JP2002315577A - Method for constructing nucleic acid library - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for constructing a nucleic acid library by utilizing Y-ligation method. SOLUTION: The nucleic acid library is constructed by repeating an operation that 3'-end terminal of a first single-stranded nucleic acid and 5'-end terminal of a second single-stranded nucleic acid are connected through T4 RNA ligase treatment and subsequently PCR and restriction enzyme treatment are performed after hybridization between each stem sequence is performed using the 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 base sequence encoding a first amino acid at the 3'-end terminal, and the second single-stranded nucleic acid having a stem sequence complementary to the above-mentioned stem sequence in the direction from the 3'-side to the 5'-side and a base sequence encoding a second amino acid at the 5'-end terminal.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for preparing 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 connecting the ends of the single-stranded region (branch portion) with RNA ligase. -A method for preparing a nucleic acid library, which utilizes a ligation method. 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.

[0002]

2. Description of the Related Art Evolutionary molecular engineering aims at obtaining useful biopolymers based on Darwin's principle of evolution. This method includes: 1) construction of a mutant library, 2)
It is performed by repeating the steps 2) to 4), which comprises the selection of a more useful molecular species, 3) introduction and amplification of a mutation into the genotype, and 4) expression of the mutant population.

[0003] When searching for a useful peptide or protein, a DNA mutant library is constructed for easy introduction of mutations and identification of the phenotype, and the protein is expressed in a form corresponding to this genotype. In many cases, a method of making a selection based on a function is adopted. Thus, the characteristics of a DNA library, which is the starting point, can be said to be one of the important factors that determine success in obtaining useful peptides or proteins.

[0004] Conventionally, DNA mutant libraries have been constructed at the nucleotide level, and there are chemical and enzymatic methods. The former employs a method in which nucleotides as raw materials are mixed at a specific ratio when DNA is synthesized using a DNA synthesizer. In the latter, DNA or R
There are methods to induce mutation by using NA polymerase under special conditions, and methods to create chimeric genes by subdividing and reconstituting multiple similar genes called DNA shuffling. Raising.

On the other hand, the present inventors are trying to construct a completely new mutant population called a block-based mutant population, and call it a block shuffling method. When constructing a DNA library encoding a protein, it has been proposed to set a hierarchical constitutional unit of the protein as a mutation unit. Attempts to create useful functional macromolecules from a mutant population in evolutionary molecular engineering can be compared to exploring a vast sequence space, starting from the conventional point mutant population of genes. It can be said that the method is equivalent to searching around the wild-type protein with a fork. On the other hand, the mutant population in the block unit corresponds to walking with a stride in the sequence space far 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 material is reached by this straddling. I can't say anything yet.

On the other hand, theoretical studies on molecular evolution have accumulated data showing that many proteins have acquired various functions through a mechanism called exon shuffling from the primary sequence and structural analysis of natural proteins. are doing. The idea is based on a significant correlation between the boundaries of the protein's modular structure and the location of the introns of the gene. In other words, various existing proteins can be regarded as products that exons as functional units and introns as evolutionary devices have developed through a shuffling mechanism.
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 the stride for searching the array space can be freely designed. The composition of the point mutation library varies depending on the lot to be constructed, and each search trial is independent and cannot be referred to each other. On the other hand,
Shuffling can be designed in consideration of a search history when determining the type and length of the block, and is excellent in accumulating know-how of block design for more efficient search.

[0007] When aiming to create a functional protein, the most basic structural unit 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, the random mutant population in nucleotide units also covers all amino acids. However, there are significant drawbacks. That is, a stop codon is always generated at a certain probability, and an immature peptide chain is generated. It is also known that the frequency of codon usage varies depending on the protein translation system. Since there is a limit to the amount of substances that can be experimentally handled, it is desirable to use efficient codons to secure a large library size. Block shuffling can avoid such problems.

The present inventors have developed a method for efficiently ligating DNA, called the Y-ligation method. This Y-ligation method uses two single-stranded DNAs.
(5'-half and 3'-half) include complementary sequences (stems) at the ends, and after hybridization, ligate the ends of the single-stranded region (branch) with RNA ligase. . FIG. 1 shows a schematic diagram of the Y-ligation method. However, no attempt has been made to apply the Y-ligation method, particularly to prepare a DNA library or a protein library.

[0009]

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.

[0010]

The present inventors have a stem arrangement and a branch arrangement in a direction from the 5 'side to the 3' side,
A first single-stranded nucleic acid having a base sequence encoding a first amino acid at a 3 ′ end, and a stem sequence and a branch sequence complementary to the stem sequence in a 3 ′ to 5 ′ direction Using a second single-stranded nucleic acid having a base sequence encoding a second amino acid at the 5 ′ end, hybridizing between each stem sequence, treating with T4 RNA ligase,
3 ′ end of first single-stranded nucleic acid and 5 ′ of second single-stranded nucleic acid
It has been found that a nucleic acid library can be prepared by repeating the operation of ligating the ends and subsequently performing PCR and restriction enzyme treatment, thereby completing the present invention.

That is, according to the present invention, the following step (1)
To (7), a method for producing a nucleic acid library is provided. (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 having a base sequence encoding the first amino acid at the 3 ′ end; From 5 '
A second single-stranded nucleic acid having a stem sequence complementary to the stem sequence in the side direction and a branch sequence, and having a base sequence encoding a second amino acid at the 5 ′ end,
(2) the first single-stranded nucleic acid and the second single-stranded nucleic acid are hybridized between the respective stem sequences; and (3) the hybridized product is treated with T4 RNA ligase to obtain the first single-stranded nucleic acid. The 3 ′ end of the single-stranded nucleic acid is linked to the 5 ′ end of the second single-stranded nucleic acid, and (4) the double-stranded nucleic acid obtained in step (3) is used as a template,
P using a primer whose 5 ′ side is modified with an affinity substance
CR is performed, and 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 are included. A single-stranded nucleic acid is prepared, and PCR is performed using the double-stranded nucleic acid amplified in step (3) as a template and a primer having a 5′-side modified with an affinity substance, and a 5′- to 3′-side primer is used. A restriction enzyme recognition sequence, a branch sequence, a base sequence encoding the first amino acid,
Preparing a double-stranded nucleic acid containing a base sequence, a branch sequence and a stem sequence encoding the amino acid of step (5);
By treating the two types of double-stranded nucleic acids obtained in the above with a restriction enzyme, a first amino acid and a second
A double-stranded nucleic acid having a base sequence encoding the amino acid of step (5) is prepared, and (6) the double-stranded nucleic acid obtained in step (5) is obtained from Prepare single-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, the following step (1)
To (7), a method for producing a nucleic acid library. (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 having a base sequence encoding the first amino acid at the 3 ′ end; From 5 '
A second single-stranded nucleic acid having a stem sequence complementary to the stem sequence in the side direction and a branch sequence, and having a base sequence encoding a second amino acid at the 5 ′ end,
(2) the first single-stranded nucleic acid and the second single-stranded nucleic acid are hybridized between the respective stem sequences; and (3) the hybridized product is treated with T4 RNA ligase to obtain the first single-stranded nucleic acid. The 3 ′ end of the single-stranded nucleic acid is linked to the 5 ′ end of the second single-stranded nucleic acid, (4a) the ligation product is converted into a single-stranded nucleic acid, and then the complementary strand is synthesized to prepare a double-stranded nucleic acid And (4b) PCR using the double-stranded nucleic acid obtained in the step (4a) as a template and a forward primer modified on the 5 ′ side with an affinity substance and a reverse primer containing a restriction enzyme recognition sequence, A double-stranded nucleic acid containing a stem sequence, a branch sequence, a base sequence encoding a first amino acid, a base sequence encoding a second amino acid, a branch sequence, and a restriction enzyme recognition sequence in the direction from the 5 ′ side to the 3 ′ side. Using the double-stranded nucleic acid prepared and amplified in step (4a) as a template, 'Perform PCR using a reverse primer having a modified side with an affinity substance, 5' forward primer and 5 containing the enzyme recognition sequence side 3 '
Preparing a double-stranded nucleic acid comprising a restriction enzyme recognition sequence, a branch sequence, a base sequence encoding a first amino acid, a base sequence encoding a second amino acid, a branch sequence and a stem sequence in the side direction; (5) The two types of double-stranded nucleic acids obtained in the step (4b) are treated with a restriction enzyme to obtain a double-stranded nucleic acid having a base sequence encoding the first amino acid and the second amino acid at the 3 ′ end or the 5 ′ end, respectively. A strand nucleic acid is prepared, (6) a single-stranded nucleic acid is prepared from the double-stranded nucleic acid obtained in the step (5) using the binding ability of the affinity substance introduced in the step (4b), and (7) ) Repeat steps (2) to (6) as many times as necessary using the single-stranded nucleic acid obtained in step (6):

As another example of the present invention, a method for preparing 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 5 ′ side to the 3 ′ side and having a base sequence encoding the first amino acid at the 3 ′ end; From 5 '
A second single-stranded nucleic acid having a stem sequence complementary to the stem sequence in the side direction and a branch sequence, and having a base sequence encoding a second amino acid at the 5 ′ end,
(2) the first single-stranded nucleic acid and the second single-stranded nucleic acid are hybridized between the respective stem sequences; and (3) the hybridized product is treated with T4 RNA ligase to obtain the first single-stranded nucleic acid. The 3 ′ end of the single-stranded nucleic acid is linked to the 5 ′ end of the second single-stranded nucleic acid, and (4) the double-stranded nucleic acid obtained in step (3) is used as a template,
A forward primer which is modified on the 5 ′ side with an affinity substance and anneals to a stem sequence of a first single-stranded nucleic acid, and a reverse primer which contains a restriction enzyme recognition sequence and anneals to a branch sequence of a second single-stranded nucleic acid PCR using
And a sequence 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 5 ′ to 3 ′ direction. A single-stranded nucleic acid is prepared and a forward primer containing a restriction enzyme recognition sequence and annealing to a branch sequence of the first single-stranded nucleic acid, using the double-stranded nucleic acid amplified in step (3) as a template, PCR using a reverse primer that anneals to the stem sequence of the second single-stranded nucleic acid, the side of which has been modified with an affinity substance,
A double-stranded nucleic acid containing a restriction enzyme recognition sequence, a branch sequence, a base sequence encoding a first amino acid, a base sequence encoding a second amino acid, a branch sequence, and a stem sequence in a direction from the 5 ′ side to the 3 ′ side. (5) treating the two double-stranded nucleic acids obtained in step (4) with a restriction enzyme,
A double-stranded nucleic acid having a base sequence encoding a first amino acid and a second amino acid at each of the 3 ′ end or the 5 ′ end is prepared, and (6) the binding ability of the affinity substance introduced in step (4) is determined. Utilizing a single-stranded nucleic acid from the double-stranded nucleic acid obtained in step (5), and (7) using the single-stranded nucleic acid obtained in step (6) to perform steps (2) to (6). ) As many times as necessary:

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, and a different single-stranded nucleic acid is used as the second single-stranded nucleic acid. A mixture of single-stranded nucleic acids having a base sequence encoding a plurality of second amino acids is used. Preferably, the length of the stem sequence is 10 to 100 bases. Particularly preferably, the stem sequence of the first single-stranded nucleic acid is GGCTCGGCGAATAC in a direction from 5 ′ to 3 ′.
TTTG (SEQ ID NO: 1), and the stem sequence of the second single-stranded nucleic acid is CCGAG in the 3 ′ to 5 ′ direction.
CGCTTATGAAAC (SEQ ID NO: 2).

[0015] Preferably, the length of the branch sequence is 10 to 100 bases. Particularly preferably, the branch sequence of the first single-stranded nucleic acid is A ′ in the direction from the 5 ′ side to the 3 ′ side.
AGATCTCTTTTT (SEQ ID NO: 3), and the branch sequence of the second single-stranded nucleic acid is TTGCCCTAGGGGGAT (SEQ ID NO: 4) in the direction from the 3 ′ side to the 5 ′ side. 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.

[0016] 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 of 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 of 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 MboII recognition sequence. Preferably, a library of nucleic acids containing a base sequence encoding 16 amino acids is prepared by repeating step (2) to step (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 yet another aspect of the present invention,
There is provided a peptide library obtained by using the nucleic acid library produced by the method for producing a nucleic acid library described above.

[0018]

Embodiments of the present invention will be described below in detail. The method for preparing a nucleic acid library of the present invention is characterized by including 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 3 ′ side, and having a base sequence encoding the first amino acid at the 3 ′ end. A main chain nucleic acid having a stem sequence and a branch sequence complementary to the stem sequence in a direction from the 3 ′ side to the 5 ′ side;
This is a step of preparing a second single-stranded nucleic acid having a base sequence encoding a second amino acid at the end.

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

The different plural kinds of amino acids include, for example, any two selected from natural 20 kinds of amino acids.
The above amino acids can be mentioned. The number of types of amino acids is not particularly limited. Natural amino acids include aliphatic amino acids (glycine, alanine), branched amino acids (valine, leucine, isoleucine), hydroxy amino acids (serine, threonine), acidic amino acids (aspartic acid, glutamic acid), and amides (asparagine, glutamine). , Basic amino acids (lysine, arginine), sulfur-containing amino acids (cysteine, methionine), aromatic amino acids (phenylalanine, tyrosine), heterocyclic amino acids (tryptophano, histidine), and imino acid (proline) However, one kind of amino acid may be selected and used as a representative from each group.
In the examples described later in this specification, glycine, isoleucine, aspartic acid, lysine, serine, cysteine, and proline are selected as seven amino acids representative of the properties of amino acids, which are examples of the present invention. It's just

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 10 to 100 bases. , More preferably 10 to 3
It is about 0 bases. The stem sequence of the 5 ′ half-strand and the stem sequence of the 3 ′ half-strand are complementary to each other, which allows the 5 ′ half-strand and the 3 ′ half-strand to hybridize under certain conditions. More specifically, since the stem sequence of the 5 'half strand (5' to 3 'direction) is complementary to the stem sequence of the 3' half strand (3 'to 5' direction), both strands hybridize. As a result, both stem sequences form a double strand, and the 5 ′ half-strand branch strand and the 3 ′ half-strand branch strand remain single-stranded, so that they form an overall Y-shape. (See FIG. 1). The name Y-ligation derives from the form of this structure. The feature of this method is that the ligation efficiency can be improved by changing the reaction for linking two DNAs from an intermolecular reaction to an intramolecular reaction. Therefore, it can be applied to low-concentration substrates.

An example of the stem sequence of the first single-stranded nucleic acid is GGCTCGC in the direction from 5 ′ to 3 ′.
GAATACTTTG (SEQ ID NO: 1) and an example of the stem sequence of the second single-stranded nucleic acid include CCGAGCCGCTTATGAAAC (SEQ ID NO: 2) in the direction from the 3 ′ side to the 5 ′ side.

The length of the branch sequence in the 5 'half strand and the 3' half strand is such that the base at the 3 'end of the 5' half strand and the base at the 5 'end of the 3' half strand are linked by T4 RNA ligase treatment. There is no particular limitation as long as it is as long as possible. In general, 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 total can be ligated with considerable efficiency. In addition, since the two chains are reacted at a stoichiometric ratio of 1: 1, the yield of the product with respect to the raw material is improved, and the cost can be reduced and the purification operation can be simplified. The length of the branch sequence is preferably about 10 to 100 bases, more preferably about 10 to 30 bases. The lengths of the 5 ′ half-strand branch sequence and the 3 ′ half-strand branch sequence may be the same or different.

The branch sequence may include a restriction enzyme recognition sequence as described later in this specification. First
As an example of the branch sequence of the single-stranded nucleic acid, AAGATCTCTTTTT (SEQ ID NO: 3) in the direction from the 5 ′ side to the 3 ′ side, and as the branch sequence of the second single-stranded nucleic acid, In direction TTGCCCTA
GGGGAT (SEQ ID NO: 4).

The 5 ′ half-strand and 3 ′ half-strand used in step (1) can be synthesized by an ordinary DNA synthesis method (a method of synthesizing using a DNA synthesizer). Further, by using the phosphorylated 5 ′ end of the 3 ′ half chain, it becomes possible to bind to the 3 ′ end of the 5 ′ half chain.

Step (2) in the method of the present invention comprises the following steps:
This is a step of hybridizing the 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
After heating at 94 ° C. for 5 minutes in a suitable buffer, for example,
By keeping the temperature at 60 ° C. for 15 minutes, 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 T4 RNA ligase, and the 3 ′ end of the first single-stranded nucleic acid and the second single-stranded nucleic acid are treated. This is a step of linking the 5 ′ end of the strand nucleic acid. When a suitable buffer for T4 RNA ligase is used as the hybridization solution, the solution containing the hybridization product can be used for the ligase reaction as it is. The product is recovered by a usual DNA purification method, and then dissolved in a buffer for T4 RNA ligase to prepare a solution for ligase reaction. In such a solution, ATP and T4RNA
The ligase reaction is performed by adding ligase and reacting at an appropriate temperature (for example, 25 ° C.) for a certain period of time (for example, 16 hours). As a result, the 3 ′ end of the first single-stranded nucleic acid is linked to the 5 ′ end of the second single-stranded nucleic acid.

The step (4) in the method of the present invention is carried out by PCR using the double-stranded nucleic acid obtained in the step (3) as a template.
A stem arrangement, a branch arrangement in a direction from the 5 ′ side to the 3 ′ side,
A base sequence encoding the first amino acid, a base sequence encoding the second amino acid, a double-stranded nucleic acid including a branch sequence and a restriction enzyme recognition sequence; and a restriction enzyme recognition sequence in a 5 ′ to 3 ′ direction. , A branch sequence, a base sequence encoding the first amino acid, a double-stranded nucleic acid including a base sequence encoding the second amino acid, a branch sequence and a stem sequence.

In the first specific example of the step (4),
(4a) After converting the ligation product into a single-stranded nucleic acid, a complementary strand is synthesized to prepare a double-stranded nucleic acid, and then (4b) Step (4a)
Using the double-stranded nucleic acid obtained in step 1 as a template, PCR was performed using a forward primer modified on the 5 ′ side with an affinity substance and a reverse primer containing a restriction enzyme recognition sequence, and the direction from the 5 ′ side to 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 is prepared in step (4a). Using the amplified double-stranded nucleic acid as a template, PCR was performed using a forward primer containing a restriction enzyme recognition sequence and a reverse primer modified on the 5 ′ side with an affinity substance, and the PCR was performed in the direction from the 5 ′ side to the 3 ′ side. Restriction enzyme recognition sequence, branch sequence, first
A double-stranded nucleic acid comprising a base sequence encoding the amino acid of the above, a base sequence encoding the second amino acid, a branch sequence and a stem sequence is prepared. In this specification, the step (4) includes the above steps (4a) and (4b).

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. In the ligation product obtained in step (3), the stem sequences of the 5 ′ half strand and the 3 ′ half strand form a double strand, and the 5 ′ half strand branch sequence, A loop structure is formed in the order of the base sequence encoding one amino acid, the base sequence encoding the second amino acid, and the branch sequence of the 3 ′ half chain. This ligation product can be made into a single-stranded nucleic acid by placing it under denaturing conditions. The single-stranded nucleic acid of the ligation product is composed of a 5 ′ half-strand stem sequence, a 5 ′ half-strand branch sequence, a base sequence encoding the first amino acid, and a second amino acid in the 5 ′ to 3 ′ direction. It has a base sequence to encode, a 3 ′ half-strand branch sequence, and a 3 ′ half-strand 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 performed by PCR using the single-stranded nucleic acid as a template, whereby a desired double-stranded nucleic acid can be amplified.

In the step (4b), using the double-stranded nucleic acid obtained in the step (4a) as a template, a forward primer modified on the 5 ′ side with an affinity substance and a reverse primer containing a restriction enzyme recognition sequence are used. PCR is performed, and a sequence 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 from the 5 ′ side to the 3 ′ side. PCR using a double-stranded nucleic acid prepared in step (4) as a template and using a double-stranded nucleic acid amplified in step (4) as a template and a forward primer containing a restriction enzyme recognition sequence and a reverse primer modified on the 5 ′ 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 branch sequence in the 5 ′ to 3 ′ direction. A step of preparing a double-stranded nucleic acid comprising a beam array.

The affinity substance in the primer used in the step (4b) can be introduced into the primer and is not particularly limited as long as it allows the preparation of a single-stranded nucleic acid in the step (7). . Examples of affinity substances include:
In this case, in step (7), single-stranded nucleic acid labeled with biotin can be recovered using streptavidin.

In the second specific example of the step (4), the first single-stranded nucleic acid obtained by modifying the 5 ′ side with an affinity substance using the double-stranded nucleic acid obtained in the step (3) as a template PCR using a forward primer that anneals to the stem sequence of No. and a reverse primer that contains a restriction enzyme recognition sequence and anneals to the branch sequence of the second single-stranded nucleic acid,
3 'direction from the side, stem arrangement, branch arrangement, 1st
A double-stranded nucleic acid containing a nucleotide sequence encoding the amino acid of the above, a nucleotide sequence encoding the second amino acid, a branch sequence, and a restriction enzyme recognition sequence, and using the double-stranded nucleic acid amplified in step (3) as a template Containing a restriction enzyme recognition sequence,
PCR was performed using a forward primer that anneals to the branch sequence of the first single-stranded nucleic acid and a reverse primer that has a 5′-side modified with an affinity substance and anneals to the stem sequence of the second single-stranded nucleic acid. A double-stranded nucleic acid comprising a restriction enzyme recognition sequence, a branch sequence, a base sequence encoding a first amino acid, a base sequence encoding a second amino acid, a branch sequence, and a stem sequence in the direction from the 5 ′ side to the 3 ′ side And (5) treating the two types of double-stranded nucleic acids obtained in the step (4) with a restriction enzyme, thereby encoding the first amino acid and the second amino acid at the 3 ′ end or the 5 ′ end, respectively. A double-stranded nucleic acid having a base sequence of

In the second specific example, the steps (4a) and (4b) in the first specific example are performed by one PCR.
Is performed. In the PCR reaction, as a result of insertion of a mutation or the like, a mutation is introduced into the final ligation product, 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. Then, step (4)
PCR for the purpose of amplifying the ligation product of a) and preparing the raw material for the next cycle of step (4b) was performed once
It was decided to carry out simultaneously by CR operation. According to the method of the second specific example, primers (stemprimer, part
PCR is performed using the primers) to perform PCR amplification of the ligation product. According to this method, the frequency of introducing mutations into a nucleic acid library prepared by repetition of cycles of base sequences encoding amino acids 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 PCRs are performed in order to produce a raw material for the next cycle, and the ligated product prepared in the steps (2) to (3) is restricted to a stem portion. This is a step of introducing an enzyme recognition sequence.

3 ′ from 5 ′ prepared in step (4)
In a double-stranded nucleic acid including a stem sequence, a branch sequence, a base sequence encoding a first amino acid, a base sequence encoding a second amino acid, a branch sequence and a restriction enzyme recognition sequence in the side direction, The restriction enzyme recognition sequence was introduced by the restriction enzyme in 5) so that the restriction enzyme recognition sequence was cleaved at the 3 ′ end of the nucleotide sequence encoding the second amino acid. Similarly, 3 ′ from the 5 ′ side prepared in step (4)
In a double-stranded nucleic acid including a restriction enzyme recognition sequence, a branch sequence, a base sequence encoding a first amino acid, a base sequence encoding a second amino acid, a branch sequence and a stem sequence in the side direction, the following steps ( A restriction enzyme recognition sequence was introduced by the restriction enzyme in 5) so that the restriction enzyme recognition sequence was cleaved at the 5 ′ end of the base sequence encoding the first amino acid.

The restriction enzyme recognition sequence in the primer used in step (4) is a recognition sequence of a restriction enzyme whose cleavage site is not included in the recognition sequence, and preferably, a recognition sequence of a restriction enzyme which cuts away from the recognition sequence. For example, a recognition sequence of MboII. As the "restriction enzyme having no cleavage site in the recognition sequence" used in the present invention, there are two types of enzymes, one that cleaves immediately outside the recognition sequence and one that cleaves 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:

[0038]

[Table 1]

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

[0040]

[Table 2]

[0041]

[Table 3]

In the step (5) in the method of the present invention, the two types of double-stranded nucleic acids obtained in the step (4) are treated with a restriction enzyme, so that a first amino acid is added to the 3 ′ end or 5 ′ end, respectively. And preparing a double-stranded nucleic acid having a base sequence encoding the second amino acid. The restriction enzyme used in step (5) is a restriction enzyme that recognizes the restriction enzyme recognition sequence introduced in step (4).

In the step (6) in the method of the present invention, utilizing the binding ability of the affinity substance introduced in the step (4),
This is a step of preparing a single-stranded nucleic acid from the double-stranded nucleic acid obtained in the step (5). A single-stranded nucleic acid having a stem sequence and a branch sequence in the direction from 5 ′ to 3 ′ and having a base sequence encoding a first amino acid and a second amino acid at the 3 ′ end by step (6) And a stem sequence complementary to the stem sequence and a branch sequence in the direction from the 3 ′ side to the 5 ′ side, and a base sequence encoding the first amino acid and the second amino acid at the 5 ′ end. A single-stranded nucleic acid is prepared. That is, each of the two types of single-stranded nucleic acids prepared in step (6) has a nucleotide sequence encoding a second amino acid added to the 3 ′ end of the first single-stranded nucleic acid prepared in step (1). And a nucleic acid obtained by adding a base sequence encoding a first amino acid to the 5 ′ end of the second single-stranded nucleic acid prepared in step (1). Step (6) provides two types of single-stranded nucleic acids for use in the next cycle.
As a library, 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 is 2, depending on the number of cycles,
It increases exponentially to 4, 8, and 16. This property is an important advantage when constructing a long shuffled product in that the number of reaction stages can be reduced.

In the present invention, the construction of a peptide / protein library is regarded as the final goal, and a codon (trinucleotide) as its minimum unit is used as a block. However, the block length can be set to any length.
It is also possible to start from a block in which multiple codons are linked. On its extension is shuffling in structural units of the protein (eg, structural units such as α-helix, β-sheet, module, domain, etc.). Although a small functional peptide is known to be composed of several amino acids, it is necessary to have a length that can form various structures to express various functions. it is conceivable that. From this point of view, 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 was prepared in the steps (2) to (4).
This is important in that it can be constructed only by turning (6) four cycles, and has a high potential for expressing functions.

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, but the number of even-numbered and odd-numbered blocks was By using a number of substrates, it is possible to construct a shuffling body having an arbitrary number of blocks.
For example, when constructing a library having a length of 12 blocks, steps (2) to (6) are performed three times, and the fourth cycle is prepared from the product of the third cycle and the product of the second cycle. A '-half strand and a 3'-half strand 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 regenerated and used by amplifying it by PCR. This greatly expands the range of the sequence space to be searched in the sense that not only the adjustment of the block length but also the possibility of docking with a library constructed from different blocks is possible.

The nucleic acid in the present invention includes DNA and RN
It is used in the broadest sense, including A, and includes analogs of DNA and RNA. In recent years, the enzymatic activity and binding activity of nucleic acids themselves, such as DNA, RNA, and analogs thereof, have been highlighted 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 creation of a new functional nucleic acid.

In the examples of the present specification, a 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 understood from the fact that the activity of RNA ligase is applied to DNA ligation, ligation of a substrate as RNA leads to an improvement in efficiency. This is achieved by including a promoter in the stem region. The final DNA library can also be converted to RNA and replaced with an RNA library. Also, by incorporating a nucleic acid analog or a modified / labeled nucleic acid in the final cycle, it is also possible to obtain a ligated substance other than a regular nucleic acid.

FIG. 2 shows an outline of one specific embodiment of the above-mentioned method of the present invention. FIG. 2 schematically illustrates the contents of the embodiment of the present specification, and does not limit the scope of the present invention. Here, M is used as a restriction enzyme.
boII is used, and biotin is used as an affinity substance. The description of each step in FIG. 2 is described below. (1) For 5′-half and 3′-half, prepare a DNA oligomer containing a common stem portion and seven trinucleotides. In the figure, a single DNA oligomer chain is used for simplification. (2) Next, the 5'-half and the 3'-half are hybridized. (3) Ligate both chains with T4 RNA ligase. (4) Amplify the ligation product by PCR.

(5) Two sets of PCRs are performed to produce a raw material for the next cycle, and a stem portion and a restriction enzyme recognition sequence are introduced into the ligated product (Pre-5'-half and Pre-3'-half). ). At this time, the primer on the stem side used is one modified with 5 ′ biotin. As the restriction enzyme, one whose cleavage portion is not included in the recognition sequence is used. (6) Unnecessary one end is cut off by restriction enzyme treatment. (7) Single-stranded DNA (5'-half and 3'-half of the next cycle) is prepared using the binding ability of biotin and streptavidin beads. DN required at this time
A is D with blocks indicated by dark gray boxes
NA chain. (8) Return to step 2 and repeat the above operations until a predetermined number of blocks are linked.

The present invention also relates to a nucleic acid library prepared by the above-described method for preparing 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 appearance of each amino acid does not generally show an 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. A peptide library can be obtained by expressing a nucleic acid library in an appropriate expression system. An expression system for preparing a peptide library includes, for example, a phage display method. The phage display method uses molecules with an affinity for the target molecule of interest to determine the number of peptides, muteins, and cDs.
It is a powerful method for screening from NA. The phage display method can produce 10 ⁸ to 10 ⁹ different recombinants, among which the affinity for antigens, antibodies, cell surface receptors, protein chaperones, DNA, metal ions, etc. It is possible to select one or more clones that have.
Library screening can be used for a number of applications because the displayed 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 the library can be regenerated by infecting bacteria, (3) The primary structure of the displayed binding peptide or protein has the advantage that it can be easily deduced by sequencing the DNA of the cloned fragment in the viral genome. The peptide library of the present invention can be expressed, for example, by bacteriophage. That is, the nucleic acid library obtained by the method of the present invention can be cloned into gene III, VI, or VIII of bacteriophage M13, and thereby expressed as a number of peptide: capsid fusion proteins.

[0053]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples. (Example 1) In this 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.

Amino acid codon Gly GGC Ile ATC Asp GAC Lys AAG Ser TCC Cys 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 procedures).

[0056] (Experimental Procedure) 1. 5′-half and 3′-half ligation Seven types of 5′-half strands and 3′-half strands (5′-terminal phosphorylated) in 30 μl of ligation buffer (5
0 mM Tris-HCl (pH 8.0), 10 mM MgCl ₂ , 10 mg / l BSA, 1 mM
hexammine cobalt chloride, and 25% polyethylene gl
ycol 6000), heated at 94 ° C. for 5 minutes, and then incubated at 60 ° C. for 15 minutes to hybridize complementary stem regions (see FIG. 3 (a), where “N” indicates nucleotides in the block portion) Is shown). Then 0.1 mM ATP and 50 units of T
4 RNA ligase (Takara) was added and reacted at 25 ° C. for 16 hours.

[0057] 2. Amplification of Ligation Product 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 DNA extracted from the gel as a template (1 μl in 50 μl of band extract) as follows. By this operation, the raw material DNA can be removed. A primer containing a sequence adjacent to the block portion (p1: TTGAAGATCTCTTTT
(SEQ ID NO: 5) and p2: TTGAACGGGATCCCCTA (SEQ ID NO: 6), each 10 pmole) were used. PCR reaction solution (50 µl)
Is 200 μM dNTP, 1 unit Taq polymerase (Gr
eignner), 50 mM Tris-HCl (pH 8.7), 2.5 mM MgCl ₂ . The PCR reaction conditions are shown below; pre-denaturation: 90 ° C.,
2 minutes; Denaturation: 90 ° C, 0.5 minutes; Annealing: 35 ° C, 1
Min; extension: 72 ° C, 0.5 min; 30 cycles.

[0058] 3. For the preparation of 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 PCR ligation is performed. Here, two types of PCR are used for preparing the 5'-half strand and for preparing the 3'-half strand.
Was done. The former used p3: GGCTCGCGAATACTTTGGGGATCTCTTT (0.1pmole) (SEQ ID NO: 7), p4: TTGAC GAAGA TCCCCTA (10pmole) (SEQ ID NO: 8), and p5: biotin-GGCTCGCGAATACTTTG (10pmole) (SEQ ID NO: 9) as primers.

The latter is used as a primer, p6: tt GAAGA tctcttt (0.1 pmole) (SEQ ID NO: 10), p7: GGCTCGCGAATACTTTGAACGGGATCCCCTA (10 pmole) (SEQ ID NO: 11), p8: biotin-GGCTCGCGAATACTTTGAA (10 pmole) (SEQ ID NO: 1)
2) 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 part in the primer sequence is the restriction enzyme MboII.
Is shown.

[0060] 4. PCR products for 3'-half strand preparation and 5'-half strand preparation were each treated with 50 units of restriction enzyme in 30 μl of buffer.
Incubated for 1 hour at 37 ° C with Mbo II (Takara).
The structure of the restriction enzyme site is shown in FIG. 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 type of enzyme is used, but it is also possible to use a combination of similar enzymes.

[0061] 5. Preparation of single-stranded DNA from PCR products
2 × Binding of manufacturing each of the restriction enzyme digests (30 [mu] l) 30 [mu] l
and Washing buffer (2 × B & W buffer, 0.2M Tris-HCl
(pH8.0), 1M NaCl, 2% Tween-20)
Wash with M NaOH, and add 1x Binding and Washing buffer (B
& W buffer, 0.1M Tris-HCl (pH8.0), 0.5M NaCl, 1% T
Ween-20) equilibrated with 60 μl streptavidin magnet beads (Dynabeads M-280 streptavidin, DynaB
eads). This suspension was stirred at room temperature for 1 hour to immobilize the PCR product. Transfer 100 μl of beads to 1 × B &
After washing once with W buffer once and twice with 100 μl of water, the non-biotin-modified DNA strand was recovered by treating twice with 25 μl of 25% aqueous ammonia at room temperature for 2 minutes. Next, add 100 μl of beads
And then treated twice with 25 μl of 25% ammonium water at 65 ° C. for 2 minutes to recover a biotin-modified DNA strand. The recovered ammonia water extract is dried, dissolved in water, and used as a raw material for the next cycle. The extract from the PCR product for preparing the 3′-half strand uses a non-biotin-modified DNA strand, and the extract from the PCR product for preparing the 5′-half strand uses the biotin-modified DNA strand (see FIG. 2). ).

[0062] 6. Recovered 3'-half strand and 5'-c
The 3′-half strand and the 5′-half strand recovered from the ligation ligation of the leaf strand were hybridized in a TRL buffer in the same manner as in the first ligation reaction, and then ligated with T4 RNA ligase. Thereafter, by repeating the same operation, the blocks were sequentially connected until a predetermined length was reached.

[0063] 7. Sequencing of shuffled product This block shuffling DNA is transferred to TA cloning kit
(TA Cloning Kit Jr., Invitrogen). Confirmation of the insert was performed by colony PCR and denaturing PAGE. Plasmid extraction kit (Wizard Plus SV Minipreps
Plasmid DNA was extracted using DNA Purification System, Promega). The DNA sequence was obtained from a DNA sequencing kit (Thermo Sequenase fluorescent labeled pri
mer cycle sequencing kit with7-deaza-dGTP, Amersha
m Parmacia Biotech) and DNA sequencer (Shimad
u, DSQ2000).

[0064] (Results and Discussion) 1. Results of gel electrophoresis analysis of the ligated product The ligated product amplified in each cycle was analyzed by denaturing PAGE and silver staining. FIG. 4 shows the results. Each product is composed of a primer region (32 base length fixed) and a linking block region (3 × 2 ⁿ
Base length, 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,
It is consistent with the analysis results. In FIG. 4, lane M is a DNA size marker, and the size is shown on the left.

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

[0066]

[Table 4]

As a result of analysis including other sequence results,
Base substitution that was considered to have occurred during PCR was confirmed. The PCR DNA polymerase currently used is Taq DNA polymerase, and it is known that this enzyme introduces 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 cleavage site of the restriction enzyme. The restriction enzyme MboII used in this example belongs to an enzyme that cuts a site far away from the recognition sequence. Separate experiments suggest that the cleavage site specificity of MboII 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 a predetermined number of blocks were included. Successfully acquired a shuffling library. No extreme bias was observed in the overall block appearance frequency as shown below.

[0069]

(Example 2) A DNA library was prepared by using the YLBS method. The following sequence was used as a starting oligonucleotide. 5'half: GGCTCGCGAATACTGCGAA GACCACCATGNNN (32mer, underlined stem region) (SEQ ID NO: 13) stem primer (biotin label): GGCTCGCGAATACTGCGAAGGCCACC
ATG (29mer) (SEQ ID NO: 14) part primer (FITC label): GCGTAGAAGATCGTGGA (17mer, 3'h
3′half: NNNTCCACGATCCTG TTCGCAGTATTCGCGAGCC ( 34mer , underlined stem region) (SEQ ID NO: 16) stem primer (biotin label): GGCTCGCGAATACTGCGAACAGGATC
GTGGA (31mer) (SEQ ID NO: 17) part primer (FITC label): CTGCGAAGACCACCATG (17mer, 5'h
alf) (SEQ ID NO: 18) The part primer contains an MboII recognition site,
The rimer is mutated to prevent cleavage by MboII.

MboII recognition site: GAAGANNNNNNNN ↓ CTTCTNNNNNNN ↑

Sequencing primers To express the YLBS library as a peptide,
Includes promoter and part of FLAG-Prosite. T7 side primer: AAGAAGGAGTTGCCACCATG (20mer) (SEQ ID NO: 19) POU side primer: TTATAGTCCAGGATCGTGGA (20mer) (SEQ ID NO: 20)

Codon Sequences Used The following codons were used in consideration of the codon usage of wheat germ and the avoidance of the recognition sequence of the restriction enzyme MboII.

[0074]

[Table 5]

The outline of the experiment is shown below. (1) 3 'half phosphorylation at the 5' end (2) 5 'half and 3' half hybridization and ligation (3) Purification of ligation product by denaturing PAGE (4) PCR amplification of ligation product (5) Restriction enzyme MboII treatment (6) Preparation of single-stranded DNA (5'half and 3'half) (7) Next YLBS cycle

(1) 3 ′ half 5 ′ end phosphorylation 3 ′ half 5 ′ end using T4 polynucleotide kinase (PNK)
The ends are phosphorylated. At this time, a solution in which 20 kinds of 3′half are mixed in an equimolar amount is used. H2O 2μl 3'half mixture 4μl (20 pmol/total 400pmol each) 10xPNK buffer 1μl 1mM ATP 1μl T4 PNK (10U / μl, Takara) 2μl Total 10μl Prepared solution was incubated at 37 ° C for 1 hour, then heated at 85 ° C for 15 minutes To inactivate the enzyme. This reaction solution has a DNA concentration of 40 pmol / μl.

(2) 5 ′ half and 3 ′ half hybridization and ligation 5 ′ half mixture 1 μl (each 2 pmol, total 40 pmol) 3 ′ half mixture 1 μl (each 2 pmol, total 40 pmol) (phosphorylated) 1 mM ATP 1 μl 2 × TRL * 5μl

2 × TRL 100 mM Tris-HCl (pH 8.0) 20 mM MgCl ₂ 20 mg / l BSA 1 mM HCC (hexammine cobalt chloride) 50% (w / v) PEG6000

After heating the prepared solution at 95 ° C. for 2 minutes,
Anneal at 10 ° C for 10 minutes. Then cool to room temperature, add 2ul of T4 RNA Ligase (25U / ul, Takara),
Incubate overnight.

(3) Purification of the ligation product by denaturing PAGE A marker dye (0.5% BPB in formam) was added to the ligation solution.
ide) Mix 10ul and fractionate by 8M urea 8% PAGE (300V, 160
mA, 50 minutes). The band of the target ligation product is cut out with a scalpel, stained by silver staining, and placed in a 1.5 ml tube. To wash the gel, add 1 ml of sterilized water, stir with a mixer for 5 minutes, and remove the sterilized water. Further, add 50ul of sterile water and crush the gel well with a gel crushing rod. After centrifugation in a tabletop centrifuge, the supernatant is used as a template for PCR. YLBS in the first cycle
The result of (Y1) is shown in FIG. Ligation product is 66mer
It is. 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 Product Along with amplifying the ligation product, two types of PCR (5′half and 3 ′) were used to prepare the raw material for the next YLBS cycle.
half) do. 10 × PCR buffer 5 μl 25 mM MgCl ₂ 5 μl Gel supernatant solution (template) 5 μl 5 ′ or 3 ′ stem primer (10 pmol / μl) 1 μl 5 ′ or 3 ′ part primer (10 pmol / μl) 1 μl 20 mM dNTP 0.5 μl Taq Polymerase (Greiner) 1 unit H ₂ O, final 50 μl

The reaction was carried out 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. After completion of PCR, a part of the reaction solution (for example, 5 μl)
And analyzed by the same PAGE as in (3) above. At this time,
When 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 amplified first, and non-specific products are amplified later. Again).

(5) Restriction enzyme MboII treatment The 5 'half and 3' half PCR products were precipitated with ethanol and 25 µl
Dissolve in H ₂ O. 5'half or 3'half 25 µl 10xL buffer 3 µl MboII (10 U / ul, Takara) 2 µl Total 30 µl Incubate the prepared solution at 37 ° C for 2 hours.

(6) Preparation of Single-Stranded DNA (5'half and 3'half) 20 μl of H ₂ O is added to each restriction enzyme digest (30 μl).
This was added to 2xBW buffer (0.2 M Tris-HCl (pH 8.0), 1 M NaCl, 2% Tw
een20) equilibrated with Dynabeads (streptavidin M-280)
Mix with 50 μl and stir with a mixer at room temperature for 1 hour.
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 (hereinafter, this operation is called bead securing). Remove the supernatant and add 1 μl of 1xBW buffer (0.1 M Tris-HCl (pH 8.0), 0.5 M NaCl, 1% Tween20).
Wash the beads by repeating the bead securing and supernatant removal twice with 200 μl of H ₂ O twice. 25 μl of 28% aqueous ammonia is added thereto, and the mixture is stirred and left at room temperature for 2 minutes. After securing the beads, collect and store the supernatant. Repeat this operation again. Store the supernatants together (non-biotin chains). After washing the beads once with 200 μl of H ₂ O, add 25 μl of 28% aqueous ammonia, stir, and heat at 65 ° C. for 20 minutes. After securing the beads, collect and store the supernatant. Repeat this operation again. Store the supernatant together (biotin chains).

The four kinds of recovered liquids are dried using a centrifugal concentrator. The next cycle of YLBS is 5 '
Half is the DNA strand labeled with biotin, and 3'half is the unlabeled DNA strand. In addition, since the remaining collected material is a measure of the recovery efficiency, it is electrophoresed 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 an electrophoretic image of the result of electrophoretic analysis of the reaction product after the second and third YLBS cycles. In FIG. 6, lanes 1 to 6 show the following. Lane 1: marker (100mer, 90, 80, 70, 60, 5 from the top)
0 ....) Lane 2: Sample subjected to Y ligation (Y2 is 72mer, Y3
Is slightly higher because of 84mer, with biotin.) Lane 3: 5'-half chain without biotin. Lane 4: 5'-half chain with biotin (Yl
Lane 5: 3′-half biotin-free strand (Y
(Lane used for ligation) Lane 6: 3'-half of biotin-attached strand A dark band around 60mer was not cleaved by MboII treatment
The DNA (the PCR product remains intact) and the dark band below it are considered unreacted DNA that was cut with MboII but not ligated.

(Results of Sequence) The ligation product after Y3 was used as a primer for sequencing (T7 side primer,
PCR amplification with POUside primer) and TA cloning kit
(invitrogen). The plasmid was recovered and sequenced. The results of the sequence are shown in FIG. As a result, four out of ten clones of an appropriate length were obtained. Furthermore, one corresponding to the used codon was one out of ten samples.

[0088]

[Table 6]

[0089]

According to the present invention, a method for constructing a nucleic acid library utilizing 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 a long peptide chain, can set the block length to an arbitrary length, and further, has an extremely biased appearance frequency of each block. Has the advantage of not being seen.

[0090]

[Sequence List] SEQUENCE LISTING <110> GenCom <120> A method for preparing a library of nucleic acids <130> A11446MA <160> 30

<210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 1 ggctcgcgaa tactttg 17

<210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 2 ccgagcgctt atgaaac 17

<210> 3 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 3 aagatctctt tt 12

<210> 4 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 4 ttgccctagg ggat 14

<210> 5 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 5 ttgaagatct ctttt 15

<210> 6 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 6 ttgaacggga tccccta 17

<210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 7 ggctcgcgaa tactttgggg atctcttt 28

<210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 8 ttgacgaaga tccccta 17

<210> 9 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 9 ggctcgcgaa tactttg 17

<210> 10 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 10 ttgaagatct cttt 14

<210> 11 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 11 ggctcgcgaa tactttgaac gggatcccct a 31

<210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 12 ggctcgcgaa tactttgaa 19

<210> 13 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 13 ggctcgcgaa tactgcgaag accaccatgn nn 32

<210> 14 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 14 ggctcgcgaa tactgcgaag gccaccatg 29

<210> 15 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 15 gcgtagaaga tcgtgga 17

<210> 16 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 16 nnntccacga tcctgttcgc agtattcgcg agcc 34

<210> 17 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 17 ggctcgcgaa tactgcgaac aggatcgtgg a 31

<210> 18 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 18 ctgcgaagac caccatg 17

<210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 19 aagaaggagt tgccaccatg 20

<210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 20 ttatagtcca ggatcgtgga 20

<210> 21 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 21 aagaaggagt tgccaccatg aaaagaccac ccccaccacc cagtccacga tcctggacta 60 taa 63

<210> 22 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 22 aagaaggagt tgccaccatg gactccctca agcactccta ccagtccacg atcctggact 60 ataa 64

<210> 23 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 23 aagaaggagt tgccaccatg cgaacactac caccacccca acctccacga tcctggacta 60 taa 63

<210> 24 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 24 aagaaggagt tgccaccatg ccatagccat acccagacct cacctccacg atcctggact 60 ataa 64

<210> 25 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 25 aagaaggagt tgccaccatg gaccaccatc cccatccacc tactccacga tcctggacta 60 taa 63

<210> 26 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 26 aagaaggagt tgccaccatg caccacctca aagctcctgc tcctccacga tcctggacta 60 taa 63

<210> 27 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 27 aagaaggagt tgccaccatg gactccaccc ttccatcacc atgactccac gatcctggac 60 tataa 65

<210> 28 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 28 aagaaggagt tgccaccatg ctcacccaac acattcacca catgtccacg atcctggact 60 ataa 64

<210> 29 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 29 aagaaggagt tgccaccatg ctcaaccaaa aagctccatt gactccacga tcctggacta 60 taa 63

<210> 30 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 30 aagaaggagt tgccaccatg aaaccaacaa ctcgatatat cctttccacg atcctggact 60 ataa 64

[Brief description of the drawings]

FIG. 1 shows a schematic diagram of a Y-ligation 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 first YLBS according to the method of the present invention.
The result of analyzing the reaction product after the cycle by electrophoresis is shown.

FIG. 6 shows the results of electrophoretic analysis of reaction products 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.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yasunori Kinoshita 10-12 Honcho, Wako-shi, Saitama F-term (reference) 4B024 AA20 CA01 DA20 HA08 HA09 HA20

Claims (16)

[Claims] 1. 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 5 ′ side to the 3 ′ side and having a base sequence encoding the first amino acid at the 3 ′ end; From 5 '
A second single-stranded nucleic acid having a stem sequence complementary to the stem sequence in the side direction and a branch sequence, and having a base sequence encoding a second amino acid at the 5 ′ end,
(2) the first single-stranded nucleic acid and the second single-stranded nucleic acid are hybridized between the respective stem sequences; and (3) the hybridized product is treated with T4 RNA ligase to obtain the first single-stranded nucleic acid. The 3 ′ end of the single-stranded nucleic acid is linked to the 5 ′ end of the second single-stranded nucleic acid, and (4) the double-stranded nucleic acid obtained in step (3) is used as a template,
P using a primer whose 5 ′ side is modified with an affinity substance
CR is performed, and 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 are included. A single-stranded nucleic acid is prepared, and PCR is performed using the double-stranded nucleic acid amplified in step (3) as a template and a primer having a 5′-side modified with an affinity substance, and a 5′- to 3′-side primer is used. A restriction enzyme recognition sequence, a branch sequence, a base sequence encoding the first amino acid,
Preparing a double-stranded nucleic acid containing a base sequence, a branch sequence and a stem sequence encoding the amino acid of step (5);
By treating the two types of double-stranded nucleic acids obtained in the above with a restriction enzyme, a first amino acid and a second
A double-stranded nucleic acid having a base sequence encoding the amino acid of step (5) is prepared, and (6) the double-stranded nucleic acid obtained in step (5) is obtained from Prepare single-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): 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 having a base sequence encoding the first amino acid at the 3 ′ end; From 5 '
A second single-stranded nucleic acid having a stem sequence complementary to the stem sequence in the side direction and a branch sequence, and having a base sequence encoding a second amino acid at the 5 ′ end,
(2) the first single-stranded nucleic acid and the second single-stranded nucleic acid are hybridized between the respective stem sequences; and (3) the hybridized product is treated with T4 RNA ligase to obtain the first single-stranded nucleic acid. The 3 ′ end of the single-stranded nucleic acid is linked to the 5 ′ end of the second single-stranded nucleic acid, (4a) the ligation product is converted into a single-stranded nucleic acid, and then the complementary strand is synthesized to prepare a double-stranded nucleic acid And (4b) PCR using the double-stranded nucleic acid obtained in the step (4a) as a template and a forward primer modified on the 5 ′ side with an affinity substance and a reverse primer containing a restriction enzyme recognition sequence, A double-stranded nucleic acid containing a stem sequence, a branch sequence, a base sequence encoding a first amino acid, a base sequence encoding a second amino acid, a branch sequence, and a restriction enzyme recognition sequence in the direction from the 5 ′ side to the 3 ′ side. Using the double-stranded nucleic acid prepared and amplified in step (4a) as a template, 'Perform PCR using a reverse primer having a modified side with an affinity substance, 5' forward primer and 5 containing the enzyme recognition sequence side 3 '
Preparing a double-stranded nucleic acid comprising a restriction enzyme recognition sequence, a branch sequence, a base sequence encoding a first amino acid, a base sequence encoding a second amino acid, a branch sequence and a stem sequence in the side direction; (5) The two types of double-stranded nucleic acids obtained in the step (4b) are treated with a restriction enzyme to obtain a double-stranded nucleic acid having a base sequence encoding the first amino acid and the second amino acid at the 3 ′ end or the 5 ′ end, respectively. A strand nucleic acid is prepared, (6) a single-stranded nucleic acid is prepared from the double-stranded nucleic acid obtained in the step (5) using the binding ability of the affinity substance introduced in the step (4b), and (7) ) Repeat steps (2) to (6) as many times as necessary using the single-stranded nucleic acid obtained in step (6): 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 the direction from the 5 ′ side to the 3 ′ side and having a base sequence encoding the first amino acid at the 3 ′ end; From 5 '
A second single-stranded nucleic acid having a stem sequence complementary to the stem sequence in the side direction and a branch sequence, and having a base sequence encoding a second amino acid at the 5 ′ end,
(2) the first single-stranded nucleic acid and the second single-stranded nucleic acid are hybridized between the respective stem sequences; and (3) the hybridized product is treated with T4 RNA ligase to obtain the first single-stranded nucleic acid. The 3 ′ end of the single-stranded nucleic acid is linked to the 5 ′ end of the second single-stranded nucleic acid, and (4) the double-stranded nucleic acid obtained in step (3) is used as a template,
A forward primer which is modified on the 5 ′ side with an affinity substance and anneals to a stem sequence of a first single-stranded nucleic acid, and a reverse primer which contains a restriction enzyme recognition sequence and anneals to a branch sequence of a second single-stranded nucleic acid PCR using
And a sequence 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 5 ′ to 3 ′ direction. A single-stranded nucleic acid is prepared and a forward primer containing a restriction enzyme recognition sequence and annealing to a branch sequence of the first single-stranded nucleic acid, using the double-stranded nucleic acid amplified in step (3) as a template, PCR using a reverse primer that anneals to the stem sequence of the second single-stranded nucleic acid, the side of which has been modified with an affinity substance,
A double-stranded nucleic acid containing a restriction enzyme recognition sequence, a branch sequence, a base sequence encoding a first amino acid, a base sequence encoding a second amino acid, a branch sequence, and a stem sequence in a direction from the 5 ′ side to the 3 ′ side. (5) treating the two double-stranded nucleic acids obtained in step (4) with a restriction enzyme,
A double-stranded nucleic acid having a base sequence encoding a first amino acid and a second amino acid at each of the 3 ′ end or the 5 ′ end is prepared, and (6) the binding ability of the affinity substance introduced in step (4) is determined. Utilizing a single-stranded nucleic acid from the double-stranded nucleic acid obtained in step (5), and (7) using the single-stranded nucleic acid obtained in step (6) to perform steps (2) to (6). ) As many times as necessary: 4. A mixture of single-stranded nucleic acids having a base sequence encoding a plurality of different types of first amino acids is used as the first single-stranded nucleic acid, and a different single-stranded nucleic acid is used as the second single-stranded nucleic acid. The method for producing a nucleic acid library according to any one of claims 1 to 3, wherein a mixture of single-stranded nucleic acids having a base sequence encoding a second amino acid of a species 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 GGCTCGCGGAATAC in a direction from 5 ′ to 3 ′.
TTTG, wherein the stem sequence of the second single-stranded nucleic acid is 3 ′
CCGAGCGCTTAT in the direction 5 'from the side
The method for producing a nucleic acid library according to any one of claims 1 to 5, which is GAAAC. 7. The method for producing a nucleic acid library according to claim 1, wherein the length of the branch sequence is 10 to 100 bases. 8. The method according to claim 8, wherein the branch sequence of the first single-stranded nucleic acid is 5 ′.
AAGATCTCTTTTT in the direction 3 'from the side
The method for producing a nucleic acid library according to any one of claims 1 to 7, wherein the branch sequence of the second single-stranded nucleic acid is TTGCCCTAGGGGAT in a direction from the 3 'side to the 5' side. 9. The method according to claim 1, 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. A method for preparing a nucleic acid library. 10. The method for producing a nucleic acid library according to claim 1, wherein the affinity substance in the primer used in step (4) is biotin. 11. The nucleic acid library according to claim 1, 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. How to make a rally. 12. The restriction enzyme recognition sequence in the primer used in step (4), which is a recognition sequence for a restriction enzyme that cuts away from the recognition sequence.
The method for producing a nucleic acid library according to any one of the above. 13. The method for producing a nucleic acid library according to claim 1, wherein the restriction enzyme recognition sequence in the primer used in step (4) is an MboII recognition sequence. 14. The library according to claim 1, wherein a library of nucleic acids containing a base sequence encoding 16 amino acids is prepared by repeating the steps (2) to (6) four times in total. A method for preparing a nucleic acid library. 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.