JP2003070483A - Method for connecting nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a substrate protein capable of expressing a relatively short peptide in a cell-free translation system. SOLUTION: This substrate protein for expressing and exhibiting a target peptide or a target protein as a fused protein comprises a globular protein composed of 30-200 amino acid residues.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for ligating nucleic acids. More specifically, the present invention relates to two different types of single-stranded or double-stranded DNA having common sequences complementary to each other.
To a ligation in the absence of a primer. The present invention also relates to a method for expressing a peptide or protein such as a peptide library using the above-described nucleic acid ligation method.

[0002]

2. Description of the Related Art Conventionally, DNA ligation has been the most basic and important technique such as gene recombination. The most commonly used method is double-stranded DN digested with a restriction enzyme.
A to T4 DNA Ligase (Sgaramella V, & Ehrlich SD (1978) E
ur J Biochem 86, 531-537). In this case, it is necessary that the DNA to be ligated does not contain a recognition site for the restriction enzyme to be used, and therefore the primary sequence of the DNA to be ligated must be known in advance.

On the other hand, in 1985, the polymerase chain reaction method (PCR method) (Saiki RK, et al., (1985) Science, 230, 13
50-1354) was reported, and DNA using this was reported.
The ligation method Overlap extension PCR method was developed by Horton et al. (Horton RM, et al., (1989) Gene, 77,
61-68). Of the two DNA fragments to be ligated, one has a complementary common sequence at the 3'end side and the other 5'end side (overlaps), so that it is ligated without using a restriction enzyme. It was an excellent method. The ligated DNA can be easily obtained by using PCR primers for amplification from both ends thereof. This is extremely useful when connecting specific DNA fragments.

[0004] In 1990, phage display method (Scott JK & Smith G), which is an evolutionary molecular engineering method for rapidly displaying proteins that display various proteins on the surface of Escherichia coli phage and specifically bind to specific target molecules.
P, (1990) Science, 249; 386-390) was commercialized and various applications started. At that time, it is necessary to connect a random sequence at the DNA level to the N-terminal side of the phage coat protein. In order to insert DNA into the genome of a phage, the length of the phage genome itself is long for PCR, so after cutting at the insertion site with a restriction enzyme without using the overlap extension PCR method, the same restriction enzyme site is used. It had the DNA at both ends inserted. If the DNA to be inserted has a random sequence, it will be cleaved if there is a restriction enzyme site in it, thus limiting the sequence of the library.

On the other hand, in vitro v developed in 1997
In the irus method, mRNA and the protein encoded by the mRNA are ligated to the 3'end of the mRNA via a spacer with puromycin in a cell-free translation system (Nemoto, N. et al. (1997) FE
BS Lett. 414, 405-408, Roberts, RW et al (1997)
Proc. Natl. Acad. Sci. USA 94, 12297-12302). In this case, since only the PCR process and the cell-free translation system process are used, it is not necessary to use a ligation method using a restriction enzyme like phage display. Conventional Overlap extension
In the PCR method, a primer replicates 10,000 times or more DNA from the original template DNA. This is not a problem when ligating several kinds of DNA, but when a library containing various sequences of 10 13 is required as in the case of the in vitro virus method, ordinary PCR can be used to diversify the original library. Significantly impairs sex. It is therefore random without compromising diversity
There is a demand for a technique for ligating DNA libraries to create mRNA.

[0006]

[Problems to be Solved by the Invention] As described above, In vit
When treating a library population having a huge sequence space in vitro like the ro virus method, a technique for maintaining the diversity of the library up to the screening process is extremely important.

According to the in vitro virus method, 1 in 10 per 1 ml
Molecules greater than the third power can be screened. This is a dramatic improvement as compared with the conventional phage display method in which the number of molecules is about 10 7 per ml. However, high copy numbers reduce the actual molecular species screened. Overlap extension PCR
By repeating the reaction for 20 cycles by the method, the copy number of 10 6 is theoretically obtained, and the in vitro virus method has the same diversity as the phage display method. That is, it was an object of the present invention to provide a method for ligating a DNA fragment encoding a target sequence such as a random sequence without increasing the copy number.

[0008]

[Means for Solving the Problems] The present inventors have conducted extensive studies to solve the above problems, and first, two different types of single-stranded or double-stranded D having common sequences complementary to each other.
When performing PCR for ligating NA, PCR reaction was performed without using a primer. In this case, in addition to the full-length double-stranded DNA that has been ligated, the complementary strand of unligated DNA remains as it is. Usually such an extra
The DNA should be purified, but since it has 100 bases or more, a simple column for removing the primer cannot be used, and it is necessary to cut out from the gel after electrophoresis. It is known that this is inefficient in work and the yield is reduced.

Therefore, in order to avoid this problem, T7R
Is RNA synthesized without any purification work by taking advantage of the fact that virus-derived RNA polymerases such as NA polymerase have high promoter specificity and specifically recognize double-stranded DNA? I checked. As a result, it was found that the desired RNA was synthesized. It was also found that the DNA coexisting at the same time can be easily removed by degrading with a DNA degrading enzyme and purifying. The present invention has been completed based on these findings.

That is, according to the present invention, two different types of single-stranded or double-stranded DNs having common sequences complementary to each other are used.
There is provided a method for ligating nucleic acids, which comprises the step of reacting A with a DNA synthase in the absence of a primer. Preferably, the reaction using the DNA synthase is Ta
Polymerase chain reaction (PC
R). Preferably, one of the two different types of single-stranded or double-stranded DNA is D containing the target sequence.
It is NA. More preferably, one of the two different types of single-stranded or double-stranded DNA is a DNA containing a target sequence, and the other DNA is a DNA encoding a support protein. Preferably, the support protein is a protein consisting of a globular protein consisting of 30 to 200 amino acid residues.

According to another aspect of the present invention, a ligated product of two kinds of DNA obtained by the above method is provided.
According to still another aspect of the present invention, (1) reacting two different types of single-stranded or double-stranded DNAs having common sequences complementary to each other using a DNA synthase in the absence of a primer Preparing a mixture containing ligated DNA and non-ligated DNA according to, and (2)
A step of performing a transcription reaction in the presence of RNA polymerase using the mixture obtained in step (1) to synthesize RNA; and a method for ligating and transcribing nucleic acids.
Preferably, after the step (2), (3) DNA is further added.
Degrading DNA with a degrading enzyme.

[0012] According to still another aspect of the present invention, there is provided RNA obtained by the above method.

According to still another aspect of the present invention, (1)
Two different types of single-stranded or double-stranded DNAs having a common sequence complementary to each other are added in the absence of the primer D
A step of preparing a mixture containing linked DNA and non-ligated DNA by reacting with NA synthase; (2) performing a transcription reaction in the presence of RNA polymerase using the mixture obtained in step (1) Provided is a method for producing a protein, which comprises the steps of synthesizing RNA; and (3) expressing the RNA obtained in step (2) in a cell-free translation system or a living cell.

According to still another aspect of the present invention, (1)
Two different types of single-stranded or double-stranded DNAs having a common sequence complementary to each other are added in the absence of the primer D
A step of preparing a mixture containing linked DNA and non-ligated DNA by reacting with NA synthase; (2) performing a transcription reaction in the presence of RNA polymerase using the mixture obtained in step (1) A step of synthesizing RNA; (3) a step of modifying the 3'end of the RNA obtained in step (2) with a nucleic acid derivative; and (4) an RNA of which the 3'end obtained in step (3) is modified with a nucleic acid derivative There is provided a method for producing a complex of a protein and a nucleic acid encoding the protein, which comprises a step of expressing the protein in a cell-free translation system or in a living cell.

Preferably, the nucleic acid derivative is a compound containing a chemical structure skeleton of puromycin, 3'-N-aminoacyl puromycin aminonucleoside, 3'-N-aminoacyl adenosine aminonucleoside, or an analog thereof. Preferably, an mRNA having a nucleic acid derivative bound to the 3'end via a spacer is used as the mRNA. Preferably, the spacer is a polymer such as polyethylene or polyethylene glycol.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. INDUSTRIAL APPLICABILITY The method for ligating nucleic acids of the present invention is used when ligating a diverse DNA library fragment containing a random sequence and a DNA fragment having a constant sequence such as a support protein in a form that does not impair the diversity. be able to. The present invention also relates to transcription using the DNA thus linked as a template.

The method of ligating nucleic acids of the present invention is characterized in that two different kinds of single-stranded or double-stranded DNAs having common sequences complementary to each other are reacted using a DNA synthase in the absence of a primer. And

The common sequences which are complementary to each other need only have such complementarity that they can be annealed under suitable conditions, and need not be completely (that is, 100%) complementary. The length of the complementary sequence is not particularly limited, but is usually about 5 to 100 bases, preferably about 5 to 50 bases.

As the DNA synthase, various DNA polymerases can be used, but Taq polymerase is preferable. In the present invention, it is preferable to ligate the DNA by polymerase chain reaction (PCR) using Taq polymerase.

The nucleic acid synthesis reaction using a DNA polymerase such as Taq polymerase can be carried out under usual conditions known to those skilled in the art. Specifically, the nucleic acid synthesis reaction can be carried out by adding two kinds of DNA fragments to be ligated, a dNTP mixture and a DNA polymerase to a suitable buffer and incubating at a suitable temperature for a certain period of time. When performing the nucleic acid synthesis reaction by PCR, DNA
Using Taq polymerase as a polymerase, for example, repeating a cycle of 95 ° C. for 30 seconds (denaturation), 54 ° C. for 2 seconds (annealing), and 74 ° C. for 30 seconds (extension) multiple times (for example, about 25 times). Can perform nucleic acid synthesis. PCR reaction conditions (temperature and time,
The number of cycles) and the like can be appropriately changed depending on the type of nucleic acid to be linked. The present invention also includes a ligated body of DNA obtained by the above method.

In a preferred embodiment of the present invention, one of the two different types of single-stranded or double-stranded DNA is a DNA containing a random sequence. The other DNA is the DNA encoding the support protein.

In the present invention, the kind of the target sequence is not particularly limited, and any sequence can be used depending on the purpose of screening. For example, a ligand capable of binding to a receptor may be a sequence encoding an invasion signal peptide or signal protein that contributes to invasion into cells, or a random sequence encoding a large number of unspecified random peptides used in screening. . The peptide or protein encoded by these target sequences recognizes the target substance mainly by protein-protein interaction. Therefore, it is preferable that the protein encoded by the target sequence is capable of sufficiently recognizing the target substance by direct or indirect interaction. Examples of the receptor include cell surface receptor proteins, antibodies, growth factors and the like.

In one of the preferred embodiments of the present invention, a target protein having a specific amino acid sequence having a specific interaction with a specific target is randomly selected for screening and further identification. A random sequence having a contiguous or discontinuous amino acid sequence can be used as the target sequence.
For example, the number of combinations of 10 randomly selected 20 kinds of amino acids is theoretically about 1 × 10 ¹³ kinds (about 10 trillion), which is considered sufficient for detecting a specific amino acid sequence by screening.

In the present invention, the number of amino acids encoded by the target sequence is not particularly limited. It is easy for a person skilled in the art to synthesize a DNA encoding a desired number of random peptides, preferably 3 to 16 by using synthetic means known to those skilled in the art. For example, a DNA encoding such a random peptide can be synthesized using a commercially available automatic DNA synthesizer or the like.

In a particularly preferred embodiment of the invention,
When a random peptide is expressed as the target peptide, the DNA encoding the random peptide (random DNA) is preferably of the formula: (NNK) n (wherein N is deoxyribonucleotide of A, G, C, or T). Nucleotides, K is a deoxyribonucleotide of either G or T, and n is the number of amino acids in these random portions.). In the present invention,
n is preferably at least 3 or more, and more preferably a number encoding 5 to 16 amino acids. However, the limitation of the number of n is not limited by the method of synthesizing the random DNA, and
There is no practical limit to the number of.

In some cases, the protein can be efficiently displayed by adding cysteine to both sides (N-terminal side and C-terminal side) of the protein encoded by the target sequence. The N-terminal side of the protein as used herein includes not only the N-terminal of the target protein but also the case where several to several tens of amino acids are separated from the N-terminal of the protein, and the C-terminal of the protein is also included.
The end side is meant to include not only the case of the C-terminal of the target protein but also the case of separating several to several tens of amino acids from the C-terminal of the protein. The term “efficient” as used herein means to reduce the influence of steric hindrance on the reactivity of proteins. When a disulfide bond is formed between the SH groups of cysteines on both sides, the protein becomes a loop structure and is presented on the surface of the fusion protein. Therefore, the effect of steric hindrance is lessened and the protein becomes more reactive.

Molecules having a loop in the molecule through cysteine include immunoglobulins of the immunoglobulin family (IgG, IgM), T cell receptors, MHC class II.
Molecules, LFA-3, ICAM-1, VCAM-1 and the like are known. Suitable protein sizes for forming loops can be designed with reference to these known molecules.

In a preferred embodiment of the present invention, the other one of the two different types of single-stranded or double-stranded DNA is a DNA encoding a support protein. As the support protein, it is preferable to use a support protein composed of a globular protein having 30 to 200 amino acid residues. More preferably, it does not contain a cysteine residue, does not have a β-sheet structure as the secondary structure of the protein, is composed of an α-helix structure, and has a N-terminal and a C-terminal separated from each other in the three-dimensional structure of the protein, which is different from other biological moieties. Support proteins that do not interact with the molecule can be used.

Specific examples of the support protein as described above include support proteins having any of the following amino acid sequences. (1) the amino acid sequence of SEQ ID NO: 9; or (2) the amino acid sequence of SEQ ID NO: 9 in which one to several amino acids are deleted, substituted, added and / or inserted. , The amino acid sequence constituting the globular protein:

In the present specification, 1 to several in the "amino acid sequence in which 1 to several amino acids are deleted, substituted, added and / or inserted in the amino acid sequence" is generally 1 to 20. , Preferably 1 to 10,
More preferably, it means 1 to 5, especially preferably 1 to 3.

The DNA used in the present invention can be easily synthesized by those skilled in the art using a commercially available automatic DNA synthesizer or the like. Further, an amino acid sequence in which 1 to several amino acids are deleted, substituted, added and / or inserted in the amino acid sequence of SEQ ID NO: 9,
A nucleic acid encoding an amino acid sequence constituting a globular protein can be similarly synthesized using a commercially available automatic DNA synthesizer or the like.

Further, according to the present invention, (1) by reacting two kinds of different single-stranded or double-stranded DNAs having a common sequence complementary to each other with a DNA synthase in the absence of a primer. A step of preparing a mixture containing ligated DNA and non-ligated DNA; and (2) synthesizing RNA by performing a transcription reaction in the presence of RNA polymerase using the mixture obtained in step (1);
Methods for ligating and transcribing nucleic acids are provided, including:

Step (1) above can be performed as described herein above. The step (2) is a step of transcribing the linked DNA obtained in the step (1) to generate RNA. The reaction mixture obtained in step (1) contains both ligated DNA and non-ligated DNA.
In the present invention, this reaction mixture is used as it is for carrying out a transcription reaction in the presence of RNA polymerase, whereby only the ligated DNA is transcribed. This is because virus-derived RNA polymerases such as T7 RNA polymerase have high promoter specificity and utilize the property of specifically recognizing double-stranded DNA. That is, in the present invention, it is preferable to use a virus-derived RNA polymerase that has high promoter specificity as described above and specifically recognizes double-stranded DNA, and it is particularly preferable to use T7 RNA polymerase. Such RNA
By using a polymerase, RNA can be synthesized without a purification operation of the reaction mixture.

By treating the reaction mixture obtained by the above transcription reaction with a DNA degrading enzyme to decompose and remove the DNA present in the mixture, only RNA is isolated while maintaining the diversity of the random sequence. can do. The present invention also includes RNA obtained by the method described above.

The present invention further provides (1) two different types of single-stranded or double-stranded DNs having common sequences complementary to each other.
A step of preparing a mixture containing ligated DNA and non-ligated DNA by reacting A with a DNA synthase in the absence of a primer; (2) RNA polymerase using the mixture obtained in step (1) Synthesizing RNA by performing a transcription reaction in the presence of
A method for producing a protein, comprising the step of expressing the RNA obtained in step (2) in a cell-free translation system or in a living cell; and (1) two different types of single strands having common sequences complementary to each other, or A step of preparing a mixture containing linked DNA and non-ligated DNA by reacting double-stranded DNA with a DNA synthase in the absence of a primer; (2) using the mixture obtained in step (1) A step of performing a transcription reaction in the presence of RNA polymerase to synthesize RNA; (3) modifying the 3 ′ end of the RNA obtained in step (2) with a nucleic acid derivative; and (4) obtaining in step (3) Provided is a method for producing a complex of a protein and a nucleic acid encoding the same, which comprises the step of expressing in a cell-free translation system or a living cell an RNA having a modified 3 ′ end modified with a nucleic acid derivative.

When a protein is translated in a cell-free protein translation system or in a living cell using the mRNA having a nucleic acid derivative bound to the 3'end as described above, the ribosome is stopped by the double chain and puro A nucleic acid derivative such as mycin can be bound to a protein by inserting it into the A site of the ribosome.

The nucleic acid derivative is not limited as long as it is a compound having the ability to bind to the C-terminal of the synthesized protein when the protein is translated in a cell-free protein translation system or in living cells. It is possible to select one whose chemical structure skeleton is similar to that of aminoacyl-tRNA at the 3'end. As a typical compound, puromycin having an amide bond, 3'-N-
Aminoacyl puromycin aminonucleoside (3 '
-N-Aminoacylpuromycin aminonucleoside, PANS-amino acid), for example, PANS-Gly in which the amino acid part is glycine,
PANS-Val in which the amino acid part is valine, PANS-Ala in which the amino acid part is alanine, and other PANS-amino acid compounds in which the amino acid part corresponds to all amino acids.

Further, 3'-N-aminoacyl adenosine amino nucleoside (3'-Aminoacyladenosine aminonuc) linked by an amide bond formed by dehydration condensation of the amino group of 3'-aminoadenosine and the carboxyl group of amino acid.
leoside, AANS-amino acid), for example, AANS-Gly for glycine in the amino acid part, AANS-Val for valine for the amino acid part, AANS-Ala for alanine in the amino acid part, and other AANS corresponding to all amino acids in the amino acid part. -Amino acid compounds can be used.

Further, nucleosides or those in which nucleosides are ester-bonded with amino acids can be used.
Furthermore, all compounds in which a substance having a nucleic acid or a chemical structure skeleton and a base similar to a nucleic acid and a substance having a chemical structure skeleton similar to an amino acid are chemically bound are all included in the nucleic acid derivative used in the present method. .

As the nucleic acid derivative, puromycin,
More preferred is a compound in which a PANS-amino acid or AANS-amino acid is linked to a nucleoside via a phosphate group. Among these compounds, puromycin derivatives such as puromycin, ribocytidyl puromycin, deoxydyl puromycin and deoxyuridyl puromycin are particularly preferable.

In the method of the present invention, it is preferable to use a nucleic acid modified product in which a nucleic acid derivative is bound to the 3'end of RNA via a spacer. As the spacer, a high molecular substance such as polyethylene or polyethylene glycol is used, and preferably polyethylene glycol is used. The length of the spacer is not particularly limited, but preferably the molecular weight is 300 to 3000, or the number of atoms in the main chain is 10 to 100 atoms.

The nucleic acid derivative as described above can be produced by a chemical bonding method known per se. Specifically, when a synthetic unit is bound by a phosphodiester bond, it can be synthesized by solid phase synthesis by the phosphoamidide method or the like which is generally used in a DNA synthesizer. When introducing a peptide bond, the synthetic unit is linked by the active ester method or the like.
When synthesizing a complex with and, a protecting group capable of supporting both synthetic methods is required.

In the method of the present invention, it is preferable to produce a protein by expressing the above-mentioned nucleic acid or a modified form thereof in a cell-free translation system or a living cell.

Transcription and translation systems for artificially producing the protein it encodes from nucleic acid are known to those of skill in the art. Specifically, a cell-free protein synthesis system in which a component having a protein synthesizing ability is extracted from an appropriate cell and the target protein is synthesized using the extract is mentioned. Such a cell-free protein synthesis system contains necessary elements for transcription / translation system such as ribosome, initiation factor, elongation factor and tRNA.

Examples of such a cell-free protein synthesis system (system derived from cell lysate) include a cell-free translation system composed of a prokaryotic or eukaryotic extract, for example, E. coli,
Rabbit reticulocyte extract, wheat germ extract and the like can be used, but any one can be used as long as it produces a target protein from DNA or RNA. As the cell-free translation system, a commercially available kit can be used. For example, a rabbit reticulocyte extract (Rabbit Reticul) can be used.
Cell Lysate Systems, Nuclease Treated, Promega) and wheat germ extract (Wheat Germ Extract, Promega). As the transcription / translation system, living cells may be used, and specifically, prokaryotic or eukaryotic cells such as Escherichia coli cells can be used. The cell-free translation system or living cell is not limited as long as protein synthesis is carried out by adding or introducing a nucleic acid encoding a protein therein.

As already mentioned above, in the present invention, a sequence encoding a peptide library or a protein library can be used as the target sequence. By screening such a library DNA functionally expressed in a form linked to a support protein according to the method for ligating nucleic acids of the present invention, and selecting a target peptide or target protein having a desired function, Functional peptides or proteins can be screened. 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.

[0047]

Example: Acquisition of sugar chain-binding peptide from random peptide library presented in mutant Pou-specific domain Peptide that binds to N-acetylglucosamine, which is one of sugar chains, in the presence of calcium (12 residues) Groups are derived from natural lectin proteins (Yamamoto, K., et a
l. (1992) J. Biochem. 111 436-439). Therefore, 5 residues in this were made into a random sequence, and this was made into the mutant Pou-spec.
It was examined whether or not a peptide that binds to the sugar chain in the presence of calcium by being fused to the N-terminal side of ific domain (SEQ ID NO: 9) can be screened. A schematic diagram of the concept of this embodiment is shown in FIG.

First, in order to prepare a mutant Pou-specific domain DNA which is a support for displaying a peptide, the following procedure was performed. D with T7 promoter and Kozac sequence
NA "T7-Kozac" (SEQ ID NO: 1; gctccgagct cattaatacg ac
tcactata gggagaccac aacggtttcc ctcttgaaat aattttgt
tt aactttaaga aggagttgcc accatg) and a DNA having a random sequence "Lec-random" (SEQ ID NO: 2 gctcaagctc ctcaa
ggtcg ccaccgcctc cggaagggtc zyxzyxzyxz yxzyxgaagg
tgtcaaattc aacgtcagtc aggtgaataa ttttatcgct catggt
ggca tctccttctt 120) and DNA "Pou" (constant number 3: gaccttgagg agcttgagca) having a constant sequence of the support.
gtttgccaag accttcaaac aaagacgaat caaacttgga ttcac
tcagg gtgatgttgg gctcgctatg gggaaactat atggaaatga
cttcagccaa actaccatct ctcgatttga agccttgaac ctcagc
ttta agaacatggc taagttgaag ccacttttag agaagtggct a
aatgatgca gaggggggag gcagctctag agctg) was organically synthesized. In SEQ ID NO: 2, the composition of each base of X, Y and Z is as follows. This is a theoretical calculation so that 20 kinds of amino acids appear evenly and the number of stop codons is small. X: A (35%), T (13%), G (32%), C (20%) Y: A (30%), T (24%), G (24%), C (22%) Z: A (0%), T (37%), G (26%), C (37%) Pou of SEQ ID NO: 3 is ttgagcttga gcgacgacct tgaggagctt
gagca (SEQ ID NO: 4) and gaggacgggg ggcggcgggg ggcagct
Polymerase chain reaction (PCR) was performed using cta gagctgcctc cccc SEQ ID NO: 5) as a primer to form a double strand (FIG. 3).

(1) Ligation of T7-Kozac and Lec-random In order to ligate T7-Kozac and Lec-random, 1 pmol of each was subjected to an extension reaction cycle under the following conditions without using a primer. Taq polymerase is EX Taq. (Takara) 2 units
The reaction conditions used are 95 ° C. for 30 seconds, 64 ° C. for 20 seconds,
25 cycles of 20 seconds at 72 ° C. The ligation product "T'7-Lec-random" was analyzed by 8M urea-denaturing acrylamide gel electrophoresis (Fig. 4). As a result, it was confirmed that almost all were linked.

(2) Linking of T'7-Lec-random and Pou In order to link the T'7-Lec-random and Pou linked in (1) above, 10 pmol of each was used under the following conditions without using a primer. An extension reaction cycle was performed. Taq polymerase is KOD Da
1.25 units of sh. (TOYOBO) are used, and the reaction conditions are 95 ° C and 3
A cycle of 0 second, 54 ° C. for 2 seconds, and 74 ° C. for 30 seconds is repeated 25 times. The ligation product "T'7-Lec-random-Pou" was analyzed by 8M urea-denatured acrylamide gel electrophoresis (Fig. 5). As a result, it was confirmed that T'7-Lec-random-Pou was linked, and 1'on the side where T'7-Lec-random and Pou were not linked.
Double-stranded DNA was also confirmed.

(3) T'7-Lec-random-Pou Transcription The product ligated in (2) above was treated with phenol to give Primer.
Purified using Remover (Edge System Science). Measure the molarity of DNA with a spectrometer and measure 2.5 μg with Ribo.
Final volume of 5 in reaction composition of Max T7 transcription kit (Promega)
The mixture was added to 0 μl and reacted at 37 ° C. for 1 hour.
Next, 2.5 units of RQ1 Rnase-free DNase (Promega) was added to this reaction solution, and the mixture was reacted at 37 ° C for 15 minutes. This was extracted with phenol and purified with Primer Remover. This 8
It was analyzed by M urea denaturing acrylamide electrophoresis. Results are shown in FIG. From the results of FIG. 6, it was found that RNA from the ligated DNA was transcribed as the main band.

Sequence of lectin-like peptide containing random 5 residues thus prepared and support protein Pou-sp
mRNA encoding ecific domain is an in vitro virus
Spacer (DNA-PEG-Puromycin) and 50 ℃ to 20 ℃
Cool for about 15 minutes until T4 RNA ligas
e (Takara) was added and reacted at 25 ° C. for 20 minutes to connect.

[0053] The spacer for in vitro virus is a resin puromycin CPG (GLEN RESEAR
3 units of spacer phosphoramidite 18 (GLEN RESEARCH: 10-1918-90) were synthesized from CH: 20-4040-01) toward the 5'side, and Amino-ModifierC6-dT (GLEN RESEARCH: 1
0-1039-90), followed by ligation, followed by DNA synthesis (ccccccgccg ccccccgtcc tc; SEQ ID NO: 6) using a normal DNA synthesis reagent.

This was purified with an RNA purification column (QIAGEN), and then in vitro vir using a wheat germ cell-free translation system.
create us virion (a combination of mRNA and protein),
In the presence of calcium, buffer A (Tris-HCl 10 mM, NaC
avidin beads with N-acetylglucosamine immobilized with 150 mM, CaCl ₂ 25 mM, pH 6.8 (EY Laboratories)
And incubated at room temperature for 1 hour. Then, after washing 4 times with buffer A, buffer B without calcium (Tris-HCl 10 mM, NaCl 150 mM, EDTA 2.5 mM, pH
It was eluted in 6.8). In vitro virus virion eluted after washing 4 times with buffer A is almost eliminated, but washing with buffer B without calcium shows in vitro virion
Since in vitro virus is eluted again, in vitro virus
It can be seen that the peptide presented by virion is a peptide that binds to this sugar chain depending on calcium (Fig. 7).

MR of eluted in vitro virus virion
NA is TrueScript II ReverseTranscriptase using a reverse transcription primer (gtcctctaga gctgcc; SEQ ID NO: 7).
(Sawasdee) reverse-transcribed under the following conditions. 20 reactions
μl scale. 90 ℃ for 2 minutes, 75 ℃ to 25 ℃ -0.0
Cool at 55 ° C / sec, 2 minutes at 25 ° C. Here, reverse transcriptase and Rnase Inhibitor were added and the reaction was carried out at 50 ° C. for 1 hour.

Then, 1 μl of this reaction solution was taken and primers (gatcccgcga aattaatacg actcactata ggg; SEQ ID NO: 8) and (gaggacgggg ggcggcgggg ggcagctcta gagctgc
Extaq Polymerase (Ta) using ctc cccc; SEQ ID NO: 5)
PCR was performed under the following conditions. 30 seconds at 95 ° C, 6
20 cycles of 4 seconds for 20 seconds and 72 degrees for 20 seconds.

As a result of sequencing the obtained sequence, a peptide having a large number of side chains easily hydrogen-bonding compared to the first library has been obtained (FIG. 8). From this, it was found that this mutant Pou-specific domain is useful as a support protein that presents a functional peptide.

[0058]

According to the present invention, D containing a random sequence
It has been found that NA can be ligated to the encoded canonical sequence DNA, such as a support protein, without loss of its diversity and subsequently transcribed into RNA. This is an extremely important method for in vitro protein screening such as the in vitro virus method.

[0059]

[Sequence list] SEQUENCE LISTING <110> GenCom <120> A process for the ligation of nulecic acids <130> A11433MA <160> 9

[0060] <210> 1 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 1 gctccgagct cattaatacg actcactata gggagaccac aacggtttcc ctcttgaaat 60 aattttgttt aactttaaga aggagttgcc accatg 96

[0061] <210> 2 <211> 120 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 2 gctcaagctc ctcaaggtcg ccaccgcctc cggaagggtc zyxzyxzyxz yxzyxgaagg 60 tgtcaaattc aacgtcagtc aggtgaataa ttttatcgct catggtggca tctccttctt 120

[0062] <210> 3 <211> 235 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 3 gaccttgagg agcttgagca gtttgccaag accttcaaac aaagacgaat caaacttgga 60 ttcactcagg gtgatgttgg gctcgctatg gggaaactat atggaaatga cttcagccaa 120 actaccatct ctcgatttga agccttgaac ctcagcttta agaacatggc taagttgaag 180 ccacttttag agaagtggct aaatgatgca gaggggggag gcagctctag agctg 235

[0063] <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 4 ttgagcttga gcgacgacct tgaggagctt gagca 35

[0064] <210> 5 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 5 gaggacgggg ggcggcgggg ggcagctcta gagctgcctc cccc 44

[0065] <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 6 ccccccgccg ccccccgtcc tc 22

[0066] <210> 7 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 7 gtcctctaga gctgcc 16

[0067] <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 8 gatcccgcga aattaatacg actcactata ggg 33

[0068] <210> 9 <211> 73 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 9 Met Asp Leu Glu Glu Leu Glu Gln Phe Ala Lys Thr Phe Lys Gln Arg   1 5 10 15 Arg Ile Lys Leu Gly Phe Thr Gln Gly Asp Val Gly Leu Ala Met Gly              20 25 30 Lys Leu Tyr Gly Asn Asp Phe Ser Gln Thr Thr Ile Ser Arg Phe Glu          35 40 45 Ala Leu Asn Leu Ser Phe Lys Asn Met Ala Lys Leu Lys Pro Leu Leu      50 55 60 Glu Lys Trp Leu Asn Asp Ala Glu Gly  65 70

[Brief description of drawings]

FIG. 1 shows a schematic diagram of the outline of the present invention.

FIG. 2 shows a schematic diagram of screening of sugar chain-binding peptides as one example of the present invention.

FIG. 3 shows the construction of DNA “T7-Kozac”, DNA “Lec-random” and DNA “Pou” with a constant sequence of support.

FIG. 4 shows the results of analysis of the ligation product “T'7-Lec-random” by 8M urea-denaturing acrylamide gel electrophoresis.

FIG. 5 shows the ligation product “T'7-Lec-random-Pou” 8
The result analyzed by the M urea denaturing acrylamide electrophoresis is shown.

FIG. 6 shows the transcription product of T'7-Lec-random-Pou at 8M.
The result analyzed by urea denaturing acrylamide electrophoresis is shown.

FIG. 7 shows the results of screening for sugar chain peptides.

FIG. 8 shows the results of sequencing the sequences obtained as a result of screening of sugar chain peptides.

Claims (14)

[Claims] 1. A method of ligating nucleic acids, which comprises a step of reacting two different types of single-stranded or double-stranded DNAs having common sequences complementary to each other using a DNA synthase in the absence of a primer. 2. The reaction using a DNA synthase is Taq.
Polymerase chain reaction (PCR) using polymerase
The method for ligating nucleic acids according to claim 1, which is 3. Two different types of single-stranded or double-stranded DN
The method for ligating nucleic acids according to claim 1 or 2, wherein one of the DNAs of A is a DNA containing a target sequence. 4. Two different types of single-stranded or double-stranded DN
One of the DNAs in A is a DNA containing a target sequence, and the other DNA is a DN encoding a support protein.
The method for ligating nucleic acids according to any one of claims 1 to 3, which is A. 5. The method for ligating nucleic acids according to claim 4, wherein the support protein is a protein consisting of a globular protein consisting of 30 to 200 amino acid residues. 6. A ligated product of two types of DNA, which is obtained by the method according to claim 1. 7. (1) A ligated DNA obtained by reacting two different types of single-stranded or double-stranded DNAs having common sequences complementary to each other with a DNA synthase in the absence of a primer. Ligating and transcribing nucleic acids, which comprises: preparing a mixture containing unligated DNA; and (2) synthesizing RNA by performing a transcription reaction in the presence of RNA polymerase using the mixture obtained in step (1) Way to do. 8. After the step (2), further (3) DN
The step of degrading DNA with an A-degrading enzyme is included.
A method for ligating and transcribing the nucleic acid according to. 9. An RNA obtained by the method according to claim 7 or 8. 10. (1) A ligated DNA obtained by reacting two different types of single-stranded or double-stranded DNAs having common sequences complementary to each other with a DNA synthase in the absence of a primer. A step of preparing a mixture containing unligated DNA; (2) a step of performing a transcription reaction in the presence of RNA polymerase using the mixture obtained in step (1) to synthesize RNA;
And (3) a method for producing a protein, which comprises a step of expressing the RNA obtained in step (2) in a cell-free translation system or a living cell. 11. (1) A ligated DNA obtained by reacting two different types of single-stranded or double-stranded DNAs having common sequences complementary to each other with a DNA synthase in the absence of a primer. A step of preparing a mixture containing unligated DNA; (2) a step of performing a transcription reaction in the presence of RNA polymerase using the mixture obtained in step (1) to synthesize RNA; (3) obtained in step (2) Modifying the 3'end of the RNA with a nucleic acid derivative; and (4) expressing the RNA obtained in step (3) with the 3'end modified with a nucleic acid derivative in a cell-free translation system or a living cell , A method for producing a complex of a protein and a nucleic acid encoding the same. 12. The nucleic acid derivative is puromycin,
The method according to claim 11, which is a compound containing a chemical structure skeleton of 3'-N-aminoacyl puromycin aminonucleoside, 3'-N-aminoacyl adenosine aminonucleoside, or an analog thereof. 13. The method according to claim 11 or 12, wherein an mRNA in which a nucleic acid derivative is bound to the 3 ′ end via a spacer is used as the mRNA. 14. The method according to claim 13, wherein the spacer is a polymer such as polyethylene or polyethylene glycol.