JP2000210090A - Oligo dna strongly combining with hcv rna and convenient production of the dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new oligo DNA which exerts a high bonding reactivity to HCV RNA, consists of a single strand oligo DNA complementary to the specific domain of the HCV RNA, shows easy RNA amplification and is used for determination, diagnosis or the like for the HCV RNA. SOLUTION: This oligo DNA is a new single strand oligo DNA having a high bonding reactivity to hepatitis C virus(HCV) RNA and containing the base sequence represented by the formula complementary to the base number 101-120 of HCV RNA, possesses the higher structure complementary to the free part in a single strand RNA such as HCV RNA or the like, enables to easily carry out the amplification or the like of RNA by using this and is useful as a reagent or the like for determination or diagnosis for HCV RNA. This oligo DNA is obtained by screening out the combination of several kinds of oligo DNA of 20-25 base length containing the sequence complementary to the cleavage domain of RNA presumed in the cleavage reaction using an enzyme which cleaves only single strand oligo DNA and RNA containing the random sequence of 6-9 base length by means of the above enzyme reaction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a simple method for producing a single-stranded oligo DNA that binds strongly to a single-stranded RNA, and to a HCV (hepatitis C virus) RNA produced using the method. It relates to a single-stranded oligo DNA to be bound. Oligo DNA produced by the production method of the present invention is used as a reagent for genetic diagnosis for performing operations such as RNA cleavage, RNA amplification and RNA detection, and as a drug for inhibiting reverse transcription and translation of RNA. Useful. In particular, the sequence of the single-stranded oligo DNA that strongly binds to HCV RNA produced by the production method of the present invention is useful for HCV RNA quantification, diagnostic reagents, and the like.

[0002]

2. Description of the Related Art By qualitatively or quantitatively determining RNA such as HCV, it is possible to measure the presence and the amount of HCV. Specific RN derived from virus, etc.
In order to quantify A, for example, a single-stranded oligo DNA that binds complementarily to the RNA is used as a primer, and the specific RNA is used as a template with this as a starting point.
An NA reverse transcription reaction is performed to form an RNA-DNA heteronuclear duplex. Subsequently, RNA of the heteronuclear double-stranded portion of RNA-DNA
A single-stranded DNA is produced by the action of an enzyme that cuts only DNA. Next, an oligo DNA called a promoter / primer having a portion complementary to the DNA and a promoter portion for RNA synthesis is added, and a DNA extension reaction is performed to make a double-stranded DNA containing the promoter appear.
Finally, an RNA synthase is allowed to act on the double-stranded DNA to amplify the specific RNA used as the starting material, and then measure the presence and amount of the amplified RNA (using the amplified RNA as the starting material). As a further example, RNA amplification is performed). Such a method has a feature that it can be performed at a relatively constant temperature as compared with a case where a DNA amplification method generally called PCR is used.

A nucleic acid sequence has the property of forming a sequence-specific complex with a nucleic acid (nucleic acid probe) having a sequence complementary to its base sequence. Therefore, utilizing such properties, single-stranded RNA can be treated with oligo D
NA is used not only for the reverse transcription reaction in the RNA amplification operation as described above, but also for the amplified RN.
It is useful as a probe for detecting A, and also as an anti-RNA agent (an agent that inhibits translation of RNA by binding to RNA), which is generally called an antisense agent. Furthermore, in the above-described RNA amplification operation and the like, not only an oligo DNA that binds to a specific RNA, but also a DNA that is complementary to its complementary oligo DNA, that is, an oligo DNA having the same sequence as the specific RNA is used. Required.

[0004]

The oligo DNA which can be used as a primer or the like for the above-mentioned RNA amplification synthesizes a sequence complementary to a specific target RNA sequence (primary structure) based on the sequence (primary structure). It is possible to manufacture it. However, knowing the sequence is often not enough.

[0005] It is known that single-stranded RNA partially takes a three-dimensional structure by forming a complex by combining sequence portions complementary in a molecule with each other in a sequence-specific manner. Therefore, it is necessary to design an oligo DNA having a sequence complementary to a portion existing in a single-stranded state, that is, a portion free of a higher-order structure. If the sequence of the cDNA is unknown, a single-stranded oligo D that binds to a specific RNA is used.
It is very difficult to produce NA.

Needless to say, there is no method for estimating the three-dimensional structure of RNA. According to crystal X-ray analysis, NMR structure analysis, and the like, it is possible to know the higher-order structure-free portion of RNA. In addition, a structure-free portion can be estimated by a method of predicting a structure formed in a single-stranded RNA molecule using a computer. As a method for experimentally estimating a higher-order structure-free portion, an experiment of a cleavage reaction using a single-stranded oligo DNA having a random sequence of 10 bases or more in length and an enzyme RNaseH was carried out, and a fragment was obtained. (J. Bio. Chem. 272, 626-).
638, 1997, et al.).

[0007] However, although various estimation methods are known, a method involving analysis requires a single-stranded RNA of several tens bases in length.
However, it is not realistic to apply these methods to an RNA sample such as a virus having a length of several hundreds of bases even if the analysis is performed. The method for estimating the higher-order structure free portion using a computer basically includes a single-strand R
This predicts the planar (secondary) structure of NA. The predicted secondary structure-free portion is a three-dimensional (tertiary) in which RNA molecules are actually formed in a liquid phase. The structure is often not free because of the sequence-specific binding of complementary parts that are spatially close but spaced apart on the secondary structure. As a matter of fact, a single-stranded oligo DN having a sequence complementary to a secondary structure free portion estimated by a computer
It is often possible that A does not bind strongly when reacted.

On the other hand, oligo DN having a random sequence
A and the method using RNaseH, which is an enzyme that cleaves only RNA in the heteronuclear double-stranded portion of RNA
Since it reflects the three-dimensional structure of single-stranded RNA in the solution in which aseH acts, it is considered to be more accurate than a method of predicting this by a computer. However, according to conventional reports, it is essential to use a nucleic acid library having a random sequence of at least 10 bases in length,
Cleavage reactions had to be performed on over one million or more very diverse sequences and the results analyzed. Therefore, a high-concentration library solution was required. In the oligo DNA containing a random sequence to be used, RNA
Can form a double-stranded chain at a plurality of sites, so that many sites are likely to be cleaved by enzymatic action, but as a result, the two were strongly bound and were cleaved, or both were weakly bound. There was also a problem that it was practically impossible to determine whether or not the connection had been cut. Furthermore, in general, the longer the chain length, the greater the bias of randomness. If the chain length is long, cleavage of the higher-order structure-free portion may occur even if the binding to a specific RNA is relatively weak. , The result is a large number of truncated bands, and specific R
It becomes difficult to select oligo DNA that truly binds NA. Therefore, this method is useful for verifying whether or not the random sequence binds to a specific RNA, but is suitable for producing a single-stranded DNA that binds strongly. Absent.

As described above, in practice, a higher-order structure-free portion of RNA is estimated, and oligo DNA having a sequence complementary to that portion is produced and used, or a sequence containing a sequence complementary to that portion is contained. At present, a plurality of types of oligo DNAs are prepared, and among them, oligo DNAs that strongly bind to a specific RNA are screened and used.

Therefore, an object of the present invention is to cleave RNA,
Provided is a method for easily producing single-stranded oligo DNA useful as a reagent for genetic diagnosis for performing operations such as NA amplification and RNA detection, and as a drug for inhibiting reverse transcription and translation of RNA. Is to do. Another object of the present invention is to provide a method for producing H
An object of the present invention is to provide a single-stranded oligo DNA that strongly binds to CV RNA.

[0011]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, completed the present invention. That is, the present invention provides a single-stranded oligo DN having a high binding reactivity to HCV RNA, which is complementary to nucleotides 101 to 120 of HCV RNA, SEQ ID NO: 1.
A, HCV having high binding reactivity to HCV RNA
Base number 97 of HCV RNA, which has high binding reactivity to single-stranded oligo DNA of SEQ ID NO: 2 and HCV RNA complementary to nucleotides 99 to 118 of RNA
The single-stranded oligo DNA of SEQ ID NO: 3 complementary to the 116th position has high binding reactivity to HCV RNA,
HCV RN, a single-stranded oligo DNA of SEQ ID NO: 4 complementary to nucleotides 95 to 114 of HCV RNA
A single-stranded oligo DNA of SEQ ID NO: 5 having a high binding reactivity to HCV RNA, which is complementary to nucleotides 248 to 267 of HCV RNA, or a nucleotide 238 of HCV RNA having high binding reactivity to HCV RNA Single-stranded oligo DN complementary to SEQ ID NO: 257
A.

[0012] The present invention also provides a nucleotide sequence, which is effective as a sense primer or a promoter primer, having the same nucleotide sequence as nucleotides 113 to 137 of HCV RNA.
A single-stranded oligo DNA, a sense primer or a promoter primer, which is effective as a sense primer or a promoter primer in SEQ ID NO: 8 identical to the nucleotide numbers 115 to 139 of HCV RNA, or a sense primer or a promoter primer Is a single-stranded oligo DNA of SEQ ID NO: 9 which is the same as nucleotides 111 to 135 of HCV RNA.

The present invention further provides a simple method for producing a single-stranded oligo DNA that is complementary to a higher-order structure-free portion in a single-stranded RNA and, as a result, strongly binds to the RNA. And a single-stranded oligo DNA containing a random sequence having a length of 6 to 9 bases and a single-stranded RNA estimated from an experiment of a cleavage reaction using an enzyme that cuts only the RNA of the RNA-DNA heteronuclear double-stranded portion. Prepare a combination of several oligo DNAs of 20 to 25 bases in length containing a sequence complementary to the cleavage region or a complementary sequence near the estimated cleavage region,
Again, this is a method for producing a single-stranded oligo DNA that binds to a single-stranded RNA selected from a screening experiment of a cleavage reaction using an enzyme that cuts only the RNA of the heteronuclear double-stranded portion of RNA-DNA.

The present invention also relates to a part of the single-stranded oligo DNA of any one of SEQ ID NOs: 1 to 6, the entirety of the single-stranded oligo DNA of any of SEQ ID NOs: 1 to 6, Single-stranded RNA cleavage, single-stranded RNA amplification, single-stranded RNA, which is a part of the single-stranded oligo DNA, or the whole of the single-stranded oligo DNA selected by the production method or a derivative of the single-stranded oligo DNA. It is a reagent for reverse transcription of strand RNA, translation inhibition of single strand RNA, or measurement of single strand RNA.

The present invention also relates to a part of the single-stranded oligo DNA of any one of SEQ ID NOs: 1 to 6, the entirety of the single-stranded oligo DNA of any of SEQ ID NOs: 1 to 6, It is a single-stranded RNA cleavage reagent in which an RNA cleavage motif is linked to a part of a single-stranded oligo DNA, all of the single-stranded oligo DNA selected by the above-mentioned production method, or a derivative of the single-stranded oligo DNA.

The present invention relates to a part of the single-stranded oligo DNA of any one of SEQ ID NOs: 1 to 6, the entirety of the single-stranded oligo DNA of any of SEQ ID NOs: 1 to 6, Single-stranded RN, which is a single-stranded oligo DNA complementary to a part of the single-stranded oligo DNA or the entire single-stranded oligo DNA selected by the above-mentioned production method or a derivative thereof.
It is a reagent for a sense primer or a promoter primer for A.

Hereinafter, the present invention will be described in detail. In the present specification, the nucleotide number of HCV RNA is based on the literature by Kato et al. (Proc. Natl. Acad. Sci. USA).
(1990) 87, 9524-9528) according to the nucleotide number of the HCV cDNA.

1. Single-stranded oligo DNAs having high binding reactivity to HCV RNA These single-stranded oligo DNAs are produced by the production method of the present invention described below, and are complementary to the higher-order structure-free portions in the HCV RNA. It is what combines.

The single-stranded oligo DNA of SEQ ID NO: 1 is HC
HCV RNA with high binding reactivity to V RNA
Are complementary to the base numbers 101 to 120. The single-stranded oligo DNA of SEQ ID NO: 2 is HCVR
It has high binding reactivity to NA and is complementary to base numbers 99 to 118 of HCV RNA. The single-stranded oligo DNA of SEQ ID NO: 3 has high binding reactivity to HCV RNA,
It is complementary from the 116th to the 116th. SEQ ID NO: 4
The single-stranded oligo DNA having a high binding reactivity to HCV RNA is complementary to nucleotides 95 to 114 of HCV RNA. The single-stranded oligo DNA of SEQ ID NO: 5 has a high binding reactivity to HCV RNA, and has nucleotides 248 to 267 of HCV RNA.
The second one is complementary. The single-stranded oligo DNA of SEQ ID NO: 6 has high binding reactivity to HCV RNA,
The second one is complementary.

The single-stranded oligo DNA of the present invention having high binding reactivity to HCV RNA is itself useful as a reagent for cleavage, amplification, reverse transcription, translation inhibition or measurement of single-stranded RNA. That is, in order to cleave HCV single-stranded RNA, an enzyme such as RNaseH that forms an RNA-DNA heteronuclear duplex with the RNA and cleaves only the RNA of the heteronuclear duplex is used. It should just work. RNa
More specifically, as the seH, reverse transcriptase (AMV-RTase) derived from avian myoblastoma virus (AMV)
Can be exemplified. Alternatively, an RNA cleavage motif that non-specifically cleaves RNA may be bound.

In order to amplify HCV RNA, the single-stranded oligo DNA of the present invention may be used in the aforementioned RNA amplification operation.
Can be exemplified as a primer for reverse transcription of a specific RNA to produce complementary DNA. Here, when performing reverse transcription of RNA in the aforementioned RNA amplification operation, if a promoter / primer in which a promoter for RNA synthesis is bound to the single-stranded oligo DNA of the present invention is used, RNA complementary to HCV RNA can be used. Can also be amplified. Furthermore, prior to the amplification operation as described above, it can be used for cutting the 5 'side of HCV RNA at an arbitrary site. In addition to the purpose of amplification, it can also be used for simply reverse transcription of HCV RNA. Furthermore, once reverse transcribed HC
The DNA having a sequence complementary to VRNA can be double-stranded and then used as a primer in PCR.

In order to inhibit translation, the single-stranded oligo DNA of the present invention may be used as an antisense agent for HCV RNA.

For the measurement reagent, the single-stranded oligo DNA of the present invention may be used as a probe for HCV RNA.

In the above, the single-stranded oligo DN of the present invention
A may use the entire sequence or a part of the sequence. When all are used, for example, a sequence containing the entire sequence of the single-stranded oligo DNA of the present invention can also be used. When using a part, HCV
In order to ensure sufficient RNA specificity and binding reactivity, it is particularly preferable to extract and use a continuous portion of 5 bases or more from any of the sequences described in SEQ ID NOS: 1 to 6. Still further, the single-stranded oligo D of the present invention
Even a derivative of NA can be used as described above. For example, a derivative in which a phosphorus atom contained in a DNA molecule is replaced with a sulfur atom, or a part of deoxyribose in a DNA molecule is replaced with ribose to achieve an effect of improving resistance to degradation in blood. First, for example, binding of a labeling substance such as an enzyme, a light-absorbing substance, a luminescent substance, a fluorescent substance, a radioisotope, and an intercalating fluorescent dye for use as a binding or probe of the RNA cleavage motif as described above, It also includes binding a linker having a suitable functional group to bind such a labeling substance.

2. Single-stranded oligo DNA having the same sequence as that of HCV RNA These single-stranded oligo DNAs are used, for example, as sense primers for HCV RNA, that is, HCV RNA.
As a primer for amplifying DNA or RNA containing a sequence complementary to the above, or by binding to a known promoter portion (sequence) for RNA synthesis, for example, It is particularly effective as a reagent called a promoter / primer used in such a case.

The single-stranded oligo DNA of SEQ ID NO: 7 is HC
It is the same as nucleotide numbers 113 to 137 of V RNA. The single-stranded oligo DNA of SEQ ID NO: 8 comprises HCV
It is the same as base numbers 115 to 139 of RNA. The single-stranded oligo DNA of SEQ ID NO: 9 is HCV RN
It is the same as base number 111 to 135 of A.

The single-stranded oligo DNA having the same sequence as the HCV RNA is used for HCV RN in addition to being used as a sense primer or a promoter primer.
It can also be used as a reagent for cleaving single-stranded RNA containing a sequence complementary to A. That is, RNA-DN between the RNA
A It is sufficient to form an heteronuclear double-strand and apply an enzyme such as RNase H that cuts only the RNA of the heteronuclear double-strand. In addition, R, which nonspecifically cleaves RNA,
An NA cleavage motif may be linked. Moreover,
It can also be used as a probe for DNA or RNA containing a sequence complementary to HCV RNA.

In the above, the single-stranded oligo DNA having the same sequence as that of HCV RNA may use the entire sequence or a part of the sequence. When all are used, for example, a sequence containing the entire sequence of the single-stranded oligo DNA of the present invention can also be used. When a part is used, a continuous portion of 5 bases or more is extracted from any of the sequences described in SEQ ID NOs: 7 to 9 in order to sufficiently ensure specificity and binding reactivity with HCV RNA. It is particularly preferred to use them. Still further, its derivatives can be used as described above. For example, a derivative in which a phosphorus atom contained in a DNA molecule is substituted with a sulfur atom, and a derivative in which a part of deoxyribose in a DNA molecule is replaced with ribose to achieve an effect of improving degradation resistance in a living body, such as , R as described above
In order to bind the NA cleavage motif or to use it as a probe, for example, to bind a labeling substance such as an enzyme, a light-absorbing substance, a luminescent substance, a fluorescent substance, a radioisotope, an intercalating fluorescent dye, It also includes attaching a linker having an appropriate functional group for attachment.

3. A simple method for producing a single-stranded oligo DNA that strongly binds to a single-stranded RNA The method for producing a single-stranded oligo DNA that strongly binds to a single-stranded RNA provided by the present invention comprises the steps of: Can be used when the primary structure is known but its primary structure is not known. For example, RNA such as HCV or HIV
(Of course, since the structure of RNA is obvious from the primary structure of cDNA, the case where the primary structure of cDNA is known) can be suitably used.

In the production method of the present invention, first, a single-stranded oligo DNA containing a random sequence having a length of about 6 bases is prepared. When the base length is 5 bases or less, it binds to an unspecified sequence portion in the RNA, and the cleavage of the RNA occurs at a very large number of sites. Because there is a possibility that the problem of
It is particularly preferable to use one having a base length. A single-stranded oligo DNA containing a random sequence is a probe for searching for a site in a single-stranded RNA where a higher-order structure is free, that is, does not form a higher-order structure, under a low temperature condition near physiological temperature. Used as Since the base of the DNA is any one of the four types of adenine, guanine, cytosine, and thymine, when a 6-base long sequence is used, 4096 types of sequences can be prepared. It is not necessary to prepare all such types of sequences. For example, if all types of single-stranded oligo DNA are prepared and made into a library, the production method of the present invention can be carried out for any single-stranded RNA. You will get. To prepare these, for example, DNA
Synthesis may be performed using an automatic synthesizer, or a commercially available random arrangement may be used.

On the other hand, the primary structure of RNA is clear,
It is also possible to use a continuous arbitrary base sequence portion in the RNA as a specific sequence and use a single-stranded oligo DNA complementary to the sequence as an oligo DNA having a random sequence in the production method of the present invention. . However, since the oligo DNA prepared in this manner is not versatile,
Each time the production method of the present invention is applied to a different RNA, it is necessary to prepare a new single-stranded oligo DNA. Moreover, as will be described later, in performing the production method of the present invention, although it seems to be complementary from the viewpoint of the primary structure, it has sufficient binding property for the higher-order structure of RNA. If, for example, it is found that the sequence contains a large number of bases not involved in the formation of a duplex with RNA in the sequence, it is necessary to carry out synthesis again. Furthermore, it is not possible to know a plurality of high-order structure-free sites in single-stranded RNA at a time. Therefore, in the production method of the present invention, it is preferable to use the former single-stranded oligo DNA containing a random sequence.

The thus prepared single-stranded oligo DNA containing a random sequence is hybridized with a single-stranded RNA whose 5 ′ end or 3 ′ end has been modified with a detectable label to obtain an RNA-DNA heteronuclear DNA. After heavy chain formation, RNA
-An enzyme that cuts only the RNA in the heteronuclear double-stranded portion of DNA is acted on. As the enzyme, an enzyme generally known to have RNaseH activity may be used. RN
Examples of the enzyme having aseH activity include reverse transcriptase (AMV-R) derived from avian myoblastoma virus (AMV).
Tase). Single-stranded RNA
Can be performed by a known method such as, for example, isotopically labeling the 5 ′ end with [γ- ³² P] ATP.

Single-stranded oligo DNA containing random sequence
Is used in a large excess with respect to RNA, but its preferred concentration is about 0.5 mM to 5 mM. This is because, when the primary structure of the single-stranded RNA is clear by using this preferred concentration range, it is possible to evaluate all types of single-stranded oligo DNA at once. In addition, the cleavage site due to the enzymatic reaction, that is, the higher-order structure in the single-stranded RNA is free, and it is expected that a number of sites presumed to have formed a double-stranded with the single-stranded oligo DNA are expected to appear, The cleavage reaction by the enzyme is carried out with an appropriate enzyme concentration and reaction time for the analysis described later. Here, the single-stranded R
When the primary structure of NA is unknown, an enzyme is allowed to act on each type of single-stranded oligo DNA, and the results are analyzed.

Next, the product of the cleavage reaction by the enzyme, that is, the cleaved RNA fragment is analyzed by a molecular weight analysis method such as denaturing polyacrylamide gel electrophoresis which can estimate the base length of the RNA fragment. For example, when performing electrophoresis, the gel used is preferably one having a long migration distance of several tens of cm, but may be about 15 cm for easier operation. The analysis method will be described taking electrophoresis as an example.
The product of the enzymatic cleavage reaction, ie, the RNA fragment, moves on the gel by a distance corresponding to its base length, and appears as a band. Therefore, if an RNA having a known base length is used as a marker and subjected to electrophoresis under the same conditions, and a calibration curve is created based on the relationship between the base length and the moving distance, the base distance of the actual cleavage product can be calculated from the actual moving distance of the cleavage product Estimate the length, and then single-stranded RN
By detecting the label at the 5 ′ or 3 ′ end of A, it becomes possible to estimate the site of cleavage in the single-stranded RNA.

When a suitable cleavage product cannot be obtained by cleavage of RNA by an enzyme, the length of single-stranded oligo DNA containing a random sequence is increased by one base at a time to increase the binding reactivity. A single-stranded oligo DNA can be produced. Those having a length of 10 bases or more are difficult to obtain a single-stranded oligo DNA having a high binding reactivity due to an increase in the ability to form a higher-order structure itself and an increase in randomness. It is preferable to use those having a length of about 9 bases, preferably up to about 8 bases.

Subsequently, a sequence complementary to the sequence in the vicinity of the estimated cleavage site was prepared, and the following procedure was performed as a candidate for a single-stranded oligo DNA having a binding reactivity with a single-stranded RNA free of higher-order structure. In order to prepare a single-stranded oligo DNA of about 3 to 10 as the number of candidates, the enzyme concentration and reaction time when RNA is cleaved by the enzyme are appropriately set, and 3 It is preferable to set such that bands derived from about 10 to about 10 cleavage reaction products appear. Of course, it is preferable that the number of candidates is large, but in order to actually prepare the candidates and carry out the following operation quickly and easily, the number of candidates can be set to about 10 as an example. If the enzyme concentration and the like are not particularly limited, the number of appearing bands, that is, the number of RNA fragments as degradation products increases, and there is a fear that selection of candidates becomes difficult. The selection can also be made based on degree). As described above, when single-stranded oligo DNAs whose single-stranded RNA sequences are unknown and whose random sequences are analyzed for the enzyme-cleaving action one by one, all operations are performed at the same enzyme concentration and enzyme reaction. It is preferable that the single-stranded oligo DNA itself, which gives rise to a dark band after being controlled so as to be a time, is a candidate.

The preparation of the candidate single-stranded oligo DNA can be performed, for example, as follows. First, its chain length is determined. The chain length is at least the length of the single-stranded oligo DNA containing the random sequence used earlier.
There is no particular limitation as long as it is at least about bases, but single-stranded RNA
The primary structure is clear, not only the binding reactivity,
When trying to prepare one with excellent binding specificity,
If necessary, the chain length can be further increased based on the known primary structure. In this case, usually 20 to 2
When the number of bases is about 5 bases, a single-stranded oligo DNA having excellent binding specificity can be produced, and the possibility of including a site free of a higher-order structure can be increased. Specifically, after determining the chain length, as a first candidate, a single-stranded oligo DNA complementary to the presumed single-stranded RNA, having a determined chain length centered on the cleavage site in the single-stranded RNA, It is preferable to use As a second candidate, the R having the determined chain length centered on the position shifted to the 5 ′ end by half the length of the determined chain length from the estimated cleavage site,
It is preferable to use a single-stranded oligo DNA complementary to NA. As a third candidate, a single strand complementary to the RNA having a determined chain length centered on a position shifted to the 3 ′ end by half the length of the determined chain length from the estimated cleavage site. It is preferable to use oligo DNA.

[0038] Three kinds of single-stranded oligo DNAs having these specific sequences are prepared, and the enzyme is again acted on as described above.
Single-stranded RNA by denaturing polyacrylamide gel electrophoresis etc.
Detect the migration distance of the band derived from the cleavage reaction product of
Single-stranded oligo DNA used as a candidate is single-stranded RNA
It is checked whether or not a double chain is formed. At this stage, it is not necessary to label the ends of the single-stranded RNA, and when performing analysis by electrophoresis, it is sufficient that the gel used is about 10 cm. If a band derived from the cleavage reaction product is confirmed, the band intensity is compared between single-stranded oligo DNAs used as candidates, and the band with the stronger band appears as one with higher binding reactivity. It can be bound to strand RNA. In the case where there is no single-stranded oligo DNA prepared as a candidate that produces a cleavage reaction product or the like, the above-described operation such as searching for a candidate again is repeated, thereby finally Thus, a single-stranded oligo DNA having high binding reactivity with single-stranded RNA can be produced.

The single-stranded oligo DNA having a high binding reactivity to HCV RNA and other RNAs, which is complementary to a specific sequence in these RNAs, produced by the above-described production method of the present invention can be used in the present invention. SEQ ID NOS: 1 to 6 provided by
In the same manner as described for the single-stranded oligo DNA, it is useful as a reagent for cleavage, amplification, reverse transcription, translation inhibition or measurement of single-stranded RNA. Here, the single-stranded oligo DNA produced by the production method of the present invention may use the entire sequence or a part of the sequence.
When using a part, it is preferable to use a continuous part of 5 bases or more. Further, for example, the single-stranded oligo DNA produced by the production method of the present invention can be derivatized. Derivatization includes substituting a phosphorus atom in a DNA molecule with a sulfur atom, substituting some deoxyribose in a DNA molecule with ribose, and binding an RNA cleavage motif or a labeling substance. .

In addition, a single-stranded oligo DNA which has high binding reactivity to HCV RNA or other RNA and which is complementary to a specific sequence in these RNAs produced by the production method of the present invention. That is, a single-stranded oligo identical to a specific sequence portion in the single-stranded RNA also has a similar sense to the RNA as described with respect to the single-stranded oligo DNA of SEQ ID NOS: 7 to 9 provided by the present invention. -Useful as a primer or promoter / primer. Also in this case, the entire array may be used, or a part of the array may be used. When using a part, it is preferable to use a continuous part of 5 bases or more. Such sequences can also be derivatized. Derivatization includes substituting a phosphorus atom in a DNA molecule with a sulfur atom, substituting some deoxyribose in a DNA molecule with ribose, and binding an RNA cleavage motif or a labeling substance. .

[0041]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

Example 1 By the following operation, a cleavage site of HCV RNA by a single-stranded oligo DNA having a length of 6 bases having a random sequence was estimated.

1. 1.5 μl of a reaction solution having the following composition
Dispense into 5 ml tubes.

200 mM Tris / HCl buffer (pH 7.5) 200 mM Potassium chloride 100 mM Magnesium chloride 1 mM EDTA 1 mM Dithiothreitol (DTT) 2.5 HCV RNA standard 10 ¹¹ whose isotope-labeled with [γ- ³² P] ATP 7.5 μl copies / μl was added to the tube. The HCV RNA standard is a 1549-base HCV2 type having nucleotide numbers from 1 to 1487, including a vector-derived sequence having a length of 62 bases at the 5 'end.

3. A single-stranded oligo DNA having a length of 6 bases containing a random sequence (manufactured by Takara Shuzo Co., Ltd., 1.5 mM) was added to 5 μl
1 was added.

4. RNaseH (manufactured by Takara Shuzo Co., Ltd .;
1U / μl) was added.

The reaction was carried out at 37 ° C. for 5 to 15 minutes.

6 7.5 μl of the reaction solution was taken and subjected to 5% polyacrylamide gel electrophoresis (15 cm gel) containing 7 M urea. At the same time, as a marker, a commercially available marker (manufactured by Novagen, trade name Perfect)
RNA Marker) with [γ- ³² P] AT
The ones labeled with P for isotope were provided.

7. After electrophoresis, the gel was dried by heating at 60 ° C. for 2 hours under reduced pressure using an aspirator.

8. The band was confirmed by exposing to an X-ray film.

FIG. 1 shows the state of the X-ray film. According to FIG. 1, between the 400-base marker and the 300-base marker and between the 200-base marker and the 100-base marker, the strongest and largest amount of a clear single cleavage reaction product is derived. A band was observed. A calibration curve was created from the distance traveled by the markers and their base lengths, and the base lengths of the cleavage products that produced each band were detected. As a result, these were estimated to be 309 and 162 base length products, respectively.
According to this example, at least in the HCV RNA,
It was presumed that there were cleavage sites near the 310th and 160th positions from the 5 'end, in other words, the vicinity of these cleavage sites was a site where the higher-order structure of HCV RNA was free.

Example 2 By the following operation, HC in the presence of RNaseH
The sequence of the single stranded oligo DNA that strongly binds to V single stranded RNA and results in cleavage was determined.

1. 5.2 μl of a reaction solution having the following composition
Dispense into 5 ml tubes.

1. 200 mM Tris-HCl (pH 7.5) 200 mM Potassium chloride 100 mM Magnesium chloride 1 mM EDTA 1 mM DTT 3.8 μl of HCV RNA standard 2 × 10 ¹¹ copies / μl was added to the tube.

3. HCV RNA cleavage site estimated in Example 1 and 10 bases from its position, 5 'end or 3'
HCV RNA, centered at three positions shifted to the ends
A total of six types of single-stranded oligo DNA having a length of 20 bases complementary to each other were synthesized, three types for each cleavage site (H
The portions complementary to these oligo DNAs in CV RNA are 248 to 267, 238 to 257, 228 to 247, 101 to 120
Th, 91st to 110th and 81st to 10th
0th). These synthetic DNAs (0.25 μM)
Were added to the tube in an amount of 5 μl each, and a solution containing no single-stranded oligo DNA and a 6-base-length single-stranded oligo DNA having a random sequence were used as control reactions.
A tube to which 5 μl (manufactured by Takara Shuzo Co., Ltd., 1.5 mM) was added was also subjected to the following operation.

4. RNaseH (manufactured by Takara Shuzo Co., Ltd .;
1 U / μl) was added to the tube in 1 μl portions.

The reaction was carried out at 37 ° C. for 15 minutes.

5. An aliquot of 7.5 μl of the reaction solution was subjected to 5% polyacrylamide gel electrophoresis (10 cm gel) containing 7 M urea.

6. After the electrophoresis, a staining solution obtained by diluting the gel 10000 times (trade name: SYBR Green, manufactured by Takara Shuzo Co., Ltd.)
II) for 30 minutes.

7. The state of staining was confirmed on a transilluminator.

FIG. 2 shows how the gel is stained. From FIG. 2, a band derived from the cleavage reaction product was observed in four of the six bases of DNA having a random sequence and the prepared six types of single-stranded oligo DNA. The observed band density (degree of blackening) varied, but HCV R
NA 248th to 267th, 238th to 25th
The one complementary to the 7th and 101st to 120th positions is a single-stranded oligo D containing a random sequence of 6 bases in length.
A darker band appeared than the band that appeared in NA. The base length of the cleavage reaction product that caused these bands to appear (if two or more bands were observed, the base length of the cleavage reaction product that caused the band with a higher degree of blackening to appear) It was found that the length of the RNA fragment generated when the RNA was cleaved by the binding of the strand oligo DNA to the estimated site in the HCV RNA was equivalent to the length from the 5 ′ end.
HCV estimated from this example using RNaseH and a single-stranded oligo DNA having a length of 6 bases including a random sequence
It was confirmed that a single-stranded oligo DNA having a length of 20 bases, which can be more strongly bonded and cleaved than the single-stranded oligo DNA containing the random sequence in the vicinity of the RNA cleavage site, can be produced. In addition, the portion complementary to the single-stranded oligo DNA in HCV RNA, in which a dark band appeared in this example, was presumed to be a high-order structure-free site. It was consistent with a structure-free site.

Example 3 The sequence of the single-stranded oligo DNA probe in which a dark band appeared in Example 2 was
RNA cleavage was observed. It was also confirmed that reverse transcriptase AMV-RTase was useful as a cleavage enzyme.

1. 0.75 μl of a reaction solution having the following composition was dispensed into a 1.5 ml tube.

400 mM Tris-acetic acid (pH 8.1) 1250 mM potassium acetate 140 mM magnesium acetate 100 mM DTT 1 mg / ml bovine serum albumin (BSA) 20 U / μl RNase inhibitor HCV RNA standard 8.1 × 10 ¹⁰ copies / μl
Was added to the tube in an amount of 3.5 μl.

3. In addition to the sequence complementary to nucleotides 101 to 120 of the HCV RNA determined in Example 2, a position shifted from the 5 'end by 2, 4, or 6 bases in the HCV RNA, ie, 99 118th from 97th, 116th from 97th, 1st from 95th
A single-stranded oligo DNA complementary to the 14th sequence was synthesized. Each of these four kinds of synthetic DNAs (2 μM) or single-stranded oligo DNAs (2 μM) used in Example 2 which are complementary to the sequence from position 81 to position 100 or from position 91 to position 110 in HCV RNA were used. 0.5 μl was added. Further, a tube to which 2.5 μl of a solution containing no DNA was added as a control reaction was also subjected to the following operation.

4. AMV-RTase (Takara Shuzo Co., Ltd.)
0.75 μl).

The reaction was carried out at 37 ° C. for 20 minutes.

6. Take the whole amount of the reaction solution and add 5M containing 7M urea.
% Polyacrylamide gel electrophoresis (10 cm gel)
Was served.

7. After the electrophoresis, a staining solution obtained by diluting the gel 10000 times (trade name: SYBR Green, manufactured by Takara Shuzo Co., Ltd.)
II) for 30 minutes.

8. The state of staining was observed on a transilluminator.

FIG. 3 shows how the gel is stained. From FIG. 3, bands derived from the cleavage action were observed in many single-stranded oligo DNAs. Although the density (degree of blackening) of these bands was variable, base number 10 of HCV RNA was
When a sequence complementary to the 1st to 120th sequence and a sequence complementary to the sequence shifted to the 5 'side by 10 or 20 bases (81th to 100th, 91 to 110th) are used The band intensity was similar to that observed in Example 2. Also from the 99th to 118
The band densities were almost the same when using single-stranded oligo DNAs complementary to the ninth, 97th to 116th, and 95th to 114th sequences. Furthermore, based on the movement distance of the band, the HCV complementary to the single-stranded oligo DNA was used.
It was confirmed that the base length of the RNA fragment as the cleavage reaction product was shortened by the extent that the sequence in the RNA was shifted to the 5 'side.

According to this embodiment, AMV-RTase is RN
It was confirmed that it can be used as a cleavage reagent having the same cleavage activity as aseH. Further, based on the single-stranded oligo DNA in which a dark band appeared in Example 2, complementary H
It was confirmed that even if the sequence in the CV RNA was shifted 2 to 6 bases to the 5 'side, the intensity of the appearing band was not significantly changed. This is based on the three single-stranded oligo DNAs prepared in Example 2, which are complementary to the HCV R
This shows that the modification of shifting the sequence in NA to the 5 ′ side or 3 ′ side is effective in producing a single-stranded oligo DNA having high binding reactivity with HCV RNA.

Example 4 A motif sequence of DNA capable of cleaving RNA was bound to a single-stranded oligo DNA by the following procedure,
RNA was cleaved without using an enzyme such as NaseH.

1. 1.5 μl of a reaction solution having the following composition
Dispense into 5 ml tubes.

1. 500 mM Tris-HCl (pH 7.5) 10 mM DTT 10 U / μl RNase inhibitor 1.5 μl of HCV RNA standard 4.7 × 10 ¹¹ copies / μl was added to the tube.

3. A single-stranded oligo DNA complementary to the sequence from base number 99 to base 106 in HCV RNA,
Motif sequence of DNA that cleaves RNA (Proc. N
atl. Acad. Sci. USA, 94, 4262-
4266 (1997)) and a single-stranded oligo DNA complementary to the sequence of base numbers 108 to 115 in the HCV RNA, which was connected in this order from the 5 ′ end, was prepared. 1.5 μl of each of the synthetic DNAs (2 μM) containing a concentration of magnesium chloride was added. The base sequence of the synthesized DNA is SEQ ID NO: 10.
It is as follows.

4. Deionized water was added to a total volume of 15 μl.

5. The reaction was carried out at 37 ° C. for 120 minutes.

6. Take the whole amount of the reaction solution and add 5M containing 7M urea.
% Polyacrylamide gel electrophoresis (10 cm gel)
Was served.

7. After the electrophoresis, a staining solution obtained by diluting the gel 10000 times (trade name: SYBR Green, manufactured by Takara Shuzo Co., Ltd.)
II) for 30 minutes.

8. The state of staining was confirmed on a transilluminator.

FIG. 4 shows how the gel is stained. From FIG. 4, it can be seen that, since the cleavage activity of the DNA motif that cleaves RNA is of the type requiring magnesium ion, when the concentration of magnesium chloride was changed to 2, 70 or 200 mM,
RN in the presence of mM or more magnesium ions
The same band as that observed in the presence of AseH or AMV-RTase was observed. The band is HCV RN
It appeared at the same position as the band that appeared when a single-stranded oligo DNA complementary to the sequence of base numbers 101 to 120 in A was used.

From this example, the single-stranded oligo DN used
A binds to a conformation-free site of HCV RNA,
It can be seen that the cleavage motif bound to the DNA acts to cut HCV RNA at the site.

Example 5 By the following operation, HCV RNA was amplified using the single-stranded oligo DNA of the present invention as a primer.

1. 18 μl of a reaction solution having the following composition was dispensed into 12 PCR tubes.

68 mM Tris-acetic acid (pH 8.1) 210 mM potassium acetate 23 mM magnesium acetate 27% sorbitol 5.1% DMSO 17 mM DTT 170 μg / ml BSA 3.4 U / μl RNase inhibitor 2.3 U / μl AMV-RTase 80 μl of mineral oil was overlaid.

3. In Example 3, a single-stranded oligo DNA complementary to the sequence of nucleotide numbers 95 to 114 in HCV RNA, which was confirmed to strongly bind to HCV RNA (in the following amplification, 3 '
In order to prevent the occurrence of an extension reaction to the side, the 3 ′ end modified with an amino group (0.1 μM) was added to 0.6 μM.
1 was added.

4. HCV RNA standard 3.8 as positive
× 10 ⁵ copies / μl were added in duplicate, and 4.4 μl of TE buffer was added in series as negative.

5. The reaction was carried out at 37 ° C. for 120 minutes.

6. Heated at 50 ° C. for 5 minutes.

7. 5.5 μl of a reaction solution having the following composition and different promoter / primers was added.

31 U / μl SP6-RNA polymerase 5.5 mM each dATP, dGTP, dCTP, dTT
P 1.1 μM antisense primer (HCV RN
A single-stranded oligo DNA complementary to the sequence from base Nos. 248 to 267 of A, SEQ ID No. 5) 1.1 μM promoter / primer Sequence identical to base sequence 113 to 137 of HCV RNA (SEQ ID No. 5) 7), a sequence containing the same sequence (SEQ ID NO: 8) as the sequence from nucleotide positions 115 to 139 of HCV RNA, a sequence containing SEQ ID NO: 12 from sequence 117 to 141 of HCV RNA, and One containing the same sequence and one containing the same sequence (SEQ ID NO: 9) as the sequence from base number 111 to base 135 of HCV RNA was reacted at SEQ ID NO: 14.8.50 ° C. for 10 minutes.

9. 20 mM ATP, GTP, CTP
And 1.5 ml of UTP.

10. The reaction was carried out at 50 ° C. for 2 hours.

11. 5 μl of the reaction solution was taken and subjected to 4% agarose gel electrophoresis (5 cm gel).

12. After the electrophoresis, a staining solution obtained by diluting the gel 10000 times (trade name: SYBR Green, manufactured by Takara Shuzo Co., Ltd.)
nII) for 30 minutes.

13. The state of staining was observed on a transilluminator.

FIG. 5 shows the state of staining of the gel. Regardless of which promoter primer was used, no band derived from the amplified HCV RNA appeared in the negative case. On the other hand, for positive samples, HCV
A band derived from RNA appeared.

The band that appeared in the positive sample of this example was the single-stranded oligo DNA of the present invention in which the 3 ′ end was modified with an amino group for pre-cleaving HCV RNA at 37 ° C., and a constant temperature of 50 ° C. HCVR under temperature conditions
This is an amplification product generated by using the single-stranded oligo DNA (promoter / primer) of the present invention for amplifying NA. As described above, by appropriately combining the single-stranded oligo DNA of the present invention, R
It can be seen that NA amplification can be performed.

[0100]

As is clear from the above description, according to the present invention, any single-stranded RN including HCV RNA can be used.
A single stranded oligo DNA is provided that is highly reactive with A and complementary to the RNA. The oligo DNA is H
The higher-order structure in single-stranded RNA such as CV RNA is complementary to a free site, and by using this, RNA can be easily amplified.

In the present invention, the single-stranded oligo D
It also provides a method for producing NA. The production method preferably comprises converting a large number of single-stranded oligo DNAs containing random sequences into any single-stranded RNA in the presence of an enzyme which cleaves only RNA of a heteronuclear double-stranded RNA.
To generate a cleavage reaction, and then analyze the cleavage reaction product to estimate a site free of a higher-order structure. Here, by making the length of the random sequence preferably about 6 bases, it is possible to evaluate at once the oligo DNAs containing all the random sequences that can be produced. Thereafter, if, for example, the single-stranded oligo DNA is to be given binding specificity for RNA, the estimated R
By producing a complementary sequence having a length of 20 to 25 bases based on the higher-order structure-free site in NA and performing the same evaluation, it was more strongly bound to single-stranded RNA, and the binding specificity was improved. It becomes possible to produce single-stranded oligo DNA.

As described above, according to the present invention, primers / probes for RNA cleavage, amplification and detection or RNA
And a single-stranded oligo DNA that strongly binds to a single-stranded RNA, which can be an important factor in the design of a reverse transcription and translation inhibitor, can be provided quickly and easily.

[0103]

[Sequence List] SEQUENCE LISTING <110> Tosoh Corporation <120> Oligo DNA that binds strongly to HCV RNA and a simple method for producing such DNA <130> PA210-9970 <150> JP 10-329874 <151> 1998- 11-19 <160> 14 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sequence complementary to base numbers 101 to 120 of HCV RNA <400> 1 ccgggagggg gggtcctgga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sequence complementary to nucleotides 99 to 118 of HCV RNA <400> 2 gggagggggg gtcctggagg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sequence complementary to nucleotides 97 to 116 of HCV RNA <400> 3 gagggggggt cctggaggct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sequence complementary to nucleotides 95 to 114 of HCV RNA <400> 4 gggggggtcc tgga ggctgc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sequence complementary to base numbers 248 to 267 of HCV RNA <400> 5 gcctttcgcg acccaacact 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sequence complementary to base number 238 to 257 of HCV RNA <400> 6 acccaacact actcggctag 20 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Sequence identical to nucleotide numbers 113 to 137 of HCV RNA <400> 7 cctcccggga gagccatagt ggtct 25 <210> 8 <211> 25 <212> DNA < 213> Artificial Sequence <220> <223> Sequence complementary to nucleotides 115 to 139 of HCV RNA <400> 8 tcccgggaga gccatagtgg tctgc 25 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Sequence complementary to nucleotides 111 to 135 of HCV RNA <400> 9 cccctcccgg gagagccata gtggt 25 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> A sequence containing a sequence complementary to nucleotide numbers 99 to 106 and 108 to 115 of HCV RNA <400> 10 aggggggggg ctagctacaa cgacctggag g 31 <210> 11 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Promoter / Primer <400> 11 atttaggtga cactatagaa tacaacctcc cgggagagcc atagtggtct 50 <210> 12 <211> 50 <212> DNA <213> Artificial Sequence < 220> <223> Promoter primer <400> 12 atttaggtga cactatagaa tacaatcccg ggagagccat agtggtctgc 50 <210> 13 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Promoter primer <400> 13 atttaggtga cactatagaa tacaaccggg agagccatag tggtctgcgg 50 <210> 14 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Promoter / Primer <400> 14 atttaggtga cactatagaa tacaacccct cccgggagag ccatagtggt 50

[Brief description of the drawings]

FIG. 1 shows about 1 isotope-labeled 5 ′ end.
A 500-base length HCV RNA is reacted with a 6-base length single-stranded oligo DNA having a random sequence to
FIG. 4 shows a state in which a reaction solution cleaved by seH was applied to an electrophoresis gel. A clear band appeared at the positions of the two arrows in the figure.

FIG. 2 is a diagram showing a state in which a reaction solution in which HCV DNA cleavage reaction was performed again using single-stranded oligo DNA prepared based on the results of Example 1 was applied to an electrophoresis gel. In addition to the first and second lanes from the right, clear bands appeared.

FIG. 3 shows a reaction solution obtained by subjecting a single-stranded oligo DNA or the like prepared based on the results of Examples 1 and 2 to an HCV RNA cleavage reaction using AMV-RTase to an electrophoresis gel. It is a state that has been done. RNase
A band appeared as in the case of using H, and a clear band appeared in each of the newly prepared single-stranded oligo DNAs.

FIG. 4 shows the results of subjecting an HCV RNA cleavage reaction using a single-stranded oligo DNA to which a cleavage motif was bound in Example 4 to an electrophoresis gel. . In the presence of a cleavage motif such as a DNA enzyme, a specific cleavage product band appeared at the position of the arrow in the figure.

FIG. 5 shows the results of amplifying HCV RNA using the single-stranded oligo DNA of the present invention. In many cases, amplification product bands appeared at the locations indicated by the arrows.

Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference)

Claims (13)

[Claims] 1. A single-stranded oligo DNA of SEQ ID NO: 1, which has high binding reactivity to HCV RNA and is complementary to nucleotides 101 to 120 of HCV RNA. 2. A single-stranded oligo DNA of SEQ ID NO: 2, which has high binding reactivity to HCV RNA and is complementary to nucleotides 99 to 118 of HCV RNA. 3. A single-stranded oligo DNA of SEQ ID NO: 3, which has high binding reactivity to HCV RNA and is complementary to nucleotides 97 to 116 of HCV RNA. 4. A single-stranded oligo DNA of SEQ ID NO: 4, which has high binding reactivity to HCV RNA and is complementary to nucleotides 95 to 114 of HCV RNA. 5. A single-stranded oligo DNA of SEQ ID NO: 5, which has high binding reactivity to HCV RNA and is complementary to nucleotides 248 to 267 of HCV RNA. 6. A single-stranded oligo DNA of SEQ ID NO: 6, which has high binding reactivity to HCV RNA and is complementary to nucleotides 238 to 257 of HCV RNA. 7. A base number 113 of HCV RNA which is effective as a sense primer or a promoter primer.
The single-stranded oligo DNA having the same SEQ ID NO: 7 as the 137th to 137th. 8. The nucleotide sequence of HCV RNA which is effective as a sense primer or a promoter primer.
The single-stranded oligo DNA having the same SEQ ID NO: 8 as the 139th to 139th positions. 9. The nucleotide sequence of HCV RNA which is effective as a sense primer or a promoter primer.
The single-stranded oligo DNA having the same SEQ ID NO: 9 as the 135th to the 135th. 10. A single-stranded oligo DNA containing a random sequence of 6 to 9 bases in length and a single-stranded oligo DNA estimated from an experiment of a cleavage reaction using an enzyme that cuts only RNA in an RNA-DNA heteronuclear double-stranded portion. Prepare a combination of several types of oligo DNA of 20 to 25 base length including a sequence complementary to the cleavage region of the strand RNA or a complementary sequence near the estimated cleavage region,
Again, a method for producing a single-stranded oligo DNA that binds to a single-stranded RNA selected from a screening experiment of a cleavage reaction using an enzyme that only cleaves RNA in the heteronuclear double-stranded portion of RNA-DNA. 11. The method according to claim 10, wherein a part of the single-stranded oligo DNA according to any one of claims 1 to 6, a whole of the single-stranded oligo DNA according to any one of claims 1 to 6, and the production method according to claim 10. A part of the single-stranded oligo DNA selected by the method, and the single-stranded oligo D selected by the production method according to claim 10.
Single-stranded RNA cleavage, amplification of single-stranded RNA, reverse transcription of single-stranded RNA, translation inhibition of single-stranded RNA, or single-stranded RNA, which is a derivative of all of NA or a single-stranded oligo DNA thereof Measuring reagent. 12. The method according to claim 10, wherein a part of the single-stranded oligo DNA according to any one of claims 1 to 6, a whole of the single-stranded oligo DNA according to any one of claims 1 to 6, and the production method according to claim 10. A part of the single-stranded oligo DNA selected by the method, and the single-stranded oligo D selected by the production method according to claim 10.
A reagent for cleaving a single-stranded RNA, wherein an RNA-cleaving motif is bound to all of NA or a derivative of the single-stranded oligo DNA thereof. 13. The production method according to claim 10, wherein a part of the single-stranded oligo DNA according to any one of claims 1 to 6, an entirety of the single-stranded oligo DNA according to any one of claims 1 to 6, and the production method according to claim 10. A single-stranded oligo DNA or a derivative thereof, which is complementary to a part of the single-stranded oligo DNA selected by the above or all of the single-stranded oligo DNA selected by the production method according to claim 10. Reagent for sense primer or promoter primer for RNA.