WO2002040664A1

WO2002040664A1 - Method of constructing nucleic acid library

Info

Publication number: WO2002040664A1
Application number: PCT/JP2001/009200
Authority: WO
Inventors: Koichi Nishigaki; Yasunori Kinoshita
Original assignee: Mitsubishi Chemical Corporation
Priority date: 2000-11-14
Filing date: 2001-10-19
Publication date: 2002-05-23
Also published as: JP2002315577A

Abstract

It is intended to establish a method of constructing a nucleic acid library by using the Y-ligation method. Namely, a method of constructing a nucleic acid library which involves the following steps (1) to (7): (1) preparing a first single-stranded nucleic acid and a second single-stranded nucleic acid each having a stem sequence and a branch sequence and a base sequence encoding an amino acid at the 3'-end; (2) hybridizing the first single-stranded nucleic acid with the second single-stranded nucleic acid between the stem sequences thereof; (3) treating the hybridized product with T4 RNA ligase to thereby ligate the 3'-end of the single-stranded nucleic acid to the 5'-end of the second single-stranded nucleic acid; (4) using the double-stranded nucleic acid obtained in the step (3) as a template, carrying out PCR with the use of primers modified in the 5'-side with a substance having affinity to thereby prepare double-stranded nucleic acids; (5) treating two double-stranded nucleic acids obtained in the step (4) with a restriction enzyme to give double-stranded nucleic acids respectively having base sequences encoding the first and second amino acids at the 3'- or 5'-end; (6) using the binding ability of the substance having affinity introduced in the step (4), preparing single-stranded nucleic acids from the double-stranded nucleic acids obtained in the step (5); and (7) using the single-stranded nucleic acids obtained in the step (6), repeating the steps (2) to (6) needed times.

Description

Method for preparing a nucleic acid library

The present invention relates to a method for producing a nucleic acid library. More specifically, the present invention provides a method for hybridizing two single-stranded nucleic acids having complementary sequences (stem portions) at their ends, and then ligating the ends of the single-stranded region (branch portion) to RNA ligase. The present invention relates to a method for preparing a nucleic acid library, wherein the method uses a Y-ligation method for ligation. The present invention relates to a nucleic acid library produced by the method for producing a nucleic acid library and a peptide library obtained by using the nucleic acid library. Background art

Evolutionary molecular engineering aims to obtain useful biopolymers based on Darwin's principle of evolution. This method consists of the following elements: 1) construction of a mutant library, 2) selection of a more useful molecular species, 3) introduction and amplification of a mutation into the genotype, and 4) expression of the mutant population. This is achieved by repeating steps 4) to 4).

When searching for a useful peptide or protein, construct a D Ν 変異 mutant library for easy introduction of mutations and identification of the phenotype, and express the protein in a form corresponding to this genotype. In many cases, a function-based selection method is used. Therefore, the characteristics of the DNA library, which is the starting point, can be said to be one of the important factors that determine the success in obtaining useful peptides or proteins.

Conventionally, DNA mutant libraries have been constructed at the nucleotide level, and there are chemical and enzymatic methods. In the former method, when synthesizing DNA using a DNA synthesizer, a method is employed in which nucleotides as raw materials are mixed at a specific ratio. The latter also uses DNA or RNA polymerase under special conditions to induce mutations. The method of generating chimeric genes by subdividing and reconstituting a plurality of similar genes, called DNA shuffling, has produced various results. The challenge is to create a completely new variant population, which is a block-wise variant population, which is called the block 'shuffling method'. When constructing a DNA library that encodes a protein, it is proposed that the hierarchical structural unit of the protein be set as a mutation unit. Attempts to create useful functional macromolecules from mutant populations in evolutionary molecular engineering can be attributed to exploring a vast sequence space, and from the conventional point mutant population of genes It can be said that the starting method is equivalent to searching for the vicinity of the wild-type protein with a short stride. On the other hand, the mutant population in block units is equivalent to walking with a stride in the sequence space far away from the wild-type protein. It can be said that the fitness of the array space is the key to the determination of whether or not the expected functional substance is reached by this stride.The shape of the topography of the array space is an important factor. It's a stage and I can't say for sure yet.

However, on the other hand, the theoretical study of molecular progression shows that many proteins have acquired various functions through the mechanism of exon shuffling from the primary sequence and structural analysis of natural proteins. Has accumulated overnight. The idea is based on the significant correlation between the boundaries of the protein's modular structure and the position of the gene's intron. In other words, various existing proteins can be seen as products that exons as functional units and introns as evolutionary devices have developed through shuffling mechanisms. This supports the effectiveness of using a block as a mutation unit, and the present inventors aim to establish a block shuffling method as a general technique applicable to engineering. One of the advantages of block shuffling is that it is possible to freely design the stride for searching the array space. The composition of the point mutation library changes depending on the lot to be constructed, and each search trial is independent and cannot be referred to each other. On the other hand, with block shuffling, it is possible to design in consideration of the search history when determining the type and length of the block. It is excellent in that it can accumulate know-how in the design of racks.

When creating functional proteins, the fundamental building block is a trinucleotide encoding an amino acid, that is, a codon. The codon-amino acid correspondence table shows that all trinucleotides encode some amino acid or stop codon. Therefore, a random mutant population in nucleotide units covers all amino acids. But there are serious drawbacks. That is, at a certain probability, a stop codon is always generated and an immature peptide chain is generated. It is also known that codon usage varies depending on the protein translation system. Since there is a limit to the amount of material that can be handled experimentally, it is desirable to use codons efficiently in order to secure a large library size. Block shuffling can avoid such problems. The present inventors have developed a method for efficiently connecting DNAs called the Y-ligation method. In this Y-ligation method, two single-stranded DNAs (5, -half and 3'-half) contain complementary sequences (stem portions) at the ends, and after hybridization, In this method, the ends of the main-strand region (branch) are ligated with RNA ligase. Figure 1 shows a schematic diagram of the Y-Ligege method. However, no attempt has been made to apply the Y-ligation method, particularly to create a DNA library or a protein library. Disclosure of the invention

The problem to be solved by the present invention is to establish a method for constructing a nucleic acid library using the Y-ligation method.

The present inventors have 5, and a stem sequence and branch arranged in a direction from the side 3 ', 3 in ⁵ end first single-stranded nucleic acid having a nucleotide sequence of the first amino acid co Solo de A second single strand having a stem sequence complementary to the stem sequence and a branch sequence in the direction from the 3 ′ side to the 5 ′ side, and having a base sequence encoding a second amino acid at the 5 ′ end After hybridization between each stem sequence using nucleic acid, T4A ligase By connecting the 3 'end of the first single-stranded nucleic acid and the 5' end of the second single-stranded nucleic acid, and then performing PCR and restriction enzyme treatment. The inventors have found that a nucleic acid library can be prepared, and have completed the present invention.

That is, according to the present invention, there is provided a method for preparing a nucleic acid library, comprising the following steps (1) to (7).

(1) a first single-stranded nucleic acid having a stem sequence and a branch sequence in the direction from the 5th side to the 3rd side and having a base sequence encoding the first amino acid at the 3rd end; A second single-stranded nucleic acid having a stem sequence complementary to the above-mentioned stem sequence and a branch sequence in the 5′-side direction and a base sequence encoding a second amino acid at the 5 ′ end is prepared. And

(2) hybridizing the first single-stranded nucleic acid and the second single-stranded nucleic acid between the respective stem sequences,

(3) treating the hybridized product with T4 RNA ligase to ligate the 3 ′ end of the first single-stranded nucleic acid and the 5 ′ end of the second single-stranded nucleic acid,

(4) The double-stranded nucleic acid obtained in step (3) is transformed into type III, and PCR is performed using a primer modified with an affinity substance on the 5, side, and the direction from 3, on the 3, side A double-stranded nucleic acid comprising a stem sequence, a branch sequence, a base sequence encoding the first amino acid, a base sequence encoding the second amino acid, a branch sequence and a restriction enzyme recognition sequence,

Convert the double-stranded nucleic acid amplified in step (3) to type III, perform PCR using primers whose 5 'side has been modified with an affinity substance, and recognize the restriction enzyme recognition sequence in the direction from 5' to 3 '. Preparing a double-stranded nucleic acid including a branch sequence, a base sequence encoding the first amino acid, a base sequence encoding the second amino acid, a branch sequence and a stem sequence, and obtaining (5) in step (4). By treating the two types of double-stranded nucleic acids with a restriction enzyme, a double-stranded nucleic acid having a nucleotide sequence encoding a first amino acid and a second amino acid at the 3 ′ end or the 5 ′ end is prepared,

(6) Using the binding ability of the affinity substance introduced in step (4), Preparing a single-stranded nucleic acid from the obtained double-stranded nucleic acid, and

(7) Repeat steps (2) to (6) as many times as necessary using the single-stranded nucleic acid obtained in step (6):

As an example of the present invention, a method for preparing a nucleic acid library including the following steps (1) to (7) is provided.

(3) treating the hybridized product with T4 RNA ligase to ligate the 3 'end of the first single-stranded nucleic acid and the 5' end of the second single-stranded nucleic acid,

(4 a) After converting the ligation product to a single-stranded nucleic acid, a complementary strand is synthesized to prepare a double-stranded nucleic acid,

(4b) Perform PCR using the double-stranded nucleic acid obtained in step (4a) as type III and using a forward primer modified on the 5 ′ side with an affinity substance and a reverse primer containing a restriction enzyme recognition sequence. , 5 'to 3' stem, branch,

Preparing a double-stranded nucleic acid comprising a nucleotide sequence encoding one amino acid, a nucleotide sequence encoding a second amino acid, a branch sequence and a restriction enzyme recognition sequence;

PCR using the double-stranded nucleic acid amplified in step (4a) as type III, and using a food primer containing a restriction enzyme recognition sequence and a reverse primer modified on the 5 ′ side with an affinity substance. The restriction enzyme recognition sequence, the branch sequence, the base sequence encoding the first amino acid, the base sequence encoding the second amino acid, the branch sequence, and the stem sequence in the direction from the 5 'side to the 3 side. Prepare double-stranded nucleic acid,

(5) The two types of double-stranded nucleic acids obtained in step (4b) are treated with a restriction enzyme to obtain bases encoding the first amino acid and the second amino acid at the 3 ′ end or the 5 ′ end, respectively. Preparing a double-stranded nucleic acid having a sequence,

(6) preparing a single-stranded nucleic acid from the double-stranded nucleic acid obtained in the step (5) by utilizing the binding ability of the affinity substance introduced in the step (4b), and

As another example of the present invention, a method for producing a nucleic acid library including the following steps (1) to (7) is provided.

(1) a first single-stranded nucleic acid having a stem sequence and a branch sequence in the direction from the 5th side to the 3rd side and having a base sequence encoding the first amino acid at the 3rd end; A second single-stranded nucleic acid having a stem sequence complementary to the above-mentioned stem sequence and a branch sequence in the 5′-side direction and having a base sequence encoding a second amino acid at the 5 ′ end is prepared. And

(4) a forward primer which forms the double-stranded nucleic acid obtained in the step (3) into a 錶 form, modifies the 5 ′ side with an affinity substance, and anneals to the stem sequence of the first single-stranded nucleic acid; PCR using a reverse primer containing a restriction enzyme recognition sequence and annealing to the branch sequence of the second single-stranded nucleic acid is performed, and the stem sequence, branch sequence, Preparing a double-stranded nucleic acid comprising a base sequence encoding the first amino acid, a base sequence encoding the second amino acid, a branch sequence, and a restriction enzyme recognition sequence;

Convert the double-stranded nucleic acid amplified in step (3) into type III, include a restriction enzyme recognition sequence, and anneal to the first single-stranded nucleic acid branch sequence, and modify the 5 and 5 sides with an affinity substance PCR using a reverse primer that anneals to the stem sequence of the second single-stranded nucleic acid was performed, and restriction enzyme recognition was performed in the direction from 5 'to 3'. A double-stranded nucleic acid including a nucleotide sequence, a branch sequence, a nucleotide sequence encoding the first amino acid, a nucleotide sequence encoding the second amino acid, a branch sequence, and a stem sequence is prepared, and obtained in step (5) (4). By treating the two types of double-stranded nucleic acids with a restriction enzyme, a double-stranded nucleic acid having a nucleotide sequence encoding a first amino acid and a second amino acid at the 3 ′ end or the 5 ′ end is prepared,

(6) using the binding ability of the affinity substance introduced in step (4) to prepare a single-stranded nucleic acid from the double-stranded nucleic acid obtained in step (5), and

Preferably, a mixture of single-stranded nucleic acids having a base sequence encoding a plurality of different first amino acids is used as the first single-stranded nucleic acid; A mixture of single-stranded nucleic acids having a base sequence encoding the second amino acid of the species is used.

Preferably, the length of the stem sequence is 10 to 100 bases.

Particularly preferably, the first single-stranded nucleic acid, the stem sequence Te Oi 5 'to 3' in the direction of the side 00 (1 ^1.000 eight Ding 80; 1 ¹ 1 ¹ chome 0 (SEQ ID NO: 1) And the stem sequence of the second single-stranded nucleic acid is CCGAGCGCTTA TGAAAC (SEQ ID NO: 2) in the direction from the 3 ′ side to the 5 ′ side.

Preferably, the length of the branch sequence is between 10 and 100 bases.

Particularly preferably, the branch sequence of the first single-stranded nucleic acid is AAGATCTCTTTT (SEQ ID NO: 3) in the direction from the 5 ′ side to the 3 ′ side, and the branch sequence of the second single-stranded nucleic acid is 3 TTGCCCTAGGGGAT (SEQ ID NO: 4) in the 'side direction.

Preferably, in step (4), the ligation product is converted into a single-stranded nucleic acid under denaturing conditions, and then amplified by PCR to prepare a double-stranded nucleic acid.

Preferably, the affinity substance in the primer used in step (4) is biotin. Preferably, the restriction enzyme recognition sequence in the primer used in step (4) is a recognition sequence for a restriction enzyme whose cleavage site is not included in the recognition sequence.

Preferably, the restriction enzyme recognition sequence in the primer used in step (4) is a recognition sequence for a restriction enzyme that cuts away from the recognition sequence.

Preferably, the restriction enzyme recognition sequence in the primer used in step (4) is an MboI I recognition sequence.

Preferably, a library of nucleic acids containing a base sequence encoding 16 amino acids is prepared by repeating steps (2) to (6) four times in total.

According to another aspect of the present invention, there is provided a nucleic acid library produced by the above-described method for producing a nucleic acid library.

According to still another aspect of the present invention, there is provided a peptide library obtained by using the nucleic acid library produced by the above-described method for producing a nucleic acid library. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic diagram of the Y-Ligege method.

FIG. 2 is a diagram showing an outline of one specific embodiment of the method of the present invention.

FIG. 3 shows the structure of the substrate of T4 RNA ligase (a) and the relationship between the recognition sequence of the introduced restriction enzyme and the cleavage site (b).

FIG. 4 shows the results of analysis of the ligation product amplified in each cycle of the method of the present invention by denaturing PAGE and silver staining.

FIG. 5 shows the result of analyzing the reaction product after the first YLBS cycle by electrophoresis according to the method of the present invention.

FIG. 6 shows the results of electrophoretic analysis of the reaction product after the second and third YLBS cycles according to the method of the present invention.

FIG. 7 shows the results of sequencing the ligation products after the third YLBS cycle. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail.

The method for producing a nucleic acid library of the present invention is characterized by comprising steps (1) to (7).

Step (1) in the method of the present invention comprises a first sequence having a stem sequence and a branch sequence in the direction from the 5 ′ side to the third side, and having a base sequence encoding the first amino acid at the 3 ′ end. A single-stranded nucleic acid, a stem sequence complementary to the above stem sequence and a branch sequence in the direction from the 3 ′ side to the 5 ′ side, and a base sequence encoding the second amino acid at the 5 ′ end. This is a step of preparing a second single-stranded nucleic acid.

In order to prepare a nucleic acid library consisting of a random amino acid sequence, that is, a base sequence encoding any amino acid different from each other, the first single-stranded nucleic acid (hereinafter, also referred to as 5, half-chain) has a different structure. Using a mixture of single-stranded nucleic acids having a base sequence encoding a plurality of first amino acids, a second single-stranded nucleic acid (hereinafter, also referred to as a 3 ′ half-strand), A mixture of single-stranded nucleic acids having a base sequence encoding an amino acid is used.

Examples of the plurality of different amino acids include, for example, any two or more amino acids selected from naturally occurring twenty amino acids. The number of kinds of amino acids is not particularly limited. Natural amino acids include aliphatic amino acids (glycine, alanine), branched amino acids (parin, leucine, isoleucine), 'hydroxyamino acids (serine, threonine), acidic amino acids (aspartic acid, glutamic acid), and amino acids. De (asparagine, glumin), basic amino acids (lysine, arginine), sulfur-containing amino acids (cystine, methionine), aromatic amino acids (phenylalanine, tyrosine), heterocyclic amino acids (tributophano, histidine), It can be classified into imino acids (proline), but one amino acid may be selected from each group and used as a representative. In the examples described later in this specification, glycine, isoleucine, and asparagine were used as the seven amino acids representative of the properties of amino acids. Acids, lysine, serine, cystine, and proline were selected, but are merely examples of the present invention.

The length of the stem sequence in the 5, half-strand and the 3, half-strand is not particularly limited as long as both strands can hybridize, and preferably from 10 to 100 bases. It is more preferably from 10 to 30 salinity.

The stem sequence of the half-strand and the stem sequence of the 3 'half-strand are complementary to each other, so that the 5, half-strand and the half-strand can hybridize under certain conditions. More specifically, since the stem sequence of the 5 'half-strand (5, 3 from the direction) is complementary to the stem sequence of the half-strand (3, 5 from the direction), both strands hybridize. that both stems sequences form a duplex, since there remains 5 ⁵ half-chain branch-chain and 3 'branch chain single strand of half chain of, as a whole forming the shape of a Y-shaped by (See Figure 1). The name Y-Lige-Shillon comes from the shape of this structure. The feature of this method is that the coupling efficiency between the two DAs can be improved by changing from the intermolecular reaction to the intramolecular reaction. Therefore, it can be applied to low-concentration substrates.

As an example of the stem sequence of the first single-stranded nucleic acid, the number is 00 in the direction from the 5, side to the 3 'side. 0_Rei_0 eight Hatcho eight (1 ¹ 1 ¹ chome 0 (SEQ ID NO: 1), as an example of a second single-stranded nucleic acid, the stem sequence 3, in the direction from the side 5 'side CC GA GCGCTTAT GAAA C ( SEQ ID NO: 2).

The length of the branch sequence in the half-strand and the half-strand can be linked by the T4 RNA ligase treatment to the base at the end of the half-strand and the base at the end of the half-strand. There is no particular limitation as long as the length is of the order. Generally, the shorter the length of the branch sequence is, the higher the ligation efficiency is, but it has been found that a substrate of about 300 nucleotides in both strands can be ligated with considerable efficiency. In addition, since both chains are reacted at a stoichiometric ratio of 1 to 1, the yield of the product with respect to the raw material can be improved, and the cost can be reduced and the purification operation can be simplified. You. The length of the branch sequence is preferably about 10 to 100 bases, more preferably about 10 to 30 bases. The length of the half-chain branch sequence and the length of the half-chain branch sequence may be the same or different.

The branch sequence may include a restriction enzyme recognition sequence as described later in this specification.

An example of a branch sequence of the first single-stranded nucleic acid is AAGATCTCTTTT (SEQ ID NO: 3) in the direction from the 5 'side to the 3' side, and a branch sequence of the second single-stranded nucleic acid is 3 ' From the 5'-side direction, there is 11 ¹ 0 ((0000 000 8 (SEQ ID NO: 4).

The 5, half-strand and the half-strand used in step (1) can be synthesized by an ordinary DNA synthesis method (a method of synthesizing using a DNA synthesizer). In addition, the use of phosphorylated 3 and 5 terminal ends of the half chain enables binding to the 3 and 5 terminal ends of the half chain.

Step (2) in the method of the present invention is a step of hybridizing the first single-stranded nucleic acid and the second single-stranded nucleic acid between the respective stem sequences.

Specifically, the 5, -half strand and 3, -half strand prepared in step (1) are heated at 94 ° C for 5 minutes in a suitable buffer, and then, for example, at 60 ° C for 15 minutes. By keeping the temperature, the complementary stem sequence can be hybridized. Hybridization conditions (buffer composition and hybridization temperature) can be appropriately set according to the length of the stem sequence, base composition, and the like.

In the step (3) of the method of the present invention, the product hybridized in the step (2) is treated with T4RNA ligase, and the 3 ′ end of the first single-stranded nucleic acid and the 5 ′ of the second single-stranded nucleic acid are treated. This is the step of connecting the terminal.

If an appropriate buffer for T4 RNA ligase is used as the hybridization solution, the solution containing the hybridization product can be used as it is for the ligase reaction. The hybridization product is recovered by standard DNA purification methods and then buffered for T4 RNA ligase. Dissolve in the buffer to prepare a solution for ligase reaction. A ligase reaction is performed by adding ATP and T4 RNA ligase to such a solution and reacting at an appropriate temperature (for example, 25 ° C) for a certain period of time (for example, 16 hours). Thereby, the 3 'end of the first single-stranded nucleic acid is linked to the 5' end of the second single-stranded nucleic acid.

Step (4) in the method of the present invention is carried out by PCR using the double-stranded nucleic acid obtained in step (3) as a type II,

Double-stranded nucleic acid containing a stem sequence, a branch sequence, a nucleotide sequence encoding the first amino acid, a nucleotide sequence encoding the second amino acid, a branch sequence, and a restriction enzyme recognition sequence in the direction from the 5 'side to the 3 side. ; And

5 Double-stranded nucleic acid containing a restriction enzyme recognition sequence, a branch sequence, a base sequence encoding the first amino acid, a base sequence encoding the second amino acid, a branch sequence, and a stem sequence in the direction from the ⁵ side to the 3 'side ;

Is a step of preparing

In the first specific example of the step (4), (4a) the ligation product is converted into a single-stranded nucleic acid, and then a complementary strand is synthesized to prepare a double-stranded nucleic acid.

(4b) Using the double-stranded nucleic acid obtained in step (4a) as type III, using a forward primer modified on the 5, side with an affinity substance and a linos primer containing a restriction enzyme recognition sequence. Perform a PCR to obtain a stem sequence, a branch sequence, a base sequence encoding the first amino acid, a base sequence encoding the second amino acid, a branch sequence, and a restriction enzyme recognition sequence in the direction from the 5 ′ side to the 3 ′ side. Preparing a double-stranded nucleic acid containing

The double-stranded nucleic acid amplified in step (4a) is converted into type II, and PCR is performed using a food primer containing a restriction enzyme recognition sequence and a reverse primer modified on the 5th side with an affinity substance. And a restriction enzyme recognition sequence, a branch sequence, a base sequence encoding the first amino acid, a base sequence encoding the second amino acid, a branch sequence and a stem sequence in the direction from the 5 ′ side to the 3 ′ side. A single-stranded nucleic acid is prepared.

In addition, when it is referred to as step (4) in the present specification, the above step (4a) and step (4b) are included. Step (4a) is a step of preparing a double-stranded nucleic acid by converting the ligation product into a single-stranded nucleic acid and then synthesizing a complementary strand. Ligation product obtained in step (3) is 5 stem sequences of the 'half-chain and 3' half chain forms a double strand, the end of the duplex, 5 ⁵ Half-chain branches sequences , A base sequence encoding the first amino acid, a base sequence encoding the second amino acid, and a half-chain branch sequence to form a loop structure. This ligation product can be converted into a single-stranded nucleic acid by placing it under denaturing conditions. The single-stranded nucleic acid of the ligation product is composed of 5, a half-chain stem sequence, a half-chain branch sequence, a half-strand branch sequence, a base sequence encoding the first amino acid, and a second sequence from the 5 ′ to the 3 ′ side. It has a base sequence that encodes the amino acid of 3, a half-chain branch sequence, and a 3, half-chain stem sequence.

In the step (4a), a complementary strand is synthesized with respect to the single-stranded nucleic acid to prepare a double-stranded nucleic acid. The double-stranded nucleic acid is preferably subjected to PCR using the single-stranded nucleic acid as a 錶 -type, whereby a desired double-stranded nucleic acid can be amplified.

In the step (4b), the double-stranded nucleic acid obtained in the step (4a) is used as a type III, and the forward primer modified on the 5, side with an affinity substance and a reverse primer containing a restriction enzyme recognition sequence are used. Perform a PCR, and in the direction from the 5 'side to the 3' side, a stem sequence, a branch sequence, a base sequence encoding the first amino acid, a base sequence encoding the second amino acid, a branch sequence, and a restriction enzyme recognition sequence. A double-stranded nucleic acid prepared in step (4) is prepared, and the double-stranded nucleic acid amplified in step (4) is converted into a type II, and a forward primer containing a restriction enzyme recognition sequence and a 5 ' PCR using a single primer and a restriction enzyme recognition sequence, a branch sequence, a nucleotide sequence encoding the first amino acid, a nucleotide sequence encoding the second amino acid, and a branch in the direction from the 5 ′ side to the 3 ′ side. Including sequence and stem sequence A step of preparing a stranded nucleic acid.

The affinity substance in the primer used in step (4b) is not particularly limited as long as it can be introduced into the primer and enables the preparation of a single-stranded nucleic acid in step (7). An example of an affinity substance is biotin, in which case in step (7) a single biotin-labeled biotin The strand nucleic acid can be recovered.

In the second specific example of the step (4), the double-stranded nucleic acid obtained in the step (3) is converted into type II, and the first side of the first single-stranded nucleic acid is modified with an affinity substance on the 5, side. PCR was performed using a forward primer that anneals to the stem sequence and a reverse primer that contains the restriction enzyme recognition sequence and anneals to the branch sequence of the second single-stranded nucleic acid. Preparing a double-stranded nucleic acid comprising a stem sequence, a branch sequence, a base sequence encoding the first amino acid, a base sequence encoding the second amino acid, a branch sequence, and a restriction enzyme recognition sequence in the direction of the side,

Convert the double-stranded nucleic acid amplified in step (3) into type III, include a restriction enzyme recognition sequence, and anneal to the first single-stranded nucleic acid branch sequence, and modify the 5 and 5 sides with an affinity substance PCR was performed using the reverse primer that anneals to the stem sequence of the second single-stranded nucleic acid, and the restriction enzyme recognition sequence, the branch sequence, and the first amino acid in the direction from 5, side to 3, side. Preparing a double-stranded nucleic acid comprising a base sequence encoding, a base sequence encoding a second amino acid, a branch sequence and a stem sequence,

(5) The two types of double-stranded nucleic acids obtained in the step (4) are treated with a restriction enzyme, so that the nucleotide sequence encoding the first amino acid and the second amino acid at the 3 ′ end or 5 ′ end respectively. To prepare a double-stranded nucleic acid.

The second specific example is characterized in that steps (4a) and (4b) in the first specific example are performed by one PCR. In the PCR reaction, mutations may be introduced into the final ligated product as a result of mutations and the like, which may cause problems such as amino acid substitution, frame shift, and insertion of a stop codon. Therefore, it is preferable to reduce the introduction of mutation by this PCR. Therefore, the PCR for the purpose of amplifying the ligated product in the step (4a) and the preparation of the raw material in the next cycle in the step (4b) were simultaneously performed by a single PCR operation. In the method of the second specific example, PCR is performed using primers (stem primer, part primer) of a stem sequence and a branch sequence (in the examples, indicated as part sequence), and the PCR width of the ligation product is determined. . By this method, the repetition of the cycle of the nucleotide sequence encoding the amino acid Thus, the frequency of introducing mutations into a nucleic acid library produced by ligation can be reduced, and as a result, the quality of the library can be improved.

The above step (4) is a step in which two sets of PCR are performed to produce a raw material for the next cycle, and the ligated product prepared in steps (2) to (3) has a stem portion and a restriction enzyme recognition site. This is the step of introducing a sequence.

A stem sequence, a branch sequence, a base sequence encoding the first amino acid, a base sequence encoding the second amino acid, a branch sequence, and the like, which are prepared in the step (4) in the direction from the 5 ′ side to the 3 ′ side. In a double-stranded nucleic acid containing a restriction enzyme recognition sequence, a restriction enzyme recognition sequence is introduced by the restriction enzyme in the next step (5) such that the restriction enzyme recognition sequence is cleaved at the 3 'end of the nucleotide sequence encoding the second amino acid. Have been.

Similarly, a restriction enzyme recognition sequence, a branch sequence, a nucleotide sequence encoding the first amino acid, and a second amino acid are prepared in the direction from 5, side to 3, prepared in step (4). For a double-stranded nucleic acid containing a base sequence, a branch sequence, and a stem sequence, a restriction enzyme recognition sequence such that the restriction enzyme in the next step (5) is cleaved at the 5 ′ end of the base sequence encoding the first amino acid Has been introduced.

The restriction enzyme recognition sequence in the primer used in step (4) is a recognition sequence for a restriction enzyme whose cleavage site is not included in the recognition sequence, and is preferably a recognition sequence for a restriction enzyme that cuts away from the recognition sequence. For example, a recognition sequence of MboII. Examples of the “restriction enzyme whose cleavage site is not included in the recognition sequence” used in the present invention include two types: an enzyme that cuts just outside the recognition sequence and an enzyme that cuts far away from the recognition sequence. In the present invention, an appropriate enzyme can be selected from the following enzymes and used.

Examples of enzymes that cleave immediately outside the recognition sequence include the following.

Show ₍

'W represents A or T,' S, represents C or G.

Of use of different types of enzymes

i j

3 'protruding type 5' protruding type

Examples of enzymes that cut far away from the recognition sequence include the following.

Table 2:

CjePI

Eci l GGCGGANNNNNNNNNJN "

Eco31 I GGTCTC 讓 ΒΠ736Ι Bsal Bso31 I

EcoA4I Eco044I

Eco57I CTGAAGNNNNNNNNNNNNNNNNJN "BspKT5I

Esp3I CGTCTCN stroke N_ BsmBI BstGZ53I

Faul CCCGCNNNN ~ NN_ BstFZ438I

Fokl GGATGN 丽 NN-one NNNN-one BstPZ418 〖

Gsul CTGGAGNNNNN NNNNNNNNJN "Bpml

HaelV ー丽 NNNN-one NNNNN orchid GAYNNNN concealed NNNNNNNNN— NNNNM

Hgal GACGCNNNNN "NNNNN_

Hphl GGTGANNNNNNN.N ^ AsuHPI

Ksp632I CTCTTCr 丽 Bco5I Bcoll6I BcoKI

BseZI Bsu6 I Eamll04I Earl

MboI I GMGAN 丽 JT

Mlyl GAGTCNN 删 Schl

Mmel TCCRAC

Mnl l CCTCNN drawing NJT

Plel GAGTCNNNN "N_ Ppsl

Ppi l "NNNNN_NN N NNGAACNNNNNCTCN NNNNNN_N NNN '

RleAI CCCACA NNNNNNNNJNN "

Sapl GCTCTTCN ~ NNN_ Vpa 32I

SfaNI GCATCNNNNN "NNNN_ BspST5I Phal

SspD5I GGTGANNNNNNNN ^

Sthl32I CCCG drawing NNN—

Stsl GGATGNNNNNNNNNN "NNNN_

Taql l GACCGANNNNNNNNNJN ", CACCC Awakening NN-1

TspRI JNCASTGNN "

Dragon 111 CAARCANNNNNNNNN

I

Step (5) in the method of the present invention comprises treating the two double-stranded nucleic acids obtained in step (4) with a restriction enzyme to thereby add a first amino acid at the 3 ′ end or the 5 ′ end, respectively. This is a step of preparing a double-stranded nucleic acid having a base sequence encoding two amino acids. The restriction enzyme used in step (5) is a restriction enzyme that recognizes the restriction enzyme recognition sequence introduced in step (4).

Step (6) in the method of the present invention is a step of preparing a single-stranded nucleic acid from the double-stranded nucleic acid obtained in step (5) by utilizing the binding ability of the affinity substance introduced in step (4). is there.

According to step (6), a single strand having a stem sequence and a branch sequence in the direction from the 5 'side to the 3 side and having a base sequence encoding the first amino acid and the second amino acid at the 3' end. A base nucleic acid having a stem nucleic acid and a stem sequence and a branch sequence complementary to the stem sequence in the direction from the 3 ′ side to the 5 ′ side, and encoding the first amino acid and the second amino acid at the 5 ′ end A single-stranded nucleic acid having a sequence is prepared. That is, each of the two types of single-stranded nucleic acids prepared in step (6) has a base sequence encoding a second amino acid at the 3, terminal of the first single-stranded nucleic acid prepared in step (1). This corresponds to the added nucleic acid and the nucleic acid obtained by adding a base sequence encoding the first amino acid to the 5 ′ end of the second single-stranded nucleic acid prepared in step (1).

Step (6) yields two types of single-stranded nucleic acids for use in the next cycle. As for the library 1, a single-stranded product was obtained in step (3), and a double-stranded DNA was obtained in step (4).

Step (7) in the method of the present invention is a step of repeating steps (2) to (6) as many times as necessary using the single-stranded nucleic acid obtained in step (6).

The number of blocks connected in this way increases exponentially to 2, 4, 8, 16 according to the number of cycles. This property is an important advantage when constructing long shuffled products in that the number of reaction stages can be reduced.

In the present invention, the construction of a peptide / protein library was considered as the ultimate goal, and codons (trinucleotides) as its minimum unit were blocked. The lock length can be set to any length. It is also possible to start from a block in which a plurality of codons are linked. On the extension is shuffling of the structural units of the protein (for example, structural units such as Hi-helix, 3-sheet, module, domain, etc.). Some small functional peptides are known to be composed of a few residues of amino acids.However, in order to express a variety of functions, they must be long enough to form various structures. Deemed necessary. From such a viewpoint, it can be said that a peptide consisting of more than ten amino acids has an optimal length for forming a flexible structure. The peptide consisting of 16 amino acids as shown in the examples of the present specification can be constructed only by rotating steps (2) to (6) for four cycles, and has a long potential having a high potential for function expression. Is important in the sense that

In the examples of the present specification, the operation was carried out so that the number of blocks of the 5′-half strand and the number of blocks of the 3′-half strand to be subjected to the ligation reaction in each cycle was the same. By using a substrate, it is possible to construct a shuffled body having an arbitrary number of blocks. For example, when constructing a library having a block length of 12, the steps (2) to (6) are performed three times, and the fourth cycle is prepared from the products of the third cycle and the products of the second cycle. The used 5'-half strand and 3'-half chain may be used.

This is one of the great advantages of the method of the present invention. That is, the library once constructed in each cycle can be arbitrarily reproduced and used by amplifying it with a PCR. This greatly expands the range of the sequence space to be searched in the sense that it can not only adjust the block length but also dock with a library constructed from different blocks.

The nucleic acid in the present invention is used in the broadest sense including DNA and RNA, and also includes analogs of DNA and RNA.

In recent years, the enzymatic activities and binding activities of nucleic acids themselves, such as DNA, RNA, and their analogs, have been closed up, and their applications are being actively pursued. The method of the present invention is suitable for applying such knowledge to block design and applying it to the creation of new functional nucleic acids. are doing.

In the examples of the present specification, DNA of 48 nucleotides was generated, but the 5'-half strand and the 3, -half strand including the raw material can be replaced with RNA. As can be seen from the application of RNA ligase activity for DNA ligation, ligation of substrate as RNA leads to improved efficiency. In this case, this can be achieved by including a promotion in the stem area.

The final DNA library can also be converted to RNA and replaced with an RNA library. In addition, by incorporating a nucleic acid analog or a modified / labeled nucleic acid in the final cycle, it is possible to obtain a linked substance other than a regular nucleic acid.

An outline of one specific embodiment of the above-described method of the present invention is shown in FIG. FIG. 2 also schematically illustrates the contents of the embodiment of the present specification, and does not limit the scope of the present invention. Here, MboII is used as a restriction enzyme, and biotin is used as an affinity substance. The description of each step in FIG. 2 is described below.

(1) Prepare DNA oligomers containing a common stem portion and 7 trinucleotides for the 5'-half and 3,1 half. The illustration is simplified for a single DNA oligomer single strand.

(2) Next, hybridize 5, -half and 3, -half.

(3) Ligate both chains with T4 RNA ligase.

(4) Amplify the ligation product with PCR.

(5) Perform two sets of PCR to produce the raw material for the next cycle and introduce the stem portion and the restriction enzyme recognition sequence into the ligated product (Pre-5, -half and Pre-3'-half) . At this time, the primer on the stem side should be modified with 5'-biotin. Use a restriction enzyme whose cleavage site is not included in the recognition sequence.

(6) Unnecessary one end is cut off by restriction enzyme treatment.

(7) Prepare single-stranded DNA (5'-half and 3, -half of next cycle) using the binding ability of biotin and streptavidin 'beads. At this time you need DNA is a DNA strand with a block indicated by a dark gray box. (8) Return to step 2 and repeat the above operations until a predetermined number of blocks are connected.

The present invention also relates to a nucleic acid library produced by the above-described method for producing a nucleic acid library.

The nucleic acid in the nucleic acid library obtained by the method of the present invention is composed of a base sequence encoding an arbitrary predetermined number of amino acids, and the frequency of occurrence of each amino acid generally does not show any extreme bias. It is characterized by.

The present invention also relates to a peptide library obtained by using the nucleic acid library prepared by the above-described method for preparing a nucleic acid library. The peptide library can be obtained by expressing the nucleic acid library in an appropriate expression system.

Examples of an expression system for preparing a peptide library include a phage display method. Phage display has become a powerful method for screening molecules with an affinity for a target molecule of interest from a number of peptides, muteins, and cDNAs. In phage Deisupurei method, it is possible to generate 10 ⁸ 10 ⁹ different recombinants, among these, antigens, antibodies, cell surface receptors, protein chaperones, DNA, to metal I O emissions Other It is possible to select one or more clones with affinity. Library-based screening can be used for a number of applications, as the presented factor is expressed on the virus surface as a capsid fusion protein. In phage display, (1) there is a physical association between phenotype and genotype, (2) virus particles isolated from a library can be regenerated by infecting bacteria (3) has the advantage that the primary structure of the presented binding peptide or protein can be easily deduced by sequencing the DNA of the cloned fragment in the viral genome.

The peptide library of the present invention is, for example, expressed by bacteriophage. Can be done. That is, the nucleic acid library obtained by the method of the present invention can be cloned into the gene III, VI, or VIII of Bacteriophage M13, and thereby expressed as a large number of peptide: capsid fusion proteins. Example

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

(Example 1)

In the present example, an attempt was made to construct a 16-block shuffling library in which trinucleotides encoding seven amino acids representing the properties of amino acids were used as blocks. The correspondence table between amino acids and trinucleotides is shown below. This codon was selected with reference to the frequency of codon usage in the translation system of wheat germ.

Ami Codon

Gly GGC

l ie ATC

Asp GAC

L y s AAG

S e r TCC

Cy s TGC

Pro CCA

Fig. 3 shows the structure of the substrate of T4 RNA ligase (a) and the relationship between the recognition sequence of the introduced restriction enzyme and the cleavage site (b) (details will be described in the experimental procedure).

(Experimental operation)

1.5, -half and 3, -half ligature

Each of the seven types of 5-, half-chain and 3,-half-chain (5, with terminal phosphorylation) in 30 ligation buffer (50 mM Tris-HCl (pH 8.0), lOmMMgCl ₂ , 10ing / l BSA, ImM hexammine cobalt chloride, and .25% polyethylene glycol 6000) were heated at 94 ° C for 5 minutes and then kept at 60 ° C for 15 minutes to hybridize the complementary stem region (Fig. 3 (a ), Where 'Ν' indicates the nucleotide in the block.) Thereafter, O.lmM ATP and 50 units of T4 RNA ligase (Takara) were added and reacted at 25 ° C for 16 hours.

2. Amplification of ligation products

The ligation product was purified by denaturing polyacrylamide gel electrophoresis (PAGE) (8 M urea 8% polyacrylamide, 300 V, 160 mA, 35 minutes). After excising the band of the target substance from the gel, the degree of purification was increased by performing PCR using the DNA extracted from the gel as a template (1: 1 of the band extract 50-1) as follows. By this operation, the raw material DNA can be removed. Primers containing sequences adjacent to the block portion (pl: TTGMGATCTCTTTT (SEQ ID NO: 5) and p2: TTGAACGGGATCCCCTA (SEQ ID NO: 6), each lOpmole) were used. The composition of the PCR reaction solution (50〃1) is 200〃MdNTP, 1 unit Taq polymerase (Greignner) ヽ 50 mM Tris-HCl (pH8.7), 2.5 mM MgCl ₂ . The PCR reaction conditions are as follows; pre-denaturation: 90 ° C, 2 minutes; denaturation: 90 ° C, 0.5 minutes; annealing: 35 ° C, 1 minute; extension: 72 ° C, 0.5 minutes 30 cycles.

3. PCR for preparing raw materials for the next cycle

PCR for introducing a restriction enzyme MboII recognition sequence and a stem portion into the amplified product of the ligation product is performed. In this case, two types of PCR were performed for preparation of 5, -half chain and preparation of 3, -half chain.

The former is a primer,

p3: GGCTCGCGAATACTTTGGGGATCTCTTT (0. lpmole) (SEQ ID NO: 7),

p4: TTGACGAAGATCCCCTA (lOpmole) (SEQ ID NO: 8),

p5: biotin-GGCTCGCGAATACTTTG (10pmole) (SEQ ID NO: 9) Was used.

In the latter case,

p6: ttGAAGAtctcttt (0.1pmole) (SEQ ID NO: 10),

p7: GGCTCGCGAATACTTTGAACGGGATCCCCTA (10pmole) (SEQ ID NO: 11),

p8: biotin-GGCTCGCGAATACTTTGAA (10pmole) (SEQ ID NO: 12)

Was used.

p5 and p8 are primers having a biotin modification at the 5 'end for the purpose of preparing single-stranded DNA to be performed later. This sequence is the same as the sequence at the 5 'end of p3 and p6. The underlined portion in the primer sequence indicates the recognition sequence of the restriction enzyme MboII.

4. Restriction enzyme treatment

PCR products for 3'-half strand preparation and 5, -half strand preparation were each incubated in a 30/1 buffer with 50 units of restriction enzyme MboII (Takara) at 37 ° C for 1 hour. -I did it. The structure of the restriction enzyme site is shown in FIG. 3 (b). In the figure, 'N' indicates the base of the block, and the recognition sequence and the cleavage site are indicated by boxes and arrows, respectively. It is necessary to cut at the brink of the connected block, and the restriction enzyme selection condition is that the cut site is not included in the recognition sequence. Here, one kind of enzyme is used, but it is also possible to use a combination of similar enzymes.

5. Preparation of single-stranded DNA from PCR products

Each restriction enzyme digest (30〃1) was mixed with 30 jl of 2X Binding and Washin buffer (2XB & W buffer, 0.2M Tris-HCl (pH8.0), 1M NaCl, 2% Tween-20). Combined, washed in advance with O.lM NaOH, and equilibrated with 1x Binding and Washing buffer (B & W buffer, 0.1 M Tris-HCl (pH 8.0), 0.5 M NaCl, 1¾ Tween-20) It was mixed with 60 zl streptavidin-magnet beads (Dynabeads M-280 streptavidin, DynaBeads). This suspension was stirred at room temperature for 1 hour to immobilize the PCR product. 100 beads ) Washed twice with lxB & W buffer of UL \ and twice with 100 l of water, and then treated twice with 25/1 25% aqueous ammonia at room temperature for 2 minutes to recover non-biotin-modified DNA chain . Next, the beads were washed twice with 100〃1 of water, and then treated twice with 25〃1 of 25% ammonia water at 65 ° C for 2 minutes to collect the biotin-modified DNA chain. The recovered aqueous ammonia extract is dried, dissolved in water and used as a raw material for the next cycle. Extracts from PCR products for 3'-half strand preparation use non-biotin-modified DNA strands, while extracts from PCR products for 5'-half strand preparation use biotin-modified DNA strands (see Figure 2). See).

6. Ligation of recovered 3, -half-chain and 5, -half-chain

The 3, -half strand and the 5, -half strand recovered from the ligation were hybridized in a TRL buffer and ligated with T4 RNA ligase in the same manner as in the first ligation reaction. Thereafter, by repeating the same operation, the blocks were sequentially connected until a predetermined length was obtained.

7. Shuffling sequencing

This block shuffling DNA was cloned using a TA cloning kit (TA Cloning Kit Jr., Invitrogen). The insert was confirmed by colony PCR and denaturing PAGE. Plasmid DNA was extracted from the transformant in which the insert was confirmed, using a plasmid extraction kit (Wizard Plus SV Minipreps DNA Purification System, Promega). DNA sequencing was performed with a DNA sequencing 'kit (Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP, Amersham Parmacia Biotech) and a DNA sequencer (Shimadu, DSQ2000).

(Results and discussion)

1. Results of gel electrophoresis analysis of the ligated product The ligation products amplified in each cycle were analyzed by denaturing PAGE and silver staining. Fig. 4 shows the results.

Each product contains a primer region (constant length of 32 bases) and a connecting block region (3 x 2 ⁿ base length, where n is the number of cycles). Therefore, a product of 38 bp in the first cycle, 44 bp in the second cycle, 56 bp in the third cycle, and 80 bp in the fourth cycle is expected, which is consistent with the analysis results. In FIG. 4, lane M is the size of DNA, and the size is shown on the left.

2. Results of DNA sequencing

The results of sequencing of a portion of the constructed DNA library are shown in Table 4 below.

Table 4:

: DNA sequence of DNA library

* As a result of analysis including mutated sequences and other sequence results, a substitution of a base which is considered to have occurred during PCR was confirmed. The currently used DNA polymerase for PCR is Taq DNA polymerase, which is known to introduce one mutation per tens of thousands of nucleotides during synthesis.

In addition, a deletion mutation that appeared to have occurred during cleavage of the restriction enzyme was also confirmed, but this is probably due to the non-specificity of the restriction enzyme cleavage site. The restriction enzyme MboI I used in this example is an enzyme that cuts away from the recognition sequence. Belong to.

Separate experiments suggest that the cleavage site specificity of Mbol I is different for both chains. In this example, the construction of a peptide library consisting of 16 amino acids was set as the final goal, and the YLBS cycle was repeated four times using seven types of trinucleotides as blocks, and shuffling including a predetermined number of blocks was performed. · Successfully acquired the library. No extreme bias was found in the overall frequency of block appearance as shown below. Codon GGC ATC GAC AAG TCC TGC CCA

(Gly) (lie) (Asp) (Lys) (Ser) (Cys) (Pro)

Appearance 7.6% 22.9 15.3% 17.8% 6Λ 6.1 1¾ 24.3¾

(Example 2)

A DNA library was prepared using the YLBS method.

The following sequences were used as starting oligonucleotides.

5, half: GGCTCGCGAATACTGCGAA GACCACCATGNNN (32mer, underlined stem region) (SEQ ID NO: 13)

stem primer (biotin label): GGCTCGCGAATACTGCGMGGCCACCATG (29mer) (SEQ ID NO: 14)

part primer (FITC label): GCGTAGMGATCGTGGA (17mer, anneal in 3 'half) (SEQ ID NO: 15)

3 'half: NNNTCCACGATCCTG TTCGCAGTATTCGCGAGCC (34mer, underlined stem region) (SEQ ID NO: 16)

stem primer (biotin label): GGCTCGCGAATACTGCGMCAGGATCGTGGA (31mer) (SEQ ID NO: 17)

part primer (FITC labeling): CTGCGMGACCACCATG (anneal on 17mer, 5 and half) (SEQ ID NO: 18) The part primer contains an MboI I recognition site, and the stem primer is mutated so as not to be cut by MboI I.

MboI I recognition site:

GAAGANNNNNNNN CTTCTNNNNNN Individual primer for sequence

To express the YLBS library as a peptide, it contains the # 7 promoter and a part of FLAG-Prosite.

T7 side primer: AAGAAGGAGTTGCCACCATG (20mer) (SEQ ID NO: 19)

POU side primer: TTATAGTCCAGGATCGTGGA (20mer) (SEQ ID NO: 20) Codon sequence used

In consideration of the codon usage of wheat germ and the avoidance of the recognition sequence of the restriction enzyme MboI I, the following codons were used.

Table 5: Amino acid codons Amino acid codons

Phe TTT 'His CAC

Leu CTC Gin CAA lie ATT Asn AAC

Met ATG Lys AAA

Val GTG Asp GAC

Ser TCC Glu GAG

Pro CCA Cys TGC

Thr ACC Trp TGG

Ala GCC Arg CGC

Tyr TAC Gly GGC The outline of the experiment is shown below.

(1) 5 'phosphorylation of 3' half

(2) 5 'half and 3' half hybridization and ligation

(3) Purification of ligation products by denaturing PAGE

(4) PCR amplification of ligation products

(5) boII restriction enzyme treatment

(6) Preparation of single-stranded DNA (5, half and 3, half)

(7) Next YLBS cycle

(1) 3, half of 5, terminal phosphorylation

Use T polynucleotide kinase (PNK) to phosphorylate the 3 and half 5 and termini. At this time, use a solution in which 20 kinds of 3 'halves are mixed in the respective molar amounts.

H20 2 1

3 'half mixture 4 1

(Each 20pmol, total400pmol)

ΙΟχΡΝΚ buffer ljul

T4 PNK (10U / / l, Takara) 2jul

Total 10 zl

After incubating the prepared solution at 37 ° C for 1 hour, heat at 85 ° C for 15 minutes to inactivate the enzyme. This reaction solution has a DNA concentration of 40 pmol / l.

(2) 5, half and 3, half hybridization and ligation

5 'half mixture 2pmol, total40pmol)

3 'half mixture for each 2pmol, total40pmol)

(Phosphorylated)

ImM ATP 1 zl 2 XTRL * 5/1

2 X TRL

lOOmM Tris-HCl (pH8.0)

20mM MgCl ₂

20mg / l BSA

lmM HCC (hexammine cobalt chloride)

50% (w / v) PEG6000

Heat the prepared solution at 95 ° C for 2 minutes and then anneal at 65 ° C for 10 minutes. After cooling to room temperature, add 2ul of T4 MA Ligase (25U / ul, Takara) and incubate at 25 ° overnight.

(3) Purification of ligation products by denaturing PAGE

Mix 10 ul of marker dye (0.5 BPB in formamide) with the ligated solution and fractionate by 8M urea 8% PAGE (300V, 160mA, 50 minutes). Stain using the silver staining method, cut out the band of the desired ligation product with a scalpel, and place in a 1.5 ml tube. To wash the gel, add 1 ml of sterile water, stir with a mixer for 5 minutes, and remove the sterile water. Add 50ul of sterile water and crush the gel well with a gel crushing rod. After centrifugation in a tabletop centrifuge, use the supernatant as a template for PCR.

Figure 5 shows the results of YLBS (Y1) at the 1st cycle. The ligation product is a 66mer. The band at the position corresponding to this is cut out. The ligation product should be 72mer for Y2 and 84mer for Y3.

(4) PCR amplification of ligation products

Amplify the ligation product and perform one type of PCR (5, half and 3, half) to prepare the starting material for the next YLBS cycle.

10 XPCR buffer 5 1

25mM MgCl ₂ 5 ^ 1 Gel supernatant solution (template) 5〃1

5, or 3, stem primer (10 pmol/l) lj

5, or 3, part primer (10 pmol/l) ijul

20mM dNTP 0.5 / 1

Taq Polymerase 1 unit

H ₂ 0 at final 50/1

The reaction was incubated at 94 ° C for 2 minutes, followed by one cycle of 94 ° C for 30 seconds, 60 ° C for 1 minute, and 72 ° C for 30 seconds. Incubated for 5 minutes. After the PCR is completed, remove a part of the reaction solution (for example, 5〃1) and analyze it by the same PAGE as in (3) above. At this time, if multiple bands appear, cut out the band of the target product and perform PCR again, or use a PCR cycle in which only the target product is amplified (the target product is first amplified and non-specific products are amplified). Tends to be amplified later).

(5) Restriction enzyme MboII treatment

The 5 'half and 3' PCR products of half precipitated with ethanol, dissolved in H ₂ 0 of 25 1.

5, half or 3 'half 25/1

10xL buffer 3 1

MboII (10U / ul, Takara)

Total 30〃1

Incubate the prepared solution at 37 ° C for 2 hours.

(6) Preparation of single-stranded DNA (5, half and 3 'half)

Add ₂₀制限 1 of H ₂₀ to each restriction enzyme digest (30 1). This was mixed with 50 l of Dynabeads (streptavidin M-280) equilibrated with 2xBW buffer (0.2 M Tris-HCl (pH 8.0), 1 M NaCl, 2% Tween20), and stirred at room temperature for 1 hour with a mixer. I do. By this operation, the PCR product binds to the beads. After stirring, the tube is centrifuged with a tabletop centrifuge, and beads are secured on the tube wall using a magnet. The work is called securing beads). The supernatant was removed, LxBW buffer 200〃 1 (0.1M Tris-HCl (pH8.0 ), 0.5M NaCl, l% Tween20) 1 times with 200 1 of H ₂ 0, twice with a bead secured The beads are washed by repeatedly removing the supernatant. To this, add 25 1 of 28% aqueous ammonia, stir, and leave at room temperature for 2 minutes. After securing the beads, collect and save the supernatant. Repeat this operation again. Store the supernatant together (non-pyotin chains). After washing with H ₂ 0 of the beads once 200/1, it is added and stirred 28% aqueous ammonia 25〃1 is heated at 65 ° C 20 min. After securing the beads, collect and save the supernatant. Repeat this operation again. Store the supernatant together (pyotin chains).

The four kinds of recovered liquids are dried using a centrifugal concentrator. In this case, in the next cycle of YLBS, 5, half is the DNA strand labeled with biotin, and 3 'half is the unlabeled DNA strand. In addition, the remaining recovered material is used as a measure of the recovery efficiency, and is run together during the next cycle of PAGE analysis.

(7) Next YLBS cycle

Since the 5 'end of the 3' half after recovery is phosphorylated, the same procedure is repeated from the hybridization in step 2.

FIG. 6 shows electrophoresis images of the results of electrophoretic analysis of the reaction products after the second and third YLBS cycles.

In FIG. 6, lanes 1 to 6 show the following.

Lane 1: Marker 1 (100mer, 90, 80, 70, 60, 50 ... from the top)

Lane 2: sample subjected to Y ligation (Y2 is 72mer, Y3 is 84mer, appears slightly above due to the use of biotin)

Lane 3 5 '-half of the chain without biotin

Lane 4 5 'half chain with biotin (remaining used for Y ligation) Lane 5 3'-half chain without biotin (remaining used for Y ligation)

Lane 6: 3'-half chain with biotin The dark band around 60mer is considered to be unreacted DNA that was not cleaved by boII treatment (the PCR product remains intact), and the dark band below it was cleaved with MboI I but did not ligate. .

(Result of the sequence)

The ligated product after Y3 was PCR-amplified with a primer for sequence (T7 side primer, POU side primer) and cloned using a TA cloning kit (invitrogen). Plasmid was recovered and a sequence was conducted. Figure 7 shows the results of the sequence. As a result, four out of ten clones of appropriate length were obtained. In addition, one of the 10 samples corresponded to the codon used.

Table 6:

Industrial applicability

According to the present invention, a method for constructing a nucleic acid library using the Y-ligation method has been established.

The method of the present invention can reduce the number of reaction stages when constructing a nucleic acid library encoding long peptide chains, can set the block length to an arbitrary length, and furthermore, the frequency of appearance of each block Has the advantage that no extreme bias is seen.

It is described in the specification of Japanese Patent Application No. 2000-0-346 467 and Japanese Patent Application No. 200-1307, which are the Japanese patent applications that form the basis of the priority claimed by the present application. The entire contents of which are incorporated herein by reference as part of the disclosure of this specification.

Claims

The scope of the claims

1. A method for preparing a nucleic acid library, comprising the following steps (1) to (7).

(4) The double-stranded nucleic acid obtained in step (3) is subjected to PCR using a primer modified with an affinity substance on the 5 ′ side in the form of 錄, and the direction is changed from the 5 ′ side to the 3 ′ side. Preparing a double-stranded nucleic acid comprising a stem sequence, a branch sequence, a base sequence encoding the first amino acid, a base sequence encoding the second amino acid, a branch sequence and a restriction enzyme recognition sequence;

Convert the double-stranded nucleic acid amplified in step (3) to type III, perform PCR using a primer whose 5 'side has been modified with an affinity substance, and recognize the restriction enzyme in the direction from the 5, 5 side to the 3' side. A double-stranded nucleic acid including a sequence, a branch sequence, a base sequence encoding the first amino acid, a base sequence encoding the second amino acid, a branch sequence and a stem sequence is prepared, and in step (5) (4), The resulting two types of double-stranded nucleic acids are treated with a restriction enzyme to prepare double-stranded nucleic acids having a base sequence encoding the first amino acid and the second amino acid at the 3, terminal or the 5, terminal, respectively. ,

(6) a single-stranded nucleic acid is prepared from the double-stranded nucleic acid obtained in step (5) by utilizing the binding ability of the affinity substance introduced in step (4), and

(7) Steps (2) to (6) are required using the single-stranded nucleic acid obtained in step (6). Repeat as many times as:

2. The method for producing a nucleic acid library according to claim 1, comprising the following steps (1) to (7).

(1) a first single-stranded nucleic acid having a stem sequence and a branch sequence in the direction from the 5 side to the 3 ′ side, and a third single-stranded nucleic acid having a base sequence encoding the first amino acid at the terminus; A second single-stranded nucleic acid having a stem sequence complementary to the above-mentioned stem sequence and a branch sequence in the 5′-side direction and having a base sequence encoding a second amino acid at the 5 ′ end. And

(4a) After converting the ligation product to a single-stranded nucleic acid, a complementary strand is synthesized to prepare a double-stranded nucleic acid, and (4b) the double-stranded nucleic acid obtained in the step (4a) PCR using a forward primer modified on the 5 'side with an affinity substance and a reverse primer containing a restriction enzyme recognition sequence was performed.From the 5' side to the 3 'side, the stem sequence, branch sequence, Preparing a double-stranded nucleic acid comprising a nucleotide sequence encoding the amino acid of the following, a nucleotide sequence encoding the second amino acid, a branch sequence and a restriction enzyme recognition sequence;

The double-stranded nucleic acid amplified in step (4a) is converted into type II, and PCR is performed using a food primer containing a restriction enzyme recognition sequence and a reverse primer modified on the 5th side with an affinity substance. And a restriction enzyme recognition sequence, a branch sequence, a base sequence encoding the first amino acid, a base sequence encoding the second amino acid, a branch sequence and a stem sequence in the direction from the 5 ′ side to the 3 ′ side. Prepare a single-stranded nucleic acid,

(5) By treating the two types of double-stranded nucleic acids obtained in the step (4b) with a restriction enzyme, a nucleotide sequence encoding the first amino acid and the second amino acid at the 3, or 5 terminal, respectively. Preparing a double-stranded nucleic acid having

(6) Using the binding ability of the affinity substance introduced in step (4b), Preparing a single-stranded nucleic acid from the double-stranded nucleic acid obtained in the above, and

3. The method for producing a nucleic acid library according to claim 1, comprising the following steps (1) to (7).

(1) a first single-stranded nucleic acid having a stem sequence and a branch sequence in a direction from the 5 ′ side to the 3 ′ side, and having a base sequence encoding the first amino acid at the 3 ′ end; A second single-stranded nucleic acid having a stem sequence complementary to the above-mentioned stem sequence and a branch sequence in the 5′-side direction, and having a base sequence coding for a second amino acid at the 5 ′ end; Prepare

(3) treating the hybridized product with T4MA ligase to ligate the 3 ′ end of the first single-stranded nucleic acid and the 5 ′ end of the second single-stranded nucleic acid,

(4) The double-stranded nucleic acid obtained in the step (3) is made into type III, and the 5-side is modified with an affinity substance, and a forward primer that anneals to the stem sequence of the first single-stranded nucleic acid, PCR using a reverse primer containing a restriction enzyme recognition sequence and annealing to the branch sequence of the second single-stranded nucleic acid is performed, and the stem sequence, the branch sequence, and the Preparing a double-stranded nucleic acid comprising a nucleotide sequence encoding one amino acid, a nucleotide sequence encoding a second amino acid, a branch sequence, and a restriction enzyme recognition sequence;

The double-stranded nucleic acid amplified in step (3) is converted into a type II, containing a restriction enzyme recognition sequence and a forward primer that anneals to the branch sequence of the first single-stranded nucleic acid, and an affinity substance on the 5 ′ side. PCR using a modified reverse primer that anneals to the stem sequence of the second single-stranded nucleic acid is performed, and the restriction enzyme recognition sequence, the branch sequence, the first Preparing a double-stranded nucleic acid comprising a base sequence encoding an amino acid, a base sequence encoding a second amino acid, a branch sequence and a stem sequence, (5) By treating the two types of double-stranded nucleic acids obtained in step (4) with a restriction enzyme, the nucleotide sequence encoding the first amino acid and the second amino acid at the 3 ′ end or at the 5th end is obtained. Preparing a double-stranded nucleic acid having

4. A mixture of single-stranded nucleic acids having a base sequence encoding a plurality of different first amino acids is used as the first single-stranded nucleic acid. 4. The method for producing a nucleic acid library according to claim 1, wherein a mixture of single-stranded nucleic acids having a base sequence encoding a second amino acid is used.

5. The method for producing a nucleic acid library according to claim 1, wherein the length of the stem sequence is 10 to 100 bases.

6. The stem sequence of the first single-stranded nucleic acid is GGC TCGCGAATACTTTG in the direction from the 5 'side to the 3' side, and the stem sequence of the second single-stranded nucleic acid is CCGAGCGCTTATGAAAC in the direction from the 3 'side to the 5' side. The method for producing a nucleic acid library according to any one of claims 1 to 5, wherein

7. The method for producing a nucleic acid library according to any one of claims 1 to 6, wherein the length of the branch sequence is 10 to 100 bases.

8. 'an AA GATCTCTTTT in the direction of the side, the second single-stranded branches sequence 3 of nucleic' first single-stranded 3 branches sequences from 5 ⁵ side of the nucleic acid 5 from the side, TTGCCCTAGGGGAT in the direction of the side The method for producing a nucleic acid library according to any one of claims 1 to 7, which is:

9. The nucleic acid library according to any one of claims 1 to 8, wherein in step (4), the ligation product is converted into a single-stranded nucleic acid under denaturing conditions, and then amplified by PCR to prepare a double-stranded nucleic acid. One manufacturing method.

10. The affinity substance in the primer used in step (4) is biotin, A method for producing a nucleic acid library according to any one of claims 1 to 9.

11. The nucleic acid library according to any one of claims 1 to 10, wherein the restriction enzyme recognition sequence in the primer used in step (4) is a recognition sequence for a restriction enzyme whose cleavage site is not included in the recognition sequence. Production method.

12. The nucleic acid library according to any one of claims 1 to 11, wherein the restriction enzyme recognition sequence in the primer used in step (4) is a recognition sequence for a restriction enzyme that cleaves away from the recognition sequence. Production method.

13. The method for producing a nucleic acid library according to any one of claims 1 to 12, wherein the restriction enzyme recognition sequence in the primer used in step (4) is a boII recognition sequence.

14. The nucleic acid according to any one of claims 1 to 13, wherein a nucleic acid library containing a base sequence encoding 16-amino acid is prepared by repeating the steps (2) to (6) four times in total. How to make a library.

15. A nucleic acid library produced by the method for producing a nucleic acid library according to any one of claims 1 to 14.

16. A peptide library obtained by using the nucleic acid library produced by the method for producing a nucleic acid library according to any one of claims 1 to 14.