similarly.
In the same manner as in Example (3-3), an mRNA protein fusion was obtained by translation and maturation in a wheat germ extract translation system (WGE), and reverse-transcribed after purification with a His-tag as in Example 4. To obtain a library of cDNA protein fusions.
On the other hand, for comparison, a “puro-linker” described in Non-Patent Document 13 was applied to the 8-mer peptide library, and a library of cDNA protein fusions was prepared according to the protocol of Non-Patent Document 13.

Each of the above-mentioned two cDNA protein fusion libraries was combined with 200 nM anti-FLAG M2 antibody (manufactured by Sigma) immobilized on magnetic beads in the same manner as in Example (5-1), and 200 μl of DTT. Was mixed in a buffer solution (SB) from which was removed, and reacted at 25 ° C. for 30 minutes. As in Example (5-2), the beads were washed 5 times (Round 1), 8 times (Round 2) and 10 times (Round 3) with an equal volume of buffer (SB) and eluted with 100 mM DTT. And then desalted.
The eluted fraction was PCR amplified and proceeded to the next round, and a total of 3 cycles were selected. After round 3, PCR products were cloned (TA cloning, Invitrogen) and 20 clones were selected and sequenced by random picking.
A conceptual diagram of these series of steps is shown in FIG. 12b.
FIG. 12 c shows the selection results when the linker-N of the present invention is used and when the Puro-linker of Non-Patent Document 13 is used.
When Linker-N was used, 80% of the FLAG-tag sequence of DYKDDDDK (SEQ ID NO: 3) could be selected after 3 cycles. The selected FLAG-tag sequence remained at 20%.

  This indicates that, during the selection process of the present invention, surplus mRNA-linker-N conjugate, mRNA protein fusion, etc., which become “noise”, could be efficiently removed from the system, and the S / N ratio could be reduced. ing.

(Example 7) Screening of VEGF-binding protein from 3F library (7-1) Preparation of 3F cDNA protein fusion library An mRNA library was prepared from a randomized 3F library (MicTx-3 template: FIG. 13).
The amino acid sequence of “3F” is as follows, and the random region is referred to as Loop1, Loop2, and Loop3 from the beginning.
LVCY (X) ₆ PGTLETCPDDFTCV (X) ₁₀ TQYCSHACAIP (X) ₇ CCQTDKCNG (SEQ ID NO: 15)
Next, the same procedure as in the above example was applied to the linker-N of the present invention, and the 3F library mRNA- (linker-N) fusion was translated in 0.5 ml of WGE lysate reaction solution to 3F-cDNA fusion. The body was made. The composition of the reaction solution is as follows.
Library mRNA-linker fusion 200 nM
WGE lysate (PROMEGA) 250μl
Amino acid mixture (PROMEGA) 40μl
Potassium acetate solution 50mM
RNase inhibitor (PROMEGA) 200unit
Protein Disulfide Isomerase (Takara Bio) 5μg
Oxidized glutathione 10 mM
Reduced glutathione 1mM
The reaction was carried out at 25 ° C. for 15 minutes.

As a result, approximately 3 × 10 ¹² molecules were obtained as a 3F-cDNA fusion.
In order to estimate the number of molecules of the cDNA protein fusion in advance, SDS-PAGE is performed using a known amount of the 3F-mRNA-linker as a concentration standard.

(7-2) In vitro selection
VEGF (PeproTech, London) was immobilized on magnetic beads (Magnosphere MS300 / Streptavidin, Takara Bio) in the same manner as in Example (5-1).
The cDNA protein fusion obtained in (7-1) above is reacted with immobilized VEGF in 200 μl of SB (selection buffer) buffer at 25 ° C. for 1 hour, followed by SB buffer and WB (wash buffer). ) Each was washed several times with buffer (1 × PBS, 100 mM NaCl and 0.5% Tween-20). A 100 mM DTT solution was administered to the washed beads, and allowed to stand at 25 ° C. for 10 minutes to obtain a supernatant containing cDNA.
The resulting supernatant was desalted with Micro-spin column (GE Health Healthcare) and amplified by PCR (denaturation (94 ° C) for 20 seconds, annealing (60 ° C) for 15 seconds and extension (72 ° C). 30 seconds). In order to apply the selection pressure, the concentration of VEGF was changed from 1 μM to 1 nM, and the number of washes was gradually increased with the WB wash. After 7 rounds (R7), the PCR product was cloned into pCR2.1 vector (TA cloning, Invitrogen) and 20 clones were randomly selected and sequenced.

(7-3) Analysis of clone sequence As a result of analyzing the sequence of the obtained 20 clones, when grouped by the amino acid sequence encoded by each, the G-1 sequence was 60%, the G-2 sequence and the G- 3 sequences were 15% each. The amino acid sequences of Loops 1 to 3 of each group are as follows.
G-1: 60% (12/20)
Loop1 = PLTRVV (sequence number 6), Loop2 = HGDHHTLSEW (sequence number 7), Loop3 = EEPTAHV (sequence number 8)
G-2: 15% (3/20)
Loop1 = WEVVLL (SEQ ID NO: 9), Loop2 = AHSVTLAHGH (SEQ ID NO: 10), Loop3 = TGPGAER (SEQ ID NO: 11)
G-3: 15% (3/20)
Loop1 = TLWLSY (SEQ ID NO: 12), Loop2 = DVPQSGTNLA (SEQ ID NO: 13), Loop3 = ELTHPVD (SEQ ID NO: 14)

(7-5) Measurement of dissociation constant The dissociation constant (Kd) was measured according to the method reported in Non-Patent Document 23. A certain amount of 3F display protein (˜1 nM) was reacted with various amounts of VEGF (0.1 nM to 1 μM) for 1 hour. The mixture was applied to a constant amount of VEGF (˜100 nM) and incubated for another 30 minutes. After washing several times, anti-His-antibody (R & D systems) was added at 1/2000 dilution and allowed to react for 1 hour. After the reaction mixture was washed several times, substrate and TMB were added. After appropriate color development, the reaction was stopped by adding 0.5 M H ₂ SO ₄ and the absorbance at 450 nm was measured.
Typical sequences of the 20 clones of G-1, G-2 and G-3 groups and their dissociation constants (Kd values) are shown in Table 4. In particular, the G-1 group has an extremely low value as a peptide of 2.1 ± 0.5 nM, and its affinity is Kd value 2.9 nM of a commercially available anti-VEGF antibody (Chen et al. JMB (1999) 293: 865-881). Is comparable.

(Example 8) Evaluation of binding specificity (8-1)
Using the R7 mRNA- (linker-N) (200 nM) obtained in Example 7, the cDNA protein fusion was prepared by translation in the same manner as in (7-1) above.
Dividing these fusions into three parts,
(1) Streptavidin beads (when nothing is bound),
(2) VEGF beads (200 nM) and (4) Fc beads (200 nM), (Fc-immobilized beads were prepared by the method described in Example (5-1) as with VEGF beads). The reaction was performed in 200 μl of SB (selection buffer) buffer at 25 ° C. for 30 minutes. As “Fc”, human IgG1-derived Fc (R & D Systems) was used.
As a control for (3), an equivalent amount (200 nM) of R7 cDNA- (linker-N) obtained by reverse transcription of R7 mRNA- (linker-N) without a translation step into protein was reacted under the same conditions as for VEGF beads. .
Each bead was washed 10 times with the same buffer and eluted with 100 mM DTT. The eluted fraction was desalted and PCR amplified under the following conditions (PCR conditions: denaturation (94 ° C) for 20 seconds, annealing (60 ° C) for 15 seconds and extension (72 ° C) for 30 seconds).
For the R7-cDNA fusion in the cases (1) to (4) above, the eluted fraction was PCR amplified and analyzed by gel electrophoresis (FIG. 14a). Other conditions such as electrophoresis conditions are the same as in Non-Patent Document 23.
Next, analysis by ELISA was performed according to Non-Patent Document 23.
The R7-cDNA fusion was divided into three parts and incubated with SA beads, 200 nM VEGF beads and 200 nM Fc beads in PBS-BSA (0.01% BSA) at 25 ° C. for 1 hour. The mixture was washed with PBS-T (0.1% Tween) and subsequently reacted with anti-His antibody (R & D system) in PBS-T at 25 ° C. for 30 minutes. The mixture was then washed several times with PBS-T, followed by the addition of the substrate, 3,3 ′, 5,5′-tetramethylbenzidine (TMB; Sigma). After color development, 0.5 M H ₂ SO ₄ was added to stop the reaction, the sample was centrifuged, and the absorbance at 450 nm was measured.
As a result, as described in detail in (8-2) below, it was clearly shown that the R7-cDNA fusion was highly bound to the VEGF beads (FIG. 14b).
Three-finger peptide AdTx1 (derived from Dendraspis angusticeps, PDB = 4IYE) that has high amino acid sequence homology with peptide G-1 and has already been subjected to three-dimensional structural analysis. As a template, an estimated three-dimensional structure diagram was created by structural modeling using molecular structure construction software “Prime” (Schroedinger) (FIG. 16).

(8-2) Results of PCR analysis
In the binding assay analyzed by PCR, the R7 library protein fusion binds less than 0.2% to the negative control without VEGF (FIG. 14a (1)). On the other hand, significant binding (2% or more; FIG. 14a (2)) was shown with VEGF beads. To verify the potential for library enrichment via nucleic acid aptamers, ie, mRNA / cDNA hybrids, R7 mRNA / cDNA hybrids were prepared by reverse transcription of mRNA-linkers and tested for binding potential. As a result, binding to VEGF via mRNA / cDNA hybrid was not observed (less than 0.2%; FIG. 14a (3)). The R7 protein fusion also showed no affinity for Fc (less than 0.2%; Figure 14a (4)), suggesting target (VEGF) specific enrichment of the 3F library. In the binding assay by ELISA, the R7 protein fusion was 5 times higher for VEGF-immobilized beads than for VEGF-free SA beads (1) and Fc-immobilized beads as a negative control (4). A strong signal was shown (Figure 14b). This result also suggests that this library was strongly specific for VEGF.

1. Sequence number 1: PS primer
2. Sequence number 2: Pv3 A primer
3. Sequence number 3: FLAG-tag
4). SEQ ID NO: 4: 8-mer peptide library primer (F)
5). SEQ ID NO: 5: 8-mer peptide library primer (R)
6). Sequence number 6: G-1 Loop1
7). Sequence number 7: G-1 Loop2
8). Sequence number 8: G-1 Loop3
9. Sequence number 9: G-2 Loop1
10. SEQ ID NO: 10: G-2 Loop2
11. Sequence number 11: G-2 Loop3
12 Sequence number 12: G-3 Loop1
13. SEQ ID NO: 13: G-3 Loop2
14 SEQ ID NO: 14: G-3 Loop3
15. SEQ ID NO: 15: CTx3-3F peptide
16. SEQ ID NO: 16: VEGF binding peptide
17. SEQ ID NO: 17: 1F-Loop I
18. SEQ ID NO: 18: 1F-Loop II
19. SEQ ID NO: 19: 1F-Loop III
20. SEQ ID NO: 20: h VEGF-A

Claims (20)

A branched nucleic acid linker composed of a main chain composed of a single-stranded DNA capable of specifically hybridizing with a target mRNA and a side chain having puromycin at the 3 ′ end;
The side chain has an oligo dA region at the other end, is covalently bonded to the modified base in the single-stranded DNA polynucleotide chain via the oligo dA, and the oligo dA region and the puromycin A spacer region is included between and
The main chain has a site that can be ligated with the mRNA at the 5 ′ end, and a region that can hybridize with the mRNA adjacent to the site, and the mRNA is reverse transcribed at the 3 ′ end. A nucleic acid linker comprising a primer site serving as a primer.   The nucleic acid linker according to claim 1, wherein the modified base is 5-methyldeoxycytosine (5-Me-dC).   The nucleic acid linker according to claim 1 or 2, wherein the oligo dA comprises a sequence having at least 12 consecutive deoxyadenines (dA).   The nucleic acid linker according to any one of claims 1 to 3, wherein the spacer sequence is a sequence containing 2 to 8 Sp.18 (Spacer 18 Phosphoramidite). A method for producing a nucleic acid linker according to claim 1,
From 3 ′ end to 5 ′ end, protein binding site to which puromycin is bound, spacer sequence, oligo dA sequence, 5-methyldeoxycytosine (5-Me-dC), region capable of hybridizing with target mRNA, and The 5-Me-dC of single-stranded DNA containing a site that can be ligated with the mRNA reacts with the phosphate group at the 5 ′ end of the DNA sequence that serves as a primer for reverse transcription of the mRNA. A manufacturing method characterized by combining them.   An mRNA-protein fusion in which a target mRNA and a protein encoded by the mRNA are linked via the nucleic acid linker according to any one of claims 1 to 4.   The mRNA-protein fusion according to claim 6, wherein the mRNA has a sequence encoding a His-tag downstream of a sequence encoding a protein. A method for producing the mRNA-protein fusion according to claim 6 or 7, comprising the following (a) to (c):
(A) annealing the nucleic acid linker according to claim 1 targeting the mRNA with the mRNA;
(B) ligating the 3 ′ end of the mRNA and the 5 ′ end of the nucleic acid linker to produce an mRNA-nucleic acid linker;
(C) A step of producing an mRNA-protein fusion by synthesizing a protein from the mRNA using a cell-free protein translation system and binding the C-terminus of the protein to puromycin in the nucleic acid linker.   In the step (c), a step of maturing the protein in the presence of a high concentration salt for 10 minutes to 2 hours is provided during or after the production of the mRNA-protein fusion. 9. The method according to 8.   An mRNA / cDNA-protein fusion comprising the nucleic acid linker according to any one of claims 1 to 4, wherein the mRNA / cDNA comprises the mRNA and cDNA complementary to the mRNA via the nucleic acid linker. An mRNA / cDNA-protein fusion in which a complex and a protein encoded by the mRNA are linked.   An mRNA / cDNA-protein fusion comprising the nucleic acid linker according to any one of claims 1 to 4, wherein the mRNA / cDNA-protein fusion is encoded by the cDNA complementary to the mRNA and the mRNA via the nucleic acid linker. A cDNA-protein fusion in which proteins are linked. An mRNA / cDNA-protein fusion comprising the nucleic acid linker according to any one of claims 1 to 4, wherein the mRNA / cDNA comprises the mRNA and cDNA complementary to the mRNA via the nucleic acid linker. A method for producing a purified mRNA / cDNA-protein fusion in which a complex and a protein encoded by the mRNA are linked, comprising the following (a) to (f):
(A) annealing the nucleic acid linker with the target mRNA;
Wherein the target mRNA is an mRNA having a sequence encoding a His-tag downstream of a sequence encoding a protein;
(B) ligating the 3 ′ end of the mRNA and the 5 ′ end of the nucleic acid linker to produce an mRNA-nucleic acid linker;
(C) producing a mRNA-protein fusion by synthesizing a protein from the mRNA using a cell-free protein translation system and binding the C-terminus of the protein to puromycin in the nucleic acid linker;
(D) binding the mRNA-protein fusion to a metal matrix having an affinity for the His-tag sequence, and washing;
(E) subjecting the mRNA-protein fusion on the surface of the metal matrix to a reverse transcription reaction to produce an mRNA / cDNA-protein fusion;
(F) washing the mRNA / cDNA-protein fusion on the surface of the metal matrix and eluting the purified mRNA / cDNA-protein fusion.   In the step (c), a step of maturing the protein in the presence of a high concentration salt for 10 minutes to 2 hours is provided during or after the production of the mRNA-protein fusion. 12. The method according to 12.   14. A method according to claim 12 or 13, characterized in that the metal matrix is Ni coated magnetic beads. An mRNA / cDNA-protein fusion comprising the nucleic acid linker according to any one of claims 1 to 4, which is encoded by the mRNA complementary to the mRNA and the mRNA via the nucleic acid linker. A method for producing a purified cDNA-protein fusion linked to a protein, comprising the following (a) to (g):
(A) annealing the nucleic acid linker with the target mRNA;
Wherein the target mRNA is an mRNA having a sequence encoding a His-tag downstream of a sequence encoding a protein;
(B) ligating the 3 ′ end of the mRNA and the 5 ′ end of the nucleic acid linker to produce an mRNA-nucleic acid linker;
(C) producing a mRNA-protein fusion by synthesizing a protein from the mRNA using a cell-free protein translation system and binding the C-terminus of the protein to puromycin in the nucleic acid linker;
(D) binding the mRNA-protein fusion to a metal matrix having an affinity for the His-tag sequence, and washing;
(E) subjecting the mRNA-protein fusion on the surface of the metal matrix to a reverse transcription reaction to produce an mRNA / cDNA-protein fusion;
(F) a step of producing a cDNA-protein fusion by degrading mRNA by allowing an RNase to act on the mRNA / cDNA-protein fusion;
(G) A step of washing the cDNA-protein fusion on the surface of the metal matrix and eluting the purified cDNA-protein fusion.   In the step (c), a step of maturing the protein in the presence of a high concentration salt for 10 minutes to 2 hours is provided during or after the production of the mRNA-protein fusion. 15. The method according to 15.   The method according to claim 15 or 16, characterized in that the metal matrix is Ni-coated magnetic beads. A method for selecting a DNA encoding a protein having a high affinity with a target substance using the mRNA / cDNA-protein fusion according to claim 10 or the cDNA-protein fusion according to claim 11. (A) to (c) and (j), or (a) to (j).
(A) a step of immobilizing the target substance on a solid surface;
(B) An mRNA / cDNA-protein fusion or cDNA-protein fusion comprising a protein that binds to a target substance by contacting the mRNA / cDNA-protein fusion or cDNA-protein fusion with an immobilized target substance. The process of selecting the body,
(C) separating a cDNA encoding a protein from the selected mRNA / cDNA-protein fusion or cDNA-protein fusion, and PCR-amplifying the obtained cDNA fragment;
(D) A template DNA containing a sequence necessary for promoting a translation reaction and a sequence encoding a His-tag is prepared using the amplified cDNA fragment, transcribed into mRNA, and any one of claims 1 to 4. Annealing the nucleic acid linker according to claim 2 and ligating the 3 ′ end of the mRNA and the 5 ′ end of the nucleic acid linker to produce an mRNA-nucleic acid linker;
(E) An mRNA-protein fusion is prepared by synthesizing a protein from the mRNA of step (d) using a cell-free protein translation system and binding the C-terminus of the protein to puromycin in the nucleic acid linker. Process,
(F) binding and washing the mRNA-protein fusion to a metal matrix having an affinity for a His-tag sequence;
(G) subjecting the mRNA-protein fusion on the surface of the metal matrix to a reverse transcription reaction to produce an mRNA / cDNA-protein fusion;
Alternatively, a step of further degrading mRNA by causing an RNase to act on the mRNA / cDNA-protein fusion to produce a cDNA-protein fusion,
(H) washing the mRNA / cDNA-protein fusion or cDNA-protein fusion on the surface of the metal matrix and eluting the purified mRNA / cDNA-protein fusion or purified cDNA-protein fusion;
(I) a step of repeating the steps (b) and thereafter;
(J) The amplified DNA fragment obtained in the step (c) or the DNA fragment obtained in the step (c) after repeating the steps (b) to (i) at least once is used as a target substance. A process of selecting DNA encoding a protein with high affinity.   The magnetic beads to which streptavidin is bound are used as the solid phase in which the target substance is biotinylated in advance in the step (a) prior to immobilization and is immobilized. the method of. A method for identifying or obtaining a protein having a high affinity for a target substance,
A method comprising the steps of recovering DNA encoding a protein having a high affinity for a target substance selected by the method according to claim 18 and 19 and determining a base sequence thereof.