JP2002291491A - Rna-dna conjugate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for linking a single-stranded RNA and a single-stranded DNA with each other, for example, to provide a new method for constructing in vitro virus genome. SOLUTION: This method for producing an RNA-DNA conjugate comprises the steps of (1) annealing a single-stranded RNA and a single-stranded DNA or a derivative thereof having sequences complementary to each other and (2) treating the annealed product with an RNA ligase to link the 3'-terminal of the single-stranded RNA and the 5'-terminal of the single-stranded DNA or the derivative thereof with each other.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for linking single-stranded RNA and single-stranded DNA and use thereof. More specifically, the present invention relates to the method of annealing RNs of single-stranded RNA and single-stranded DNA having complementary sequences to each other.
The present invention relates to a nucleic acid ligation method characterized by treating with A ligase and use thereof.

[0002]

2. Description of the Related Art Nucleic acid cleavage and ligation is one of the most important basic techniques in genetic engineering. As a method for linking two kinds of nucleic acids, conventionally, two methods using a restriction enzyme are used.
Ligation of main-strand nucleic acids was the main, most of which used DNA ligase.

[0003] On the other hand, RNA is mainly ligated using T4 RNA ligase to synthesize artificial rRNA (Bruce AG, Uhlenb
ech OC: Biochemistry, (1982) 21 (5) 855-61) and full length c
A method of adding a primer to the 5 'end of mRNA for DNA preparation (Troutt AB, et al .: Proc Natl Acad Sci.
USA (1992) 89 (20): 9823-5).

Recently, as a technique of evolutionary molecular engineering, m
Technology for covalently binding RNA and its encoded protein in a cell-free translation system (in vitro virus method) (N
emoto, N., et al. (1997) FEBS Lett. 414, 405-408) appeared, and it became necessary to ligate DNA to mRNA and its 3 'end. Since RNA is generally single-stranded, a method is considered in which DNA to be linked and DNA serving as a splint complementary to the sequence of the RNA are hybridized and ligated with DNA ligase. However, this is not preferable in terms of efficiency, and is a major problem in using the in vitro virus method.

On the other hand, in order to connect single-stranded DNAs, Nishigaki et al. Increased the concentration effect by giving a complementary sequence to some of them and hybridizing them, thereby increasing the concentration effect of T4RN.
A method for efficiently ligating single-stranded DNAs using A ligase has been devised (also referred to as a Y-ligation method) (K. Nishigaki, (1998) Molecular Diversity,
4: 187-190). However, single-stranded RNA and single-stranded D
It has not been reported that NA is linked using the Y-ligation method.

[0006]

SUMMARY OF THE INVENTION The present invention provides a single-stranded RN
An object of the present invention is to provide a novel method for linking A with a single-stranded DNA, for example, providing a novel method for constructing an in vitro virus genome. Another object of the present invention is to provide a method for efficiently producing an RNA-DNA conjugate in a short time. Another object of the present invention is to provide a method for efficiently producing a protein-RNA conjugate by subjecting the RNA-DNA conjugate obtained by the above method to a cell-free translation system.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, annealed a single-stranded RNA having a sequence complementary to each other to a single-stranded DNA or its derivative. It has been found that an RNA-DNA conjugate can be efficiently produced in a short time by treating with RNA ligase later, and the present invention has been completed.

That is, according to the present invention, (1) a step of annealing a single-stranded RNA having a sequence complementary to each other to a single-stranded DNA or a derivative thereof; and (2) treating the annealing product with RNA ligase And single-stranded RNA
Connecting the 3 ′ end of the DNA to the 5 ′ end of the single-stranded DNA or a derivative thereof.

According to a preferred embodiment of the present invention, (1) a protein containing a coding sequence encoding a
Annealing of a single-stranded RNA having an annealing sequence and a branch sequence in the 3 ′ to 3 ′ direction and a single-stranded DNA or a derivative thereof having a sequence complementary to the annealing sequence and a branch sequence in the 3 ′ to 5 ′ direction And (2) treating the annealing product with RNA ligase to ligate the 3 ′ end of the single-stranded RNA to the 5 ′ end of the single-stranded DNA or a derivative thereof.
A method for producing an A-conjugate is provided.

[0010] Preferably, the single-stranded RNA is an mRNA or an mRNA library. Preferably, single-stranded RN
A is characterized by having (1) a promoter sequence, (2) a DNA sequence recognized by ribosomes during translation, and (3) a sequence encoding a target protein.

Preferably, as a single-stranded DNA or a derivative thereof, a single-stranded DNA having a nucleic acid derivative bound to the 3 ′ end is used.
A derivative of NA is used. Preferably, the nucleic acid derivative is
A compound containing a chemical structural skeleton of puromycin, 3'-N-aminoacylpuromycin aminonucleoside, 3'-N-aminoacyladenosine aminonucleoside, or an analog thereof. Preferably, as the single-stranded DNA or a derivative thereof, a single-stranded DNA derivative having a nucleic acid derivative bound to the 3 ′ end via a spacer is used.
Preferably, the spacer is a polymer such as polyethylene or polyethylene glycol.

Preferably, a sequence which acts as a primer at the time of reverse transcription of the single-stranded RNA is present at the 3 'end of the single-stranded DNA or a derivative thereof. Preferably, single-stranded D
A derivative of NA includes a single-stranded DNA sequence complementary to a sequence at the 3 ′ end of the single-stranded RNA, and a primer for reverse transcription of the single-stranded RNA at the 3 ′ end of the DNA sequence. A nucleic acid construct in which the sequence and a spacer sequence having a nucleic acid derivative at the end are linked in a branched state is used. The RNA ligase is preferably T4 RNA ligase.

According to still another aspect of the present invention, there is provided an RNA-DNA conjugate obtained by the method of the present invention. According to still another aspect of the present invention, there is provided a method for producing a DNA conjugate by subjecting an RNA-DNA conjugate obtained by the method of the present invention to a reverse transcription reaction.

According to still another aspect of the present invention, RNA and RNA are introduced, wherein the RNA-DNA conjugate obtained by the method of the present invention is introduced into a protein translation system to translate the RNA into a protein. The present invention provides a method for producing an RNA-protein complex comprising a protein encoded by According to still another aspect of the present invention, there is provided an RNA-protein complex produced by the above method.

According to still another aspect of the present invention, the RNA-protein complex of the present invention is subjected to a reverse transcription reaction, wherein the DNA comprises a DNA and a protein encoded by the DNA. A method for producing a protein complex is provided. According to still another aspect of the present invention, there is provided a DNA-protein complex produced by the production method described above.

[0016]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in more detail. The method for producing an RNA-DNA conjugate according to the present invention comprises: (1) a step of annealing a single-stranded RNA having a sequence complementary to each other to a single-stranded DNA or a derivative thereof; and (2) an RN of the annealing product.
A step of treating with A ligase to ligate the 3 ′ end of the single-stranded RNA to the 5 ′ end of the single-stranded DNA or a derivative thereof:
It is characterized by including.

In the method of the present invention, the single-stranded R
NA and single-stranded DNA (or a derivative thereof) need to have sequences complementary to each other. By annealing single-stranded RNA and single-stranded DNA having mutually complementary sequences under suitable conditions, the two can be associated with each other, and then treated with RNA ligase, whereby both can be efficiently linked. .

The method of the present invention is an extension of the method of linking DNAs by the Y-ligation method.
In the present invention, by using an RNA-DNA conjugate obtained by converting one of the nucleic acids to be ligated into RNA for a new use, a novel and efficient in vitro virus g
enome (Nemoto, N., et, al. (1997) FEBS Lett. 414,40
5-408.) Can be provided.

In the method of the present invention, the 3 'end of RNA and the 5' end of DNA having a sequence complementary to the sequence in the RNA are covalently linked by RNA ligase by the Y-ligation method. The RNA used in the method of the present invention is a single-stranded RNA, more preferably an RNA containing a coding sequence encoding a protein, and annealing at the 3 ′ end in the 5 ′ to 3 ′ direction. It is preferable to have an array and a branch array.

The term "branch sequence" as used herein refers to a sequence which does not anneal to each other when the annealing sequences in single-stranded RNA and single-stranded DNA or a derivative thereof are annealed, but exists in a single-stranded state. is there. Single chain RN
The length of A and the length of the branch sequence in single-stranded DNA or a derivative thereof is not particularly limited as long as both can be ligated by RNA ligase treatment. In general, the shorter the length of the branch array, the higher the coupling efficiency, but the upper limit is not particularly limited. The length of the branch sequence is preferably 1
To 100 bases, more preferably about 1 to 10 bases. The length of the branch sequence in both nucleic acids may be the same or different.

As used herein, the term “annealing sequence” refers to a sequence that can anneal to DNA to be bound, and a sequence complementary to the annealing sequence in RNA is:
It will be present in the DNA to be ligated. The length of the annealing sequence is not particularly limited as long as it is long enough to allow both strands to hybridize, but is generally about 10 to 50 bases, more preferably about 10 to 30 bases.

The single-stranded RNA and single-stranded DNA or a derivative thereof bound by the method of the present invention have complementary sequences to each other, so that they can be annealed under certain conditions. More specifically, the annealing sequence in the single-stranded RNA and the sequence complementary to the annealing sequence in the single-stranded DNA or its derivative hybridize to form a double-stranded structure. that time,
Since the branch sequence in the single-stranded RNA and the branch sequence in the single-stranded DNA or a derivative thereof remain single-stranded,
These parts will generally form a Y-shape (see the top figure in FIG. 1). The name Y-ligation is derived from the form of this structure. The feature of this method is that the reaction for linking two kinds of nucleic acids is changed from an intermolecular reaction to an intramolecular reaction, whereby the linking efficiency can be improved. Therefore, it can be applied to low-concentration substrates.

In the method of the present invention, first, the above-mentioned single-stranded RNA having a sequence complementary to each other is annealed with a single-stranded DNA or a derivative thereof. Annealing is carried out by dissolving the above-mentioned two single-stranded nucleic acids in an appropriate buffer (preferably a buffer for RNA ligase in terms of the convenience of the subsequent operation) and gradually decreasing the temperature from a high temperature to a low temperature. be able to. Such a temperature change can also be performed using a PCR device or the like. An example of the annealing condition is a condition of cooling from 94 ° C. to 25 ° C. over 10 minutes, but this is only an example,
Temperature and time can be appropriately changed. Annealing conditions (such as the composition of the buffer, the annealing temperature, and the annealing time) can be appropriately set according to the length of the annealing sequence, the base composition, and the like.

The molar ratio of single-stranded RNA to single-stranded DNA or a derivative thereof in the annealing reaction is not particularly limited as long as the annealing reaction proceeds, but from the viewpoint of reaction efficiency, it is 0.5: 1 to 1: 2. It is preferably about 0.5.

After annealing of the single-stranded RNA with the single-stranded DNA or its derivative, the annealing product is treated with RNA ligase, and the 3 ′ end of the single-stranded RNA and the single-stranded DN
The 5 'end of A or its derivative is linked. The RNA ligase used in the present invention may be any one capable of linking two single-stranded nucleic acids, and preferably T4 RNA ligase can be used.

Note that RN was used as a solution for annealing.
If an appropriate buffer is used as the A ligase buffer, the solution containing the annealing product can be used for the ligase reaction as it is; otherwise, the annealing product is recovered by a conventional nucleic acid purification method. Then, it is dissolved in a buffer solution for RNA ligase to prepare a solution for ligase reaction.

The conditions for the ligation reaction (ligase reaction) may be any conditions under which the activity of the RNA ligase to be used is exhibited, and for example, a suitable buffer solution (for example, T4 RNA ligase bu
ffer (50mM Tris-HCl, pH7.5, 10mM MgCl ₂ , 10mM DTT, 1
mM ATP) or the like, or a reaction at 25 ° C. for 30 minutes after repeating a cycle of 25 ° C. for 30 minutes and 45 ° C. for 2 minutes. The temperature and the reaction time shown here are only examples and can be appropriately changed so as to increase the reaction efficiency.

After the reaction, by purifying the reaction product by a conventional method such as phenol extraction and ethanol precipitation,
An RNA-DNA conjugate can be obtained. The thus obtained RNA-DNA conjugate itself is also within the scope of the present invention.

The type of the single-stranded RNA used in the present invention is not particularly limited.
RNA expressed in vitro from NA may be used. Also, not all of the nucleic acids that are the components of the single-stranded RNA need to be ribonucleotides, and only some of them may be of the RNA type. The other regions are ribonucleotides, deoxyribonucleotides, or PNA type. May be. Further, a peptide or a sugar or the like may be bonded.

The length of the single-stranded RNA used in the present invention is not particularly limited as long as a ligation reaction is possible. Generally, the length of single-stranded RNA is about several tens to several tens of kilobases, for example, about 10 to 50,000 bases, and more preferably about 20 to 10,000 bases. is there.

The single-stranded RNA used in the present invention preferably contains a protein-encoding sequence.
Preferably, it is a NA or mRNA library.
When introducing the RNA-DNA conjugate obtained by the method of the present invention into a transcription / translation system, the single-stranded RN
A preferably contains (1) a promoter sequence, (2) a DNA sequence recognized by ribosomes during translation, and (3) a sequence encoding a target protein.

The type of the promoter sequence is not particularly limited as long as it can be appropriately selected as appropriate for the expression system to be applied. For example, a T7 promoter sequence recognized by RNA polymerase of Escherichia coli virus T7 and the like can be mentioned.

The DNA sequence recognized by the ribosome during translation includes a DNA sequence corresponding to the RNA sequence (Kozak sequence) recognized by the eukaryotic ribosome during translation and the DNA sequence recognized by the prokaryotic ribosome. Shine-Dalgarno sequence and the like. The type of the sequence encoding the target protein is not particularly limited, and can be appropriately selected depending on the purpose.

The single-stranded DNA or a derivative thereof used in the present invention includes a single-stranded DN prepared from a naturally-derived DNA.
A, a single-stranded DNA prepared by genetic recombination technology, or a single-stranded DN prepared by chemical synthesis.
A may be used. Further, it is not necessary that all of the nucleic acids that are components of the single-stranded DNA be deoxyribonucleotides,
Only a part thereof may be of the DNA type, and the other region may be of the ribonucleotide, deoxyribonucleotide or PNA type. Further, a peptide or a sugar or the like may be bonded. The length of the single-stranded DNA or a derivative thereof used in the present invention is not particularly limited as long as a ligation reaction is possible. Generally, single-stranded DNA
Has a length of about several bases to several tens of kilobases, for example, about 10 bases to 50,000 bases, and more preferably about 20 bases to 10,000 bases.

In the method of the present invention, it is preferable to use, as the single-stranded DNA or a derivative thereof, a single-stranded DNA derivative having a nucleic acid derivative bound to the 3 ′ end. When a protein is translated in a cell-free protein translation system or a living cell using such a single-stranded DNA derivative,
The ribosome is stopped by the double strand, and puromycin can bind to the protein by entering the A site of the ribosome (see FIG. 1).

The nucleic acid derivative is not limited as long as it is a compound having the ability to bind to the C-terminus of the synthesized protein when the protein is translated in a cell-free protein translation system or living cells. Those whose 3 ′ terminus has a similar chemical skeleton to aminoacyl-tRNA can be selected. As a typical compound, puromycin having an amide bond (Puromycin), 3′-N-
Aminoacylpuromycin aminonucleoside (3 '
-N-Aminoacylpuromycin aminonucleoside, PANS-amino acid), for example, PANS-Gly in which the amino acid portion is glycine,
Examples include PANS-Val in which the amino acid portion is valine, PANS-Ala in which the amino acid portion is alanine, and PANS-amino acid compounds in which the amino acid portion corresponds to all amino acids.

Further, 3'-N-aminoacyladenosine aminonucleoside (3'-N-aminoacyladenosine aminonucleoside linked by an amide bond formed by dehydration condensation of the amino group of 3'-aminoadenosine and the carboxyl group of the amino acid.
leoside, AANS-amino acid), for example, AANS-Gly in which the amino acid portion is glycine, AANS-Val in which the amino acid portion is valine, AANS-Ala in which the amino acid portion is alanine, and AANS in which the amino acid portion corresponds to each amino acid of all amino acids -Amino acid compounds can be used.

Also, nucleosides or nucleosides and ester bonds of amino acids can be used.
Furthermore, all compounds chemically bonded to a nucleic acid or a substance having a chemical structural skeleton and a base similar to a nucleic acid and a substance having a chemical structural skeleton similar to an amino acid are included in the nucleic acid derivative used in the present method. .

As nucleic acid derivatives, puromycin,
Compounds in which a PANS-amino acid or AANS-amino acid is linked to a nucleoside via a phosphate group are more preferred. Among these compounds, puromycin derivatives such as puromycin, ribocytidyl puromycin, deoxydilpuromycin, and deoxyuridyl puromycin are particularly preferred.

In the method of the present invention, it is preferable to use, as the single-stranded DNA or a derivative thereof, a single-stranded DNA or a derivative thereof having a nucleic acid derivative bound to the 3 ′ end via a spacer. As the spacer, a polymer 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,
It has a molecular weight of 300 to 3000, or the number of atoms in the main chain is 10 to 100.

The single-stranded DNA derivative as described above is
It can be produced by a chemical bonding method known per se. Specifically, in the case where the synthesis unit is linked by a phosphodiester bond, the synthesis unit can be synthesized by solid phase synthesis by a phosphoramidite method generally used for a DNA synthesizer. When a peptide bond is introduced, the synthetic units are linked by an active ester method or the like. However, when a complex with DNA is synthesized, a protecting group compatible with both synthesis methods is required.

[0042] RNA is included in the RNA-DNA conjugate obtained by the method of the present invention. By subjecting this to a reverse transcription reaction, a conjugate consisting of only DNA can be produced. That is, after the above-described ligation reaction between RNA and DNA by the Y-ligation method, a reaction is performed using a reverse transcriptase, whereby a DNA conjugate can be produced by a transcription reaction from RNA to DNA. When such a reverse transcription reaction is intended, it is preferable that a sequence that acts as a primer at the time of reverse transcription of the single-stranded RNA is present at the 3 ′ end of the single-stranded DNA or a derivative thereof. By allowing such a primer sequence to exist, a transcription reaction can be performed without newly adding a primer.

According to a preferred embodiment of the present invention, the single-stranded D
A derivative of NA includes a single-stranded DNA sequence complementary to a sequence at the 3 ′ end of the single-stranded RNA, and a primer for reverse transcription of the single-stranded RNA at the 3 ′ end of the DNA sequence. A nucleic acid construct in which the sequence and a spacer sequence having a nucleic acid derivative at the end are linked in a branched state can be used. In addition, since such a nucleic acid construct has a T-shaped structure, in this specification, T-Spacer
Also called. FIG. 1 shows a specific example of such a T-Spacer. As used herein, the term “primer sequence for reverse transcription of single-stranded RNA” refers to a nucleic acid construct of the present invention obtained by ligation of a nucleic acid construct (T-Spacer) with single-stranded RNA. When introduced into a reaction system, it means a base sequence that acts as a primer sequence for initiating a reverse transcription reaction, and is generally preferably composed of a sequence complementary to the sequence of a single-stranded RNA. .

Furthermore, according to the present invention, RNA and RNA are characterized in that the RNA-DNA conjugate obtained by the method of the present invention is introduced into a transcription / translation system to translate single-stranded RNA into a protein. The present invention provides a method for producing an RNA-protein complex comprising a protein encoded by the above, and an RNA-protein complex produced by the production method. Transcription / translation systems for artificially producing the protein it encodes from nucleic acids are known to those skilled in the art. Specifically, there is a cell-free protein synthesis system in which a component having a protein synthesis ability is extracted from an appropriate cell, and a target protein is synthesized using the extract. Such cell-free protein synthesis systems include ribosomes, initiation factors,
Elements required for transcription / translation systems such as elongation factors and tRNA are included.

Examples of such a cell-free protein synthesis system (system derived from a cell lysate) include a cell-free translation system composed of a prokaryotic or eukaryotic extract.
Rabbit reticulocyte extract, wheat germ extract and the like can be used, but any may be used as long as it produces the 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
(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 organisms, for example, E. coli cells and the like can be used. The cell-free translation system or living cell is not limited as long as protein synthesis is performed by adding or introducing a nucleic acid encoding a protein into the cell-free translation system or living cell.

In the present invention, the RNA and the DNA encoded by the RNA are transcribed by introducing the RNA-DNA conjugate into the above-described transcription / translation system and translating the single-stranded RNA into a protein, and then removing the ribosome. An RNA-protein complex consisting of a protein can be produced.

Furthermore, according to the present invention, the R
A method for producing a DNA-protein complex comprising DNA and a protein encoded by the DNA, which comprises subjecting the NA-protein complex to a reverse transcription reaction, and a DNA-protein complex produced by the production method A body is provided. That is, by treating an RNA-protein complex comprising RNA and a protein encoded by the RNA with reverse transcriptase, DN
Reverse transcription to A occurs, and a DNA-protein complex consisting of DNA and the protein encoded by the DNA is produced. R obtained as described above
The NA-protein complex and the DNA-protein complex provide useful materials for analyzing the function of nucleic acids and the like.

[0048]

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. FIG. 1 shows a schematic diagram of the construction of an in vitro virus genome using the Y-ligation method according to the present invention.

Example 1: Ligation of Thioredoxin mRNA to DNA fragment and reverse transcription from this DNA fragment (1) Construction of transcription DNA and preparation of mRNA DNA sequence recognized by RNA polymerase of Escherichia coli virus T7 having high transcription efficiency (T7 promoter sequence) and D, which is recognized by eukaryotic ribosomes during translation.
Recognized by NA sequence (Kozak sequence) and prokaryotic ribosome (Shine-Dalgar sequence: Shine-Dalgar
No), and a DNA encoding Thioredoxin was constructed downstream thereof as follows.

First, a T7 promoter sequence (Rosenberg,
AH, et al., Gene, 56, 125-135 (1987)) and single-stranded DNA containing Kozak consensus sequence and Shine-Dalgarno sequence
(SEQ ID NO: 1) was organically synthesized, and this was used as a template to give DN.
Polymerase chain reaction (PCR) was performed using the A primer (SEQ ID NO: 2) and a primer encoding a part of thioredoxin (SEQ ID NO: 3). DNA synthase is KO
D Taq Polymerase (TOYOBO) was used. Regarding the PCR conditions, a cycle of 95 ° C. for 20 seconds, 68 ° C. for 2 seconds, and 74 ° C. for 15 seconds was repeated 30 times. This PCR product was subjected to phenol extraction with a primer remover (edge science) and ethanol precipitation to remove the primer.

On the other hand, a polymerase chain reaction was carried out by using the pTrxFus plasmid (manufactured by Invitrogene) carrying thioredoxin as a template and the antisense primer (SEQ ID NO: 4) of SEQ ID NO: 3 and the DNA primer (SEQ ID NO: 5). The DNA region encoding thioredoxin was amplified. PCR conditions were 95 ° C for 20 seconds, 68 ° C for 20 seconds,
The cycle of 74 ° C. for 20 seconds was repeated 25 times. After phenol extraction, the PCR product was precipitated with ethanol using a primer remover. The DNA primer (SEQ ID NO: 5)
MRNA that is more suitable as a substrate for T4 RNA ligase
It is designed to have.

The above two PCR products are overlap-extended (Ov
erlap extension) method (Horton RM, et al. (1989) Gene
77, 61-68), and a T7 promoter sequence-Kozak consensus sequence-Shine-Dalgarno sequence-Thioredoxin was prepared with two primers (SEQ ID NO: 2 and SEQ ID NO: 5). In all PCRs, DNA synthase is KODT
aq Polymerase (TOYOBO) was used.

The DNA prepared by the above-mentioned method was
10 μg per μl, RNA synthesis kit Ribomax Large Sc
mR using ale RNA Production System (Promega)
Transferred to NA. To increase the translation efficiency, a cap analog (RNA capping Analog; manufactured by Gibco BRL) was added to a final concentration of 7.2 mM to modify the 5 ′ side of the mRNA. To remove cap analogs and excess NTP (nucleotide triphosphate), remove primers (Primer Rem
over: Edge Biosystems) to perform ethanol precipitation.

(2) Preparation of DNA for Ligation RT-thio (SEQ ID NO: 6) was synthesized by Nippon Flour Milling. RT
-thio has a part of thioredoxin as an antisense sequence.

(3) Annealing conditions for mRNA and RT-thio Thioredoxin mRNA and RT-thio were mixed at a ratio of 1: 1.5 (molar ratio), and a T4 RNA ligase buffer (50 mM Tris-HCl,
Dissolved in pH 7.5, 10 mM MgCl2, 10 mM DTT, 1 mM ATP) and used DMSO (Dimethyl sulfoxid
e) was added to a final concentration of 5%. Annealing was performed by cooling to 94 ° C to 25 ° C over 10 minutes using a PCR device.

(4) Ligation reaction by T4 RNA ligase 2 T4 RNA ligase was added to the solution annealed in (3).
5 units plus 10 units of T4 polynukureotide kinase, 25
The reaction was carried out at 150C for 15 minutes. After the reaction, Rneasy Mini (QIAGEN
Was performed to purify the ligation product.

(5) Confirmation of ligation reaction To confirm the ligation efficiency, 6% acrylamide 8M urea denaturing gel electrophoresis, 65 ° C., 220 V,
Flow the sample under the conditions of 120 mA for 50 minutes,
en (Amersham pharmacia), and
r (Bio Rad). The results are shown in FIG. FIG.
In, the left lane shows the molecular weight marker, the middle lane shows the result of electrophoresis of the original mRNA, and the right lane shows the result of electrophoresis of the ligation product. When electrophoresis was performed under denaturing conditions, the ligation product had a higher molecular weight than the original mRNA, confirming that the ligation reaction was performed.

(6) Confirmation of reverse transcription by RT-thio It was confirmed whether the purified mRNA ligated with RT-thio could actually reverse transcribe. MRNA containing 8 pmol of RT-thio bound to the 3 'end was treated with AMV Reverse Transcriptase.
(Promega) for reverse transcription. Then, half RNase H
(Takara) It was digested with 2 units, and it was confirmed whether there was reverse transcribed DNA. The results are shown in FIG. In FIG. 3, lane M shows a molecular weight marker, lane 1 shows a result of electrophoresis of mRNA before ligation, and lane 2 shows a result of electrophoresis of a product obtained by treating a product obtained by reverse transcription of a ligation product with RNase H. Lane 3 shows the result of electrophoresis of the product obtained by reverse transcription of the ligation product. In lane 2, a band corresponding to the reverse transcript was observed, confirming that the reverse transcription reaction was performed.

Example 2: Ligation to Thioredoxin mRNA
In vitro virus virion formation by the resulting hybrid spacer The hybrid spacer is PEG (Polyethylene glycol) and dCdC-puro as spacers on the 3 'side of the RT-thio used in Example 1.
Mycin is chemically synthesized.
It functions as a rus spacer. The specific manufacturing method is as follows. Pu using a DNA synthesizer
After synthesizing dCdC from romycinCPG (GLEN RESEARCH) based on the usual phosphoramidite method, Spacer-18 (GLEN RESEARCH)
SEARCH) was also synthesized by the phosphoramidite method. At this time, the length of PEG is changed by changing the number of Spacer-18. Next, by performing normal DNA synthesis, Hybri
The spacer is synthesized.

(1) Ligation reaction of mRNA and Hybri spacer and its confirmation In order to confirm that the ligation reaction can be performed under the same conditions as in Example 1 also with the above-described RT-thio primer-processed Hybri spacer, the same as in Example 1 was used. The ligation reaction was performed according to the procedure described in. The result of electrophoresis of the reaction product is shown in FIG. In FIG. 4, the left lane shows the molecular weight marker, the middle lane shows the result of electrophoresis of the original mRNA, and the right lane shows the result of electrophoresis of the ligation product. When electrophoresis was performed under denaturing conditions, the ligation product had a higher molecular weight than the original mRNA, confirming that the ligation reaction was performed. The ligation reaction product was purified using RNeasy Mini (QIAGEN),
Used as irus genome.

(2) Confirmation of In Vitro Virus Virion Formation Regarding in vitro virus virion formation, four different PEGs (specifically,
A Hybri spacer having 5, 6, 7, or 8 Spacer-18) was prepared, and a ligation product of mRNA and Hybri spacer was prepared based on (1).

[0062] 1 µg of mRNA to which each of these spacers is attached and 1 µg of mRNA
MBS 35S Met (Amersham) was added to the wheat germ cell-free translation system, reacted at 30 ° C. for 45 minutes, and the final concentration was 20 mM MgC
l _2, 600 mM KCl the salt to be added and refrigerated overnight at -20 ° C.. Next free 35SMet that is not incorporated into translation products
Micro BioSpin Coloumn-6 (Biolad
After purification using EDTA, EDTA was added to a final concentration of 100 μM. As a result, the ribosome with added mRNA is completely separated, and the mRNA and protein are bound in vitro
Only s virion remains.

The translation product purified as described above was subjected to 15% SD
The result confirmed by S-polyacrylamide gel electrophoresis is shown in FIG. In FIG. 5, lane 1 shows a structure in which five spacer C-18 (Gren Research) as a spacer are linked, lane 2 shows six, lane 3 shows seven, and lane 4 shows eight. As can be seen from the results in FIG.
The connection was most efficient when a spacer in which five acerC-18 (Gren Research) were connected (lane 1) was used.

Example 3: In vitro vir using T-Spacer
Us virion formation and reverse transcription (1) Method for preparing T-Spacer The following modified DNA was synthesized with a DNA synthesizer as a raw material of T-Spacer. DNA1: (thiol) (Spc) (Spc) (Spc) (Spc) CC (ZFP) DNA2: (Pso) TACGCCAGCTGCACCCCCCGCCGCCCCCCG (At) CCGC DNA3: CCCGG (Ft) GCAGCTGGCGTATAAAAAAAAAAAAAAAAAAAAA
AAAAA DNA4: CCCGGTGCAGCTGTTTCATC (Bt) CGGAAACAGCTGCACCCCC
CGCCGCCCCCCG (At) (Ft) (Spc) (Spc) (Spc) (Spc) CC (ZFP) DNA5: (thiol) CGCT sequence (thiol) is 5'-Thiol-modifier C6, (Spc) is Sp
acer 18, (Bt) is Biotin-dT, (Ft) is Fluorescein-d
T, (At) is Amino-modifier C6 dT, (Pso) is Psoralen C6
(All above are Glenn Research), (ZFP) is Z-phenylalany
l-puromycin is shown respectively.

(A) T-splint3FA DNA2 (12 nmol) and DNA3 (12 nmol) were added to a TBS buffer (25
mM Tris-HCl, pH 7.0, 100 mM NaCl)
After heating at 85 ° C. for 40 seconds, it was allowed to cool at room temperature. After standing on an ice bath for 5 minutes, light irradiation was performed for 8 minutes with a handy UV lamp (365 nm), and the reaction product was purified by reverse phase HPLC. This DNA 24 n
was dissolved in 15 μl of a 0.1 M disodium hydrogen phosphate aqueous solution, 4 μl of a 5 mM DMF solution of EMUS (crosslinking agent; Dojindo) was added, and the mixture was stirred for 20 minutes at room temperature. DNA 1 (20 nmol) in this solution
Was dissolved in 40 μl of 0.1 M phosphate buffer (pH 7.1), and the mixture was further stirred at room temperature for 16 hours. The target compound bound via a cross-linking agent was isolated by reverse-phase high-performance liquid chromatography (reverse-phase HPLC), dissolved in 50 mM phosphate buffer (pH 8.0), and the chymotrypsin solution was added at a weight ratio of enzyme to substrate of 10%.
%, And left at 36 ° C. for 1 hour. Reversed phase
Purification by HPLC yielded T-splint3FA (FIG. 6).

(B) T-splint4FB DNA 4 (5 nmol) was dissolved in 0.1 M disodium hydrogen phosphate aqueous solution
Dissolve in 15 μl and add 10 parts of Sulfo-KMUS (crosslinking agent; Dojindo).
5 μl of a mM DMF solution was added, and the mixture was stirred at room temperature for 20 minutes. DNA 5 (60 nmol) is further added to this solution with 0.1 M phosphate buffer (pH 7.
1) A solution dissolved in 40 μl was added, and the mixture was further stirred at room temperature for 16 hours. The target product in which DNA4 and DNA5 were bound via a cross-linking agent was purified by reverse-phase HPLC, and digested with chymotrypsin in the same manner as T-splint3FA. Purify again by reversed-phase HPLC and use T-splint
4FB was obtained (FIG. 6).

(2) Construction of DNA for transcription and preparation of mRNA A DNA sequence (T7 promoter sequence) recognized by RNA polymerase of Escherichia coli virus T7 having high transcription efficiency, and recognized by eukaryotic ribosomes during translation. It has a DNA sequence (Kozak sequence) and is recognized by prokaryotic ribosomes (Shine-Dalgarno sequence: Shine-Dalgarno), and a part of Oct-1 (POU), FLAG sequence, T-Spa
DNA encoding a sequence (Y-tag) for linking with cer
(SEQ ID NO: 7 below) was constructed. GATCCCGCGA AATTAATACG ACTCACTATA GGGAGACCAC AACGGTTTCC CTCTTGAAAT AATTTTGTTT AACTTTAAGA AGGAGATTCC ACCATGGACC TTGAGGAGCT TGAGCAGTTT GCCAAGACCT TCAAACAAAG ACGAATCAAA CTTGGATTCA CTCAGGGTGA TGTTGGGCTC GCTATGGGGA AACTATATGG AAATGACTTC AGCCAAACTA CCATCTCTCG ATTTGAAGCC TTGAACCTCA GCTTTAAGAA CATGGCTAAG TTGAAGCCAC TTTTAGAGAA GTGGCTAAAT GATGCAGAGG GGGGAGGCAG CGATTACAAG GATGACGATG ACAAGGGCGG AAGCGGACGG GGGGCGGCGG GAAA (SEQ ID NO: 7)

The prepared DNA was added in an amount of 10 μg per 100 μl of the reaction solution.
And the RNA synthesis kit Ribomax Large Scale RNA Produ
mRNA was transcribed using the ction System (Promega). Cap analog (RNA Capping)
Analog; Gibco BRL) was added to a final concentration of 7.2 mM to modify the 5 'end of the mRNA. To remove cap analogs and excess NTP (nucleotide triphosphate), remove primers (Primer Remover; Edge Biosys
tems) for ethanol precipitation.

(3) between mRNA and T-Spacer (T-splint3FA)
Ligation Ligation of the T-Spacer (T-splint3FA) prepared in the above (1) with the mRNA prepared in the above (2) was performed in Example 1.
Was carried out according to the method described in (1). Specifically, it is as follows. The mRNA prepared in the above (2) and the T-Spacer (T-splint3FA) prepared in the above (1) were mixed at a ratio (molar ratio) of 1: 1.2-1.5, and a T4 RNA ligase buffer (50 mM Tris-HC) was mixed.
l, pH 7.5, 10 mM MgCl ₂ , 10 mM DTT, 1 mM ATP)
DMSO (Dimethyl sulfo)
xide) was added to a final concentration of 5%. The obtained mixture was annealed by cooling it to 94 ° C to 25 ° C over 10 minutes using a PCR device.

Subsequently, in the above-mentioned annealed solution,
T4 Polynucleotide Kinase (Takara) and T4 RNA ligase
(Manufactured by Takara) was added thereto and reacted at 25 ° C. for 30 minutes.
After the reaction, the ligation product was purified using the RNeasy Mini Kit (QIAGEN). To check the ligation efficiency, run the sample under the conditions of 4% acrylamide 8M urea denaturing gel,
en (Amersham pharmacia) stained with Molecular Imager
(Bio Rad). In addition, the fluorescence (Fluorescein) introduced into the spacer was also confirmed. FIG. 7 shows the results.

In FIG. 7, lane 1 shows the result of electrophoresis of the original mRNA, and lane 2 shows the result of electrophoresis of the ligation product. When electrophoresis was performed under denaturing conditions, the ligation product had a higher molecular weight than the original mRNA, confirming that the ligation reaction was performed. This ligation product is
Named rus genome.

(4) In using T-Spacer (T-splint3FA)
In vitro virus virion formation In vitro virus genome is actually in vitro virus viri
It was confirmed whether on could be formed. In vitro virus gen
ome 4 pmol was reacted and translated at 26 ° C. for 30 minutes using a wheat germ cell-free translation system PROTEIOS (TOYOBO), and the final concentration was 40 mM MgCl ₂ to bind the translated peptide to puromycin (virionization). A salt was added so as to obtain 1 M KCl, and the mixture was reacted at 26 ° C for 1 hour. To confirm the efficiency of Virionization, the sample was run on a 5M urea-denatured 5% SDS-PAGE gel at 20 mA. Fluorescence introduced into T-Spacer (Fl
uorescein) and imaged with a Molecular Imager (Bio Rad). FIG. 8 shows the results. In FIG. 8, the left lane shows the result of electrophoresis of the sample before the translation reaction. The right lane shows the electrophoresed sample after virion formation.

(5) Confirmation of Reverse Transcription Reaction Using T-Spacer (T-splint3FA) It was confirmed whether or not the in vitro virus genome could actually reverse transcribe. 2pmol In vitro virus genome with TrueSc
Reverse transcription was performed using ript II Reverse Transcriptase (sawady). Thereafter, half was digested with 2 units of RNase H (Takara), and it was confirmed whether there was reverse-transcribed DNA. The sample was electrophoresed by 4% acrylamide 8M urea denaturing gel electrophoresis, stained with Vistra Green (Amersham pharmacia),
Images were taken with an olecular Imager (Bio Rad). Also, Space
The fluorescence (Fluorescein) introduced into r was also confirmed. FIG. 9 shows the results.

In FIG. 9, lane 1 shows in vitro vir
The lane 2 shows the results of electrophoresis of the us genome.
o The product obtained by electrophoresing the product obtained by reverse transcription of the virus genome is shown. Lane 3 shows the product obtained by electrophoresing the product obtained by treating the product obtained by reverse transcription of the in vitro virus genome with RNase H.
The presence of a band corresponding to the reverse transcript confirmed that the reverse transcription reaction was performed.

(6) I using T-spacer (T-splint3FA)
Confirmation of reverse transcription reaction after in vitro virus virion formation After in vitro virus virion was formed and purified from the translation system, it was confirmed whether reverse transcription could be actually performed using T-spacer. 8 pmol of in vitro virus genome by the above method
Micro BioSpin to virion and exchange buffer
After desalting using Column-6 (Bio-Rad), 1 M NaCl, 100 mM T
ris-HCl (pH8.0), 10mM EDTA, 0.25% Triton-X100, and adjust Biotinylated Oligo (dT) Probe (Prom
egaNOTE-conjugated MAGNOTEX-SA (Takara) 5 μl and 4 ° C, approx.
Let it join for 1 hour. After that, take the supernatant and wash bufferA
(1M NaCl, 100mM Tris-HCl (pH8.0), 0.25% Triton-X10
0) Wash 3 times with 20 μl, bufferB (500 mM NaCl, 100 mM Tris-
HCl (pH 8.0), 0.25% Triton-X100) Wash once with 20 μl, b
ufferC (250 mM NaCl, 100 mM Tris-HCl (pH 8.0), 0.25% T
riton-X100) Wash once with 20 μl, then add 3 μl with 10 μl of Dep water.
Elution was performed several times to confirm whether in vitro virus could be purified.

Next, 1 and 2 of the eluted fraction were mixed, and TrueScript
Reverse transcription was performed using II Reverse Transcriptase (sawady). In addition, in vitro as a negative control
virus genome, in vitr as positive control
o Polymerase chain reaction using reverse transcribed virus genome (Fig. 9) and sense primer (SEQ ID NO: 8) and antisense primer (SEQ ID NO: 9)
(PCR) was performed. DNA synthase is TaKaRa Ex Taq (TAK
ARA) was used.

To confirm the results, the sample was electrophoresed on a 6M urea-denatured 6% polyacrylamide gel at 250 V, stained with Vistra Green (Amersham Pharmacia), and
Images were taken with an olecular Imager (Bio Rad). Figure 1 shows the results
0 is shown.

In FIG. 10, lane 1 shows in vitro v
irus genome, lane 2 shows the result of reverse transcription of the in vitro virus genome, and lane 3 shows the result of electrophoresis of a sample obtained by performing PCR using in vitro virus purified and reverse transcribed after virion as a template. In vitro virus purified and reverse transcribed after virion is
In the same way as PCR was performed using the reverse transcribed template as the template, the target DNA could be amplified, so it was purified after virion and in vitro virus was used using T-sapcer.
It was confirmed that reverse transcription could be performed. SEQ ID NO: 8, 5′-GTT TAA CTT TAA GAA GGA GTT GCC AC
C ATG −3 ′ SEQ ID NO: 9, 5′-TTT CCC GCC GCC CCC CGT CCG CTT CC
G CCC TTG TCA TCG TCATCC TTG TAA TC −3 '

[0079]

According to the present invention, RNA can be efficiently prepared in a short time.
-It has become possible to provide a method for producing a DNA conjugate. Further, according to the present invention, it has become possible to efficiently produce a conjugate of a protein and RNA. That is, according to the method of the present invention, it is possible to synthesize in vitro virus genome, whose production efficiency was low with the conventional method, in a short time with an efficiency of 90% or more.
By translating the A-conjugate using a cell-free translation system, it became possible to improve the binding efficiency between protein and RNA by a factor of 10 or more. In addition, by adding a primer sequence for reverse transcription of RNA to DNA, reverse transcription can be performed as it is, and it can be stabilized by forming a conjugate of protein and DNA. The method of the present invention can be widely used for obtaining various new functional proteins in evolutionary molecular engineering and for analyzing protein interactions in post-genome.

[0080]

[Sequence List] SEQUENCE LISTING <110> GenCom <120> RNA-DNA conjugate <130> A21011A <160> 9

<210> 1 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 1 gatcccgcga aattaatacg actcactata gggagaccac aacggtttcc ctctagaaat 60 aattttgttt aactttaaga aggagatgcc accat

<210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 2 gatcccgcga aattaatacg actcactata ggg 33

<210> 3 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 3 caggtgaata attttatcgc tcatggtggc atctccttc 39

<210> 4 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 4 gaaggagatg ccaccatgag cgataaaatt attcacctg 39

<210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 5 tttcccgccg ccccccgtc 19

<210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 6 ccccgccagg ttagcgtcga ggaactcttt 30

<210> 7 <211> 374 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 120 gccaagacct tcaaacaaag acgaatcaaa cttggattca ctcagggtga tgttgggctc 180 gctatgggga aactatatgg aaatgacttc agccaaacta ccatctctcg atttgaagcc 240 ttgaacctca gctttaagaa catggctaag ttgaagccac ttttagagaa gtggctaaat 300 gatgcagagg ggggaggcag cgattacaag gatgacgatg acaagggcgg aagcggacgg 360 ggggcggcgg gaaa 374

<210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 8 gtttaacttt aagaaggagt tgccaccatg 30

<210> 9 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 9 tttcccgccg ccccccgtcc gcttccgccc ttgtcatcgt catccttgta atc 53

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the construction of an in vitro virus genome using the Y-ligation method according to the present invention.

FIG. 2 shows mRNA and RT before ligation.
FIG. 9 is a view showing the result of electrophoresis of mRNA after ligation with -thio.

FIG. 3 shows the results of electrophoresis of a reverse transcript of mRNA after ligation (lane 3) and a product obtained by treating it with RNase H (lane 2).

FIG. 4 shows mRNA before ligation and Hy.
FIG. 4 shows the results of electrophoresis of mRNA after ligation with a bri spacer.

FIG. 5 shows the results of electrophoresis of a translation product obtained by adding a ligation product to a cell-free translation system on a 15% SDS-polyacrylamide gel.

FIG. 6 shows a schematic diagram of an example of the nucleic acid construct (T-Spacer) of the present invention.

FIG. 7 shows mRNA and T-Spacer (T-splint3F) of the present invention.
It is a figure which shows the result of the ligation with A).

FIG. 8 shows in vitro vir containing T-Spacer of the present invention.
The result of having formed in vitro virus virion using us genome is shown.

FIG. 9 shows in vitro vir containing T-Spacer of the present invention.
The result of reverse transcription of us genome is shown.

FIG. 10 shows an in vitro culture containing the T-Spacer of the present invention.
In vitro virus virion prepared using virus genome
Shows the results of reverse transcription.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Miwa Shiratori 2-12-3 Naruse, Machida-shi, Tokyo Poplargaoka Bldg. 6, Room 306 F-term (reference) 4B024 AA20 CA02 HA20 4B064 AG32 CA21 CC24 CD30 4H045 AA10 BA10 BA54 FA70

Claims (17)

[Claims] 1. a step of annealing a single-stranded RNA having a sequence complementary to a single-stranded DNA or a derivative thereof; and (2) treating the annealing product with an RNA ligase to obtain a single-stranded RNA. 3 'end of RNA and single-stranded D
A method for producing an RNA-DNA conjugate, comprising: linking the 5 ′ end of NA or a derivative thereof. (1) a single-stranded RNA containing a coding sequence encoding a protein and having an annealing sequence and a branch sequence in the 5 'to 3' direction at the 3 'end, and a 3' to 5 'direction Annealing a single-stranded DNA having a sequence complementary to the annealing sequence and a branch sequence or a derivative thereof; and (2) annealing the annealing product to R
A method for producing an RNA-DNA conjugate, comprising: treating the 3 ′ end of single-stranded RNA with the 5 ′ end of single-stranded DNA or a derivative thereof by treating with NA ligase. 3. The method according to claim 1, wherein the single-stranded RNA is an mRNA or an mRNA library. 4. A single-stranded RNA comprising: (1) a promoter sequence; and (2) a D sequence recognized by a ribosome during translation.
The method according to any one of claims 1 to 3, comprising an NA sequence and (3) a sequence encoding a target protein. 5. A single-stranded DNA or a derivative thereof,
The method according to any one of claims 1 to 4, wherein a single-stranded DNA derivative having a nucleic acid derivative bound to its 3 'end is used. 6. The nucleic acid derivative is puromycin, 3′-
N-aminoacyl puromycin aminonucleoside,
The method according to claim 5, which is a compound having a chemical structural skeleton of 3'-N-aminoacyladenosine aminonucleoside or an analog thereof. 7. As a single-stranded DNA or a derivative thereof,
The method according to any one of claims 1 to 6, wherein a single-stranded DNA derivative having a nucleic acid derivative bound to the 3 'end via a spacer is used. 8. The method according to claim 7, wherein the spacer is a polymer such as polyethylene or polyethylene glycol. 9. The method according to claim 1, wherein a sequence that acts as a primer at the time of reverse transcription of the single-stranded RNA is present at the 3 ′ end of the single-stranded DNA or a derivative thereof.
The method according to any one of the above. 10. A derivative of a single-stranded DNA comprising a single-stranded DNA sequence complementary to a sequence at the 3 ′ end of single-stranded RNA, wherein the single-stranded DNA is RNA
The method according to any one of claims 1 to 9, wherein a nucleic acid construct in which a primer sequence for reverse transcription and a spacer sequence having a nucleic acid derivative at the end are linked in a branched state. 11. The method according to claim 1, wherein the RNA ligase is T4 RNA ligase. An RNA-DNA conjugate obtained by the method according to any one of claims 1 to 11. 13. A method for producing a DNA conjugate by subjecting the RNA-DNA conjugate obtained by the method according to claim 1 to a reverse transcription reaction. 14. An RNA and a RNA, wherein the RNA-DNA conjugate obtained by the method according to any one of claims 1 to 11 is introduced into a protein translation system to translate the RNA into a protein. A method for producing an RNA-protein complex comprising an encoded protein. 15. An RNA-protein complex produced by the production method according to claim 14. 16. A DN, comprising subjecting the RNA-protein complex according to claim 15 to a reverse transcription reaction.
A and D consisting of the protein encoded by the DNA
A method for producing an NA-protein complex. 17. A DNA-protein complex produced by the production method according to claim 16.