JPH11137296A - Determination of base sequence of dna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for favorably determining the base sequence of a DNA without inhibiting its transcription due to RNA polymerase even in the case of using a compression inhibition ribonucleotide derivative as a ribonucleotide in order to improving sequence reading accuracy. SOLUTION: This method for determining the base sequence of a DNA comprises the following procedure: in the presence of at DNA fragment containing an RNA polymerase and a promoter sequence therefor, a ribonucleoside-5-triphosphate is reacted with 3-deoxyribonucleoside-5-triphosphate to form a nucleic acid-transcribed product, which is then separated, and from the resulting fraction, the aimed sequence of the nucleic acid is read out; wherein at least one kind of nucleic acid transcription initiator selected from oligoribonucleotides bearing no phosphate group on the 5-terminal and oligoribonucleotides bearing mono- or diphosphate group on the 5-terminal is used.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for determining the base sequence of DNA using an RNA polymerase and an initiator for a nucleic acid transcription reaction by the RNA polymerase. In particular, the present invention provides a more accurate reading of the sequence by suppressing compression during the sequence,
The present invention relates to a method for determining a nucleotide sequence of DNA that can smoothly initiate a nucleic acid transcription reaction by RNA polymerase when a compression-suppressing ribonucleotide derivative is used.

[0002]

2. Description of the Related Art The polymerase chain reaction (PCR) method is an excellent method, and its use is expanding year by year [Rand
all K. Saiki et al. (1988) Science 239, 487-491].
In the PCR method, one molecule of a DNA fragment can be amplified. A method in which the product amplified by the PCR method is sequenced without cloning (direct sequencing method) is also a useful method [Corinne Wong et al. (1988)
Nature, 330,384-386]. This method does not require library preparation and screening of the library, and is a rapid method that can simultaneously obtain sequence information of many samples.

However, the direct sequence method has two major problems. One is the primer that could not be incorporated and 2 'deoxyribonucleoside 5
Triphosphates (2'dNTPs) remain in the reaction system, which hinder the sequencing reaction.
Therefore, in the conventional method, these remaining primers and 2 ′
dNTPs had to be removed from the PCR product before sequencing. There are various methods for purifying a PCR product, for example, an electrophoresis purification method, an ethanol precipitation method, a gel filtration method, and an HPLC purification method [for example, Dorit RL et al. (1991) Current Protocols in M
olecular Biology, Vol. 11, John Wiley and Sons, Ne
w York, 15.2.1-15.2.11. " However, both methods are complicated.

[0004] A second problem is the rapid renaturation of the PCR product. PCR product is double-stranded DNA
If the primer is regenerated, it will not be a single-stranded template (template) and will prevent annealing between the primer and the single-stranded template. Methods for minimizing regeneration include, for example, quenching after denaturation, biotilation of one primer and adsorption of the PCR product to a streptavidin coating, use of exonucleases, acemetric PCR, etc. It has been reported. For example, Barbara Bachmann et al., 1990, Nucleic Acid Res., 1
8, 1309-. However, most of these methods require a long time and are very troublesome.

Therefore, as a new method for solving these problems, the present inventor has proposed a method without removing unreacted primer and 2 ′ deoxyribonucleoside 5 triphosphate (2 ′ dNTPs) remaining in a PCR reaction system.
In order to avoid the problem of rapid regeneration of PCR reaction products, it is not necessary to carry out denaturation at all, and a completely new DNA
[WO96 / 14434]. This method uses RNA polymerase such as T7 RNA polymerase and RN
A terminator of the transcription reaction (eg, 3 ′ deoxyribonucleoside 5 ′ triphosphate, 3 ′ dNTP
s) is a direct transcription sequencing method. According to this method, the base sequence of the DNA product amplified by the polymerase chain reaction is converted into a primer and 2 ′ deoxyribonucleoside 5 triphosphate (2 ′ dNT).
Ps) can be directly used for sequencing without having to be removed. Furthermore, since the denaturation itself is not performed at all, the PC
This is a very excellent method because the problem of rapid regeneration of the R product can be avoided.

[0006]

However, the present inventor has further studied the above method, and found that there is a problem to be solved in order to obtain more accurate base sequence data. In the above nucleotide sequence determination method, an RNA polymerase such as T7 RNA polymerase is reacted in a mixture of ribonucleoside 5 triphosphates and 3 ′ deoxyribonucleotide. In this reaction, a ribonucleotide having a base corresponding to the template sequence and 3 ′ deoxyribonucleotide are sequentially incorporated into the ribonucleotide sequence, whereby a polyribonucleotide is synthesized. The base sequence of DNA is determined by separating the obtained polyribonucleotide (nucleic acid transcription product) and reading the sequence of the nucleic acid from the obtained separated fraction.

However, a polyribonucleotide fragment is
For example, when separating according to the length of fragments by electrophoresis, if the mobility is not uniform between the fragments, the separation of adjacent fragments becomes insufficient. As a result, the signals of the sequences (for example, the fluorescence emission peaks) obtained from the adjacent fragments may overlap with each other, preventing reading of the sequence. Such a phenomenon is called compression. Compression is well known for sequencing with DNA polymerase. However, it was not known that compression appeared in a sequence using RNA polymerase. As a result of the study of the present invention,
In particular, it was found that the longer the chain length of the polyribonucleotide, the easier it is for the polyribonucleotide to form a higher-order structure, and that compression tends to occur.

In the case of determining a huge amount of gene sequences as in a genome project, the longer the length of a DNA sequence that can be read in one sequence, the higher the efficiency. Furthermore, if the full-length cDNA can be read as it is, the function of the gene can be easily understood. Therefore, the demand for sequencing of longer-chain DNA will increase in the future. In such a case, the above-described compression deteriorates the accuracy of reading the sequence, and is a serious problem.

Accordingly, the present inventors have proposed various methods for determining the base sequence of DNA using a DNA polymerase in order to provide a method for determining the base sequence of DNA in which the occurrence of compression is suppressed and the accuracy of sequence reading is improved. investigated. As a result, they have found that the above object can be achieved by using a compression-suppressing ribonucleotide derivative capable of suppressing compression during sequencing as ribonucleoside, which is a raw material of a transcription reaction by RNA polymerase. As the compression-suppressing ribonucleotide derivative, for example, a ribonucleotide having a base analog (base analog) instead of a base is used.

[0010] However, when a ribonucleotide having such a base analog is used, the initiation of a transcription reaction by RNA polymerase is inhibited, and in some cases, sequencing cannot be performed. As a result of various studies, the inhibition of transcription initiation was confirmed by this sequence method using RNA polymerase and R
It was found that the transcriptional activity of NA polymerase was due to promoter dependence. That is, transcription by the RNA polymerase is carried out by the RNA polymerase that recognizes the promoter sequence and has a D sequence homologous to the promoter sequence.
It begins by starting to synthesize the NA sequence. However, when the ribonucleotide incorporated at the first position in the DNA sequence was the above-mentioned compression-suppressing ribonucleotide derivative, the incorporation by RNA polymerase was not performed well, and as a result, transcription initiation was inhibited.

Therefore, an object of the present invention is to provide a method for determining a base sequence of a DNA using an RNA polymerase, in which a compression-suppressing ribonucleotide derivative is used as a ribonucleotide for the purpose of improving the accuracy of sequence reading. An object of the present invention is to provide a method capable of favorably determining the base sequence of DNA without inhibiting transcription by RNA polymerase.

[0012]

SUMMARY OF THE INVENTION The present invention provides a method for preparing ATP, GTP, and CTP in the presence of an RNA polymerase and a DNA fragment containing a promoter sequence for the RNA polymerase.
Ribonucleoside 5 triphosphates consisting of TP and UTP or their derivatives and 3dATP, 3dGTP, 3dC
TP, one or two or more 3 deoxyribonucleoside 5 triphosphates (hereinafter referred to as 3dNTP derivatives) consisting of 3dUTP and derivatives thereof are reacted to obtain a nucleic acid transcription product, and the resulting nucleic acid transcription product is separated. A method for determining a nucleotide sequence of DNA for reading a nucleic acid sequence from the obtained separated fraction, comprising oligoribonucleosides having no phosphate group at the 5 terminus and mono- or 5-terminus at the 5 terminus as an initiator of the nucleic acid transcription reaction. The present invention relates to a method comprising using at least one nucleic acid transcription initiator comprising oligoribonucleotides having a diphosphate group.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The method for determining the nucleotide sequence of DNA according to the present invention comprises the steps of: providing an RNA polymerase and a DNA fragment containing a promoter sequence for the RNA polymerase;
Reacting a ribonucleotide (NTP) with a 3 deoxyribonucleotide (3dNTP) derivative, and reading the sequence of the nucleic acid from the separated fraction of the obtained nucleic acid transcription product. As the initiator of the nucleic acid transcription reaction, It is characterized in that at least one nucleic acid transcription initiator consisting of oligoribonucleosides having no phosphate group at the 5 terminal and oligoribonucleotides having a mono or diphosphate group at the 5 terminal is used. Further, the method of the present invention is particularly useful when a compression-suppressing ribonucleotide derivative is used as one or more of the NTPs.

Nucleic acid transcription initiator Examples of the nucleic acid transcription initiator include ribonucleoside,
Ribonucleoside 5 monophosphate, ribonucleoside 5 diphosphate, general formula N ¹ (N) _n (wherein N ¹
Is ribonucleoside, ribonucleoside 5 monophosphate, or ribonucleoside 5 diphosphate, N is ribonucleoside 5 monophosphate, and n is an integer of 1 or more), and General formula N ² (N) _n (wherein N ² is a group represented by the following formula (1), and N is a ribonucleoside
5 monophosphate, and n is an integer of 1 or more).

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More specifically, examples of the nucleic acid transcription initiator include guanosine, guanosine 5 monophosphate (GMP), and guanosine 5 diphosphate (GD
P), general formula N ¹ (N) _n-1 G (where N ¹ is ribonucleoside, ribonucleoside 5 monophosphate, or ribonucleoside 5 diphosphate, and N is ribonucleoside 5 monophosphate) , N is an integer of 1 or more, and G is guanosine 5 monophosphate) and an oligoribonucleotide represented by the general formula N
² (N) _n-1 G (wherein N ² is a group represented by the above formula (1), N is ribonucleoside 5 monophosphate, n is an integer of 1 or more, and G is guanosine 5 monophosphate).

More specifically, examples of the nucleic acid transcription initiator include adenosine, adenosine 5 monophosphate (AMP), and adenosine 5 diphosphate (AD
P), general formula N ¹ (N) _n-1 A (where N ¹ is ribonucleoside, ribonucleoside 5 monophosphate or ribonucleoside 5 diphosphate, and N is ribonucleoside 5 monophosphate) , N is an integer of 1 or more, and A is adenosine 5 monophosphate), and an oligoribonucleotide represented by the general formula N ² (N) _n-1 A (wherein N ² represents the following formula (1) ), N is ribonucleoside 5 monophosphate, n is an integer of 1 or more, and A is adenosine 5
Which are monophosphates).

[0017] Specific examples of the nucleic acid transcription initiator include cytidine and cytidine 5 monophosphate (CM).
P), cytidine 5 diphosphate (CDP), general formula N ¹ (N) _n-1 C (where N ¹ is ribonucleoside, ribonucleoside 5 monophosphate, or ribonucleoside 5 diphosphate; Is a ribonucleoside 5 monophosphate, n is an integer of 1 or more, and C is cytidine 5 monophosphate), and an oligoribonucleotide represented by the general formula N ² (N) _n-1
C (wherein, N ² is a group represented by the following formula (1);
Is ribonucleoside 5 monophosphate, n is an integer of 1 or more, and C is cytidine 5 monophosphate).

Further, specific examples of the nucleic acid transcription initiator include uridine, uridine 5 monophosphate (UM
P), uridine 5 diphosphate (UDP), general formula N ¹ (N) _n-1 U (where N ¹ is ribonucleoside, ribonucleoside 5 monophosphate, or ribonucleoside 5 diphosphate; Is ribonucleoside 5 monophosphate, n is an integer of 1 or more, and U is uridine 5 monophosphate), and an oligoribonucleotide represented by the general formula N ² (N) _n-1
U (wherein N ² is a group represented by the following formula (1);
Is ribonucleoside 5 monophosphate, n is an integer of 1 or more, and U is uridine 5 monophosphate).

The above general formulas N ¹ (N) _n , N ¹ (N) _n-1 G,
N ¹ (N) _n-1 A, N ¹ (N) _n-1 C, N ¹ (N) _n-1 U, N
² (N) _n , N ² (N) _n-1 G, N ² (N) _n-1 A, N ² (N)
_n-1 C, N ² (N) In _n-1 U, a ribonucleoside represented by N ¹ , ribonucleoside 5 monophosphate,
The base of ribonucleoside 5 diphosphate is not particularly limited, and can be appropriately selected from guanine, adenine, cytosine, and uridine. Further, there is no particular limitation on the type of base of ribonucleoside 5 monophosphate represented by N and the base sequence when n is 2 or more. Further, n has no upper limit in terms of the function of the initiator, but in consideration of availability and the like, n is practically at most about 10 or less, preferably 5 or less.

Induction of compression-suppressing ribonucleotides
Conductor The method of the present invention is particularly useful when using a compression-suppressing ribonucleotide derivative as described above.
A compression-suppressing ribonucleotide derivative is a compound that is a ribonucleotide derivative and can suppress compression during sequencing. The compression-suppressing ribonucleotide derivative can be selected, for example, from ribonucleotides having a base analog (base analog) instead of a base. Base analogs may be natural or synthetic. The synthetic product may be, for example, one in which a part of the carbon constituting the purine or pyrimidine ring is substituted with nitrogen, or one in which a part of the nitrogen constituting the purine ring or pyrimidine ring is substituted with carbon. it can.
Alternatively, those obtained by introducing various substituents into a purine ring or a pyrimidine ring can also be used.

Examples of the compression-suppressing ribonucleotide derivative which is a ribonucleotide having a base analog include deazaribonucleoside 5-triphosphates. Furthermore, examples of the deazaribonucleoside 5 triphosphates include 7-deazaribonucleoside 5 triphosphates and 3-deazaribonucleoside 5 triphosphates. 7-Deazaribonucleoside 5 triphosphates include 7-deaza ATP, 7-deaza GTP and derivatives thereof, and 3-deazaribonucleoside 5 triphosphates include 3-deaza CTP, 3 -Deaza UTP and their derivatives.

Another example of a compression-suppressing ribonucleotide derivative is deaminoribonucleoside 5
Triphosphates can be mentioned. Amino groups are present in the bases of ribonucleotides except for uracil. GTP has an amino group at the 2-position of the purine ring, 6-position of the ATP-purine ring, and CTP has an amino group at the 4-position of the pyrimidine ring. The above deaminoribonucleoside 5
Triphosphates are ribonucleotides from which these amino groups have been eliminated, and include N ² -deamino GTP, N ^6-
Mention may be made of deamino ATP and N ⁴ -deamino CTP and their derivatives. Note that N ² -deaminoguanine is the same substance as inosine, and N ² -deamino GTP
Can also be abbreviated as ITP.

Other examples of the compression-suppressing ribonucleotide derivative include a derivative in which one or two of the hydrogens of the amino group present in the base of the ribonucleotide are substituted with an organic group other than hydrogen. Examples of derivatives include N-alkyl substituted ribonucleosides
5 Triphosphates (where the alkyl has 1 to 1 carbon atoms)
6 lower alkyl and the substitution is mono- or di-substituted). However, the substituent may be selected from organic groups other than the alkyl group. Examples of the N-alkyl-substituted ribonucleoside 5-triphosphates include N ² -monomethyl-substituted GTP, N ⁴ -monomethyl-substituted CTP, N ⁶ -monomethyl-substituted ATP, and derivatives thereof.

In the method of the present invention, ATP, GT
Of the four types of ribonucleoside 5 triphosphates composed of P, CTP and UTP or their derivatives, a compression-suppressing ribonucleotide derivative is used instead of the ribonucleoside 5 triphosphates for the base that easily causes compression. If necessary, a compression-suppressing ribonucleotide derivative can be used for two or more ribonucleoside 5-triphosphates.

When the deazaribonucleoside 5 triphosphate is used as a compression-suppressing ribonucleotide derivative for one type of base among the four types of ribonucleoside 5 triphosphates, the combination is [7-deaza ATP, GTP , CTP, UTP],
[ATP, 7-deaza GTP, CTP, UTP], [A
TP, GTP, 3-deaza CTP, UTP], [AT
P, GTP, CTP, 3-deaza UTP]. Deaza NTP can be replaced by another compression-suppressing ribonucleotide derivative. In addition, 7-Deaza NT
Compression-suppressing ribonucleotide derivatives such as P are commercially available, and commercially available products are available. In addition, two or more kinds of compression-suppressing ribonucleotide derivatives can be used in combination, or the same base can be used in combination with a compression-suppressing ribonucleotide derivative and ordinary ribonucleoside 5 triphosphates.

When a compression-suppressing ribonucleotide derivative is used as a part or all of ribonucleoside 5 triphosphates which are GTP or its derivative, guanosine, guanosine is used as a nucleic acid transcription initiator.
5 monophosphate (GMP), guanosine 5 diphosphate (GDP), oligoribonucleotides represented by general formula N ¹ (N) _n-1 G, or general formula N ² (N) _n-1 G
It is appropriate to use oligoribonucleotides represented by the following formulas in combination. In this case, the above-mentioned compression-suppressing ribonucleotide derivative is, for example, 7-deaza GTP, N ²
-Deamino GTP, or N ² -monomethyl substituted GTP.

When a compression-suppressing ribonucleotide derivative is used as a part or all of ribonucleoside 5 triphosphates which are ATP or its derivative, adenosine, adenosine 5 monophosphate (AMP), adenosine 5 5 diphosphate (ADP), oligoribonucleotides represented by general formula N ¹ (N) _n-1 A, or general formula N ² (N)
It is appropriate to use oligoribonucleotides represented by _n- 1A in combination. In this case, the compression-suppressing ribonucleotide derivative is, for example, 7-deaza ATP,
It can be N ⁶ -deamino ATP or N ⁶ -monomethyl substituted ATP.

When a compression-suppressing ribonucleotide derivative is used as a part or all of ribonucleoside 5 triphosphates which are CTP or its derivative, cytidine or cytidine is used as a nucleic acid transcription initiator.
5 monophosphate (CMP), cytidine 5 diphosphate (CDP), oligoribonucleotides represented by the general formula N ¹ (N) _n-1 C, or represented by the general formula N ² (N) _n-1 C It is appropriate to use oligoribonucleotides in combination. In this case, compression suppression ribonucleotide derivatives are, for example, 3-deaza CTP, N ⁴ - deamino CTP, or N ⁴ - may be a monomethyl-substituted CTP.

When a compression-suppressing ribonucleotide derivative is used as a part or all of ribonucleoside 5 triphosphates which are UTP or its derivative, uridine, uridine may be used as a nucleic acid transcription initiator.
5 monophosphate (UMP), uridine 5 diphosphate (UDP), oligoribonucleotides represented by general formula N ¹ (N) _n-1 U, or general formula N ² (N) _n-1 U
It is appropriate to use oligoribonucleotides represented by the following formulas in combination. In this case, the compression-suppressing ribonucleotide derivative can be, for example, 3-deaza UTP.

DNA base sequencing method D including a promoter sequence for RNA polymerase
A method for enzymatically synthesizing a nucleic acid transcription product using RNA polymerase using an NA fragment as a template, a method for separating a nucleic acid transcription product, and a method for reading a nucleic acid sequence from the separated fractions are in principle. Are known methods.
Accordingly, any of the known methods, conditions, devices, and the like can be appropriately used in these respects.

The DNA fragment used as a template also contains a promoter sequence for RNA polymerase.
No restrictions. For example, it may be a DNA product in which a DNA fragment containing a promoter sequence has been amplified by the polymerase chain reaction. Further, from the amplified DNA product, the primer used in the polymerase chain reaction and / or 2 deoxyribonucleoside 5 triphosphate and
The nucleic acid transcription production reaction in the method of the present invention can be performed without removing the / or derivative thereof. D above
As the polymerase chain reaction for NA amplification, a method widely used as a PCR method can be used as it is. The DNA fragment containing the promoter sequence is obtained by ligating the DNA fragment to be amplified with the promoter sequence and then cloning the cloned DNA using an appropriate host.
It can also be an NA fragment. That is, in the present invention,
There are no particular restrictions on the DNA sequence to be amplified, primers, conditions for amplification, and the like.

For example, as a reaction system of a polymerase chain reaction for amplifying a DNA fragment containing a promoter sequence, 10 to 50 ng of genomic DNA or 1 pg of cloned DNA, 10 μM of each primer,
μM of each 2 ′ deoxyribonucleoside 5 ′ triphosphate (dATP, dGTP, dCTP, dTTP)
Can be carried out using, for example, Taq polymerase or the like as a DNA polymerase in a volume of 20 μl containing

However, either one of the primers for the polymerase chain reaction or the amplified insert DN
A needs to include a promoter sequence for the RNA polymerase described below. The direct transcription sequence method is obtained by using a primer having a phage promoter sequence in one of two types of primers in a PCR method, or by having a phage promoter sequence in amplified insert DNA. PCR
The product can be subjected to in vitro transcription using an RNA polymerase driven by the promoter. The promoter sequence for RNA polymerase depends on the RNA used.
It can be appropriately selected according to the type of the polymerase.

In the method of the present invention, a promoter sequence is included.
A nucleic acid transcript such as an RNA transcript is synthesized from a DNA fragment. D
Since the NA fragment contains a promoter sequence for an RNA polymerase, the promoter sequence is recognized by the aforementioned RNA polymerase to synthesize a nucleic acid transcript such as an RNA transcript.

The synthesis of a nucleic acid transcript such as an RNA transcript is carried out by using A
Ribonucleoside 5 'triphosphate (NT) consisting of TP, GTP, CTP and UTP or their derivatives
P) (where one of them is a compression-suppressing ribonucleotide derivative) and one or more 3 ′ dNTP derivatives. 3'dNT
The P derivative is herein referred to as 3 ′ dATP, 3 ′
dGTP, 3'dCTP, 3'dUTP and their derivatives are used as a generic term. As ribonucleoside 5 'triphosphates (NTPs), a part thereof is ATP.
At least four compounds having different bases are required for the synthesis of a transcript, including the case of derivatives such as

By incorporating a 3 'dNTP derivative into the 3' end of RNA or nucleic acid which is a transcription product, a 3 'hydroxy group is lost and synthesis of RNA or nucleic acid is inhibited. As a result, RNA or nucleic acid fragments of various lengths whose 3 ′ end is a 3 ′ dNTP derivative are obtained.
Such ribonucleoside analogs are obtained for each of the four types of 3 ′ dNTP derivatives having different bases. By preparing four types of this ribonucleoside analog, it can be used for determination of RNA or nucleic acid sequence [Vladimir D. Axelred er al. (1985) Biochemist
ry Vol. 24, 5716-5723].

One or more 3 ′ dNTP derivatives can be used in one nucleic acid transcription reaction. 1
When one nucleic acid transcription reaction is performed using only the 3 ′ dNTP derivative of the species, four transcription products having different bases of the 3 ′ dNTP derivative at the 3 ′ end are obtained by performing the nucleic acid transcription reaction four times. In one nucleic acid transcription reaction, 3'd
A transcription product is obtained which is a mixture of various RNA or nucleic acid fragments having the same NTP derivative and different molecular weight. The resulting four transcription products can be independently subjected to separation and sequence reading described below. Also, 4
Alternatively, two or more of the different transcription products can be mixed and the mixture subjected to separation and sequence reading.

In one nucleic acid transcription reaction, two or more 3 ′ dN
When the TP derivative is used at the same time, in one reaction product,
Two or more types of transcription products having different bases of the 3 ′ dNTP derivative at the 3 ′ end will be included. This can be subjected to separation and sequence reading described below. It is preferable to use two or more 3 ′ dNTP derivatives at the same time in the nucleic acid transcription reaction because the number of nucleic acid transcription reaction operations can be reduced.

Further, transcription of a nucleic acid such as RNA is performed by an RNA polymerase in the presence of four types of ribonucleoside 5 'triphosphates having different bases, and terminated by a 3' dNTP derivative. As a result, for each base, an RNA or nucleic acid ladder
Is generated for the sequence. In the present invention,
Nucleic acid transcription is performed using four types of ribonucleosides 5 'with different bases.
It is preferable to carry out in the presence of triphosphates, to separate them, and to read the sequence of four bases at once (simultaneously).

RNA polymerase The RNA polymerase used in the method of the present invention is a wild-type R
Either NA polymerase or mutant RNA polymerase may be used. However, a mutant RNA in which at least one amino acid of the wild-type RNA polymerase has been modified so as to increase the ability to incorporate the 3dNTP derivative as compared to the ability of the corresponding wild-type RNA polymerase
Preferably it is a polymerase. Here, “wild-type RNA polymerase” refers to all naturally occurring RNA.
An amino acid other than a modified RNA polymerase, which comprises a polymerase, and which is intended to increase the ability to incorporate 3 deoxyribonucleotides or their derivatives as compared to the ability of the corresponding wild-type RNA polymerase May further have mutations, insertions or deletions. That is, wild type RN
R obtained by artificially modifying A polymerase for other purposes
NA polymerase is also included in the above “wild-type RNA polymerase”. However, it is appropriate that such mutation, insertion or deletion of amino acids is performed within a range that maintains the activity as an RNA polymerase.

As the “wild-type RNA polymerase”,
Examples include RNA polymerases derived from T7 phage, T3 phage, SP6 phage, and K11 phage. However, it is not limited to these RNA polymerases. In the present invention, the “wild-type RNA polymerase” is a naturally-occurring heat-resistant RNA polymerase, or a naturally-occurring RNA polymerase that has been artificially modified to have heat resistance (ie, mutation or insertion of an amino acid). Or missing). However, the modification for imparting heat resistance is RN
It is appropriate that the reaction is performed within a range that maintains the activity as A polymerase. The mutant RNA polymerase of the present invention using a heat-resistant RNA polymerase as the “wild-type RNA polymerase” also becomes heat-resistant. As a result, for example, in combination with the PCR method, in situ using the PCR product as a template, ie, in parallel with the PCR, the R
It is also possible to synthesize NA fragments.

T7 RNA polymerase is known as a highly specific promoter-specific RNA polymerase. For the nucleotide sequence and production method of T7 RNA polymerase, see Davanloo et al., Proc. Natl. Acad. Sci. USA., 81: 203.
5-2039 (1984). Further, regarding mass production, see Zawadzki et al., Nucl. Acids Res., 19: 194
8 (1991). This phage-derived RN
A polymerase, unlike RNA polymerase of Escherichia coli and higher organisms, can perform a transcription reaction using only a single polypeptide (Chamberlin et al., Nature, 228: 227-23).
1,1970). Therefore, it has become an excellent material for analyzing the transcription mechanism, and many mutants have been isolated and reported. Further Sousa et al., Nature, 364: 593-599, 1993
Shows the results of the crystal analysis.

Furthermore, T cells that infect E. coli as other highly specific promoter-specific RNA polymerases
Three well-known three phages, SP6 phage infecting Salmonella and K11 phage-derived RNA polymerase infecting Klebsiella pneumoniae are well known. The above four RNA polymerases have the primary structure of amino acids,
Very similar, such as the sequence of the promoter.

[0044] The mutant RNA polymerase may have an increased ability to incorporate 3 deoxyribonucleotides or their derivatives, as compared to the ability of the corresponding wild-type RNA polymerase. As described above, in the wild-type RNA polymerase, the incorporation of 3 deoxyribonucleotides is lower than that of ribonucleotides, which has hindered the use in the nucleotide sequencing method. In contrast, mutant RNA polymerases have been modified to increase their incorporation capacity for 3 deoxyribonucleotides or their derivatives, preferably at least twice as much as the wild type. The uptake of 3 deoxyribonucleotides tends to decrease particularly when a 3 deoxyribonucleotide derivative having a fluorescent label attached to 3 deoxyribonucleotides is used.
NA polymerase can also improve incorporation of such 3 deoxyribonucleotide derivatives.

The mutant RNA polymerase is obtained by modifying at least one amino acid of the corresponding wild type RNA polymerase. Such amino acid modifications
It can be insertions or deletions as well as amino acid mutations. In addition, the amino acid mutation is, for example, replacing at least one of the naturally occurring amino acids with tyrosine. Further, the naturally occurring amino acid to be substituted can be, for example, phenylalanine. However, it is not limited to phenylalanine,
Any amino acid substitution that can increase the ability to incorporate 3 deoxyribonucleotides or other ribonucleotide analogs for the corresponding ribonucleotide may be used.

Examples of the mutant RNA polymerase include mutant T7 RNA polymerase F644Y and L665P / F667Y. Here, the number is a number from the N-terminus of the polymerase protein, for example, F667 indicates that the 667th amino acid residue of this polymerase is F, and the description of F667Y indicates that the 667th amino acid residue F It means that Y was substituted. They retain sufficient RNA synthesis activity, furthermore greatly improve the ability to incorporate 3'dNTPs, and significantly reduce the strong bias observed in the wild type. By using mutant T7 RNA polymerase F644Y and L665P / F667Y having such excellent properties,
It enables sequencing of transcription products at a practical level beyond sequencing using NA polymerase. Mutant T7 RNA polymerase F644Y, E. coli pT7RF644Y (DH5α) and pT7RL665P / F667Y (DH producing L665P / F667Y)
5α) has the International Depository No. 5998 (FERM-BP
No. 5998) and No. 5999 (FERM-BP-5999) were deposited on July 2, 1997.

The above mutant RNA polymerase is RN
A nucleic acid molecule encoding A polymerase is provided, and a nucleic acid molecule at one or more sites in the nucleotide base sequence is prepared.
It can be prepared by mutating the nucleic acid molecule to mutate one or more bases, and then recovering the modified RNA polymerase expressed by the mutated nucleic acid molecule. Preparation of a nucleic acid molecule encoding an RNA polymerase, introduction of a mutation into the nucleic acid molecule, and recovery of the modified RNA polymerase are all performed by:
It can be performed using a known method.

The mutant T7 RNA polymerase can be constructed, for example, by the following method. Using an expression vector containing the T7 RNA polymerase gene as a template
An expression plasmid is constructed in which a region between the restriction enzyme Hpa I and Nco I sites corresponding to the C-terminal side of the T7 RNA polymerase gene is mutated by PCR using the PCR method. Then
Escherichia coli DH5α was transformed using this expression plasmid, and isopropyl-β-D-thiogalactopyranoside (IPT
When G) is added, the mutant T7 RNA polymerase protein can be expressed in a large amount.

Inorganic Pyrophosphatase In the method of the present invention, the nucleic acid transcription production reaction is preferably performed in the presence of inorganic pyrophosphatase. The difference in peak height (signal intensity) obtained corresponding to each labeled ribonucleotide is reduced to improve the accuracy of sequence reading, thereby enabling more accurate sequence data to be obtained.

Pyrophosphorolysis occurs when the amount of pyrophosphate produced by DNA synthesis increases, and serves to promote the reaction in a direction in which the synthesized DNA product is degraded. As a result, pyrophosphorolysis inhibits sequencing in the dideoxy sequencing method using DNA polymerase. On the other hand, it has been known that when inorganic pyrophosphatase is used in the dideoxy sequencing method using a DNA polymerase, pyrophosphorolysis is inhibited and stable sequence data can be obtained [Japanese Patent Laid-Open No. 4-5060].
02]. Pyrophosphorolysis is also effective in a sequencing method using RNA polymerase. That is, by performing the nucleic acid transcription production reaction in the presence of inorganic pyrophosphatase, the difference in peak height (signal intensity) obtained for each labeled ribonucleotide can be reduced, and more stable sequence data can be obtained. can get.

Inorganic pyrophosphatase (EC. 3.6.1.1)
Is available as a commercial product, for example, as INORGANIC PYROPHOSPHATASE from Sigma and as pyrophosphatase from Boehringer.
The amount of the inorganic pyrophosphatase used depends on the activity of the inorganic pyrophosphatase and the RNA polymerase. For example, it is appropriate to set the range of 10 ^-6 to 10 ^-2 units per 1 unit of the RNA polymerase. is there.

Separation and Reading of Nucleic Acid Transcription Products In the method of the present invention, RNA or nucleic acid transcription products are separated. This separation can be appropriately performed by a method capable of separating a plurality of product molecules having different molecular weights contained in the transcription product according to the molecular weight. An example of such a separation method is an electrophoresis method. In addition, HPLC and the like can be used. The conditions of the electrophoresis method and the like are not particularly limited, and the electrophoresis can be performed by a conventional method. An RNA or nucleic acid sequence can be read from a band (RNA or nucleic acid ladder) obtained by subjecting the transcription product to electrophoresis.

The RNA or nucleic acid ladder can be read by labeling ribonucleoside 5 ′ triphosphates (NTPs), which are terminators used in the transcript reaction. The reading of the RNA or nucleic acid ladder can also be performed by labeling the 3 ′ dNTP derivative used in the transcript reaction. As a sign,
For example, a fluorescent label or a radioactive or stable isotope can be mentioned, but a fluorescent label is preferable in terms of safety and operability. In addition, without using a label as described above, the sequence of the transcription product can be read by measuring the mass of each transcription reaction product separated by electrophoresis or the like with a mass spectrometer.

Specifically, for example, a labeled 3 ′ dN
TP derivatives, more specifically labeled 3'dAT
Using P, 3 ′ dGTP, 3 ′ dCTP and 3 ′ dUTP, by detecting the radioactive or stable isotope or fluorescence of the band obtained by subjecting the transcription product to electrophoresis,
The sequence of the transcription product can be read. By labeling the 3 ′ dNTP derivative in this way, there is no variation in the radioactive intensity or the fluorescent intensity between any of the bands, and the measurement is facilitated. Further, detection of a radioactive or stable isotope or a ladder that generates fluorescence can be performed, for example, by appropriately using an apparatus used for a DNA sequence sink. Also, by using a radioactive or stable isotope or fluorescently labeled ATP, GTP, CTP and UTP, detecting the radioactive or stable isotope or fluorescence of the band obtained by electrophoresis, the sequence of the transcription product can be determined. Can be read.

Furthermore, 3′d labeled with different fluorescence
ATP, 3'dGTP, 3'dCTP and 3'dUTP
Using 3 'dATP, 3' dGTP, 3 'dC
It is also possible to read the sequence of RNA or nucleic acid by detecting the four kinds of fluorescence of the band obtained by subjecting a mixture of various transcription product fragments, which are TP or 3 ′ dUTP, and labeled differently, to electrophoresis. it can. In this method, four types of 3 ′ dNTPs are labeled with different fluorescence. In this manner, by applying a mixture of four types of transcription products having different 3′-ends to electrophoresis, four different types of bands emitting fluorescence according to (3′-dNTP at the 3′-end) are obtained. And identify the differences in fluorescence at a time
The sequence of any kind of RNA or nucleic acid can be read.

As the fluorescently labeled 3 ′ dNTP, WO 96
/ 14434 and JP-A-63-152364.
Deoxyribonucleotide derivatives can be used. Also, the fluorescent label is preferably a fluorescent dye or the like, which produces detectable luminescent radiation following stimulation by energy absorption from a suitable source such as an argon laser.

From the RNA or nucleic acid sequence read as described above, the DNA sequence used as a transcription template can be determined. When a ladder was formed for each base, RNs obtained from four types of ladders were obtained.
The DNA sequence used as a transcription template can be determined by integrating the information of A or the nucleic acid sequence. When two or more bases form a ladder simultaneously (when two or more base bands coexist in the same ladder)
The DNA sequence used as a transcription template can be determined by combining the RNA or nucleic acid sequence information obtained from each ladder. In particular, when ladders are formed simultaneously for four types of bases (in the case where bands of four types of bases coexist in the same ladder), a transcription template is obtained from RNA or nucleic acid sequence information obtained from one ladder. Can be determined.

[0058]

According to the present invention, it is possible to provide a method for determining the base sequence of DNA which suppresses the occurrence of compression and improves the accuracy of sequence reading. Furthermore, considering the high processability of RNA polymerase, the method for determining a nucleotide sequence of the present invention has a DN
It enables the base sequence determination more than the base sequence determination method using A polymerase. Further, according to the method of the present invention, the PC
Without purifying the R product, the base sequence of the DNA of the PCR product can be determined as it is. This is due to the characteristic of RNA polymerase that the 2 'dNTP remaining in the reaction mixture is not consumed as a reagent in the RNA transcription reaction in the presence of 3' dNTP for sequencing.

Further, in the method of the present invention, since the transcription reaction of RNA is used, there is no need to use a single-stranded template DNA as in the ordinary DNA sequencing method, no primer is required, and further, the sequencing primer is not used. No denaturing step is required for hybridization. Therefore, it is not affected by the regeneration of the PCR product,
The nucleotide sequence of NA can be determined. In the method for determining a nucleotide sequence of DNA of the present invention, when a mutant RNA polymerase having heat resistance is used as the RNA polymerase, it can be used together with the step of performing the PCR method,
As a result, it is possible to more quickly determine the nucleotide sequence of DNA.

When inorganic pyrophosphatase is used in the method for determining a nucleotide sequence of DNA of the present invention, the corresponding 3′-deoxyribonucleotide or its derivative is hardly incorporated into the polyribonucleotide sequence as compared with ribonucleotides. In ribonucleotides and 3'-
Among the deoxyribonucleotides, it is possible to eliminate the bias on the incorporation ability of ribonucleotides and the like, such as a difference in the manner of incorporation into the sequence, depending on the type of base, thereby obtaining more stable sequence data.

[0061]

The present invention will be described in more detail with reference to the following examples. Example 1 Example of sequencing reaction using PCR product as template Human thyroid-stimulating hormo as a template for PCR reaction
ne (hTSH-β) cDNA obtained by subcloning into a BS750-derived plasmid having a T7 promoter was used. Using 100 fg of the plasmid having the hTSH-β cDNA, the L220 primer (5'-T
AA CAA TTT CAC ACA GGA AAC A-3 ') and 1211 primers
(5'-ACG TTG TAA AAC GAC GGC CAG T-3 ')
PCR reaction with μl [(94 ° C for 2 minutes) once, (94 ° C for 1 minute, 55 ° C for 1 minute)
Min, 72 ° C for 1.5 minutes) 30 times, 72 ° C for 5 minutes]. This PCR
The T7 promoter is located downstream of the 1211 primer of the PCR product produced by the reaction.

The sequence transcription reaction was performed in Melton, DA,
[Nucleic Acids Res., 12: 7035-7056 (1984)]. 1 μl of the above PCR product (about 10
ng) was used for the sequencing reaction. The reaction was carried out as a derivative of 3 ′ dNTP using a dye terminator 4 μM R6G-3 ′ dATP [5-carboxyrhodamine 6G-labeled 3′-deoxyadenone-5-trifoam] synthesized with reference to the method described in WO96 / 14434. Sulfate (n = 4)], 4 μM R110-3′dGTP [5-carboxyrhodamine 110-labeled 3′-deoxyguanosine-5-triphosphate (n = 4)], 80 μM XR-3′dCTP [5-carboxy -X-rhodamine labeled 3′-deoxycytidine-5-triphosphate (n = 4)], 20 μM TMR-3 ′ dUTP [5-carboxytetramethylrhodamine labeled 3′-deoxyuridine-5-triphosphate (n = 4)] was used. 500 μM 7-deaza GTP, 500 μM UTP, 250 μM ATP, 250 μM CTP, 200 μM
M GMP (nucleic acid transcription initiator), 8 mM MgCl ₂ , 2 mM spermidine
-(HCl) ₃ , 5 mM DTT, 40 mM Tris / HCl pH 8.0 (BRL, Gibco) under the conditions of mutant T7 RNA polymerase F644Y25
Add units to make a total reaction volume of 10 μl, also at 37 ° C.
For 1 hour.

[0063]

Embedded image 5-carboxyrhodamine 6G-labeled 3'-deoxyadenone-5
-Triphosphate (n = 4)

[0064]

Embedded image 5-carboxy rhodamine 110 labeled 3'-deoxyguanosine-5-triphosphate (n = 4)

[0065]

Embedded image 5-carboxy-X-rhodamine labeled 3'-deoxycytidine-
5-triphosphate (n = 4)

[0066]

Embedded image 5-carboxytetramethylrhodamine labeled 3'-deoxyuridine-5-triphosphate (n = 4)

Next, to remove unreacted dye terminator remaining in the reaction product, Sephadex G-
The transcript was purified by a gel filtration method using 50 columns (manufactured by Pharmacia Biotech), and the purified product was evaporated to dryness using a centrifugal evaporator. The dried reaction product is transferred to ABI PRISM 377D of PerkinElmer Japan, Inc.
According to NA S equencing System Operation Manual Ver.1.0,
Formamide / EDTA / Blue dextrane loading buffer 6μ
2 μl of the solution, using an ABI 377 DNA Sequencer and an analysis program (Sequencing Analysis Ver. 3.0) using a denaturing gel for sequencing analysis containing 6 M urea / 4% Long Ranger ^™ acrylamide solution (FMC). Analyzed.

FIG. 1B shows the result. In FIG.
For comparison, the results using GTP instead of 7-deaza GTP are also shown (a). In (a) using GTP, adjacent peaks do not overlap, as shown in (b), when 7-deaza GTP is used, even in a place where adjacent peaks overlap and a reading error occurs, as shown in (b). It can be seen that reading is possible. On the other hand, GMP
In the system to which (nucleic acid transcription initiator) was not added, almost no transcription occurred, and the nucleotide sequence could not be read at all.

Example 2 Investigation of Influence of Initiator Type and Concentration pBLUESCRIPT KS (+) as a template
dsDNA which had been cut with uII and made linear was used. The reaction conditions are as follows. 40mM Tris / HCl pH8.0 (BR
L, Gibco), 8 mM MgCl ₂ , 5 mM DTT, 2 mM spermidine
(HCl) ₃ , 25 U of T7 RNA polymerase, 25 U each
0 μM ATP, UTP and CTP, and 0.2 μC
In the presence of i [α- ³² P] UTP (3000 Ci / mmol), the total volume was adjusted to 10 μl and reacted at 37 ° C. for 1 hour. As a transcription initiator, CG (cytidine 5 monophosphate (CMP) -guanosine 5 monophosphate (GMP) dimer), GMP, or GDP was used at a concentration of 400 μM. Furthermore, instead of GTP, a substrate (compression-suppressing ribonucleotide derivative)
7-Deaza GTP or N ² -deaminoguanine (inosine) as the concentration, 200, 400, 80
0 and 1600 μM.

The reaction product was converted to formamide / EDTA loading bu
After dissolving in ffer and denaturing, a portion was electrophoresed on a 6M urea / 4% acrylamide gel. The gel after the electrophoresis was dried, and the radioactivity of the transcript was analyzed using BAS2000 image analysis (Fuji Photo Film). FIG. 2 shows the results when 7-deaza GTP was used as the substrate (compression suppressing ribonucleotide derivative), and FIG. 3 shows the results when N ² -deaminoguanine (inosine) was used.

[Brief description of the drawings]

FIG. 1 shows a sequence result using GTP as NTP (a) and a sequence result using 7-deaza GTP as NTP (b).

FIG. 2 shows the results of analyzing the radioactivity of a transcript when 7-deaza GTP is used as a substrate (compression suppressing ribonucleotide derivative).

FIG. 3 shows the results of analyzing the radioactivity of a transcript when N ² -deaminoguanine (inosine) is used as a substrate (compression-suppressing ribonucleotide derivative).

Claims (11)

[Claims] 1. A ribonucleoside 5 triphosphate comprising ATP, GTP, CTP and UTP or a derivative thereof and 3dATP, 3dGTP, 3dCTP in the presence of an RNA polymerase and a DNA fragment containing a promoter sequence for the RNA polymerase. A nucleic acid transcription product is obtained by reacting one or more kinds of 3 deoxyribonucleoside 5 triphosphates (hereinafter referred to as 3dNTP derivatives) composed of 3dUTP and derivatives thereof, and separating the obtained nucleic acid transcription product to obtain A method for determining a nucleotide sequence of DNA for reading a nucleic acid sequence from a separated fraction obtained, comprising oligoribonucleosides having no phosphate group at the 5 terminus and mono- or di-mononucleotides at the 5 terminus as an initiator of the nucleic acid transcription reaction. Initiation of transcription of at least one nucleic acid consisting of oligoribonucleotides having a phosphate group Wherein the use of. 2. A ribonucleoside derivative capable of suppressing compression during sequencing (hereinafter referred to as a compression-suppressing ribonucleotide derivative) as the ribonucleoside 5 triphosphates.
The method described in. 3. The nucleic acid transcription initiator is a ribonucleoside,
Ribonucleoside 5 monophosphate, ribonucleoside 5 diphosphate, general formula N ¹ (N) _n (wherein N ¹
Is ribonucleoside, ribonucleoside 5 monophosphate, or ribonucleoside 5 diphosphate, N is ribonucleoside 5 monophosphate, and n is an integer of 1 or more), and General formula N ² (N) _n (wherein N ² is a group represented by the following formula (1), and N is a ribonucleoside
5 is monophosphate, and n is an integer of 1 or more.) 1 or 2 or more selected from the group consisting of oligoribonucleotides
The method described in. Embedded image 4. A compression-suppressing ribonucleotide derivative is used as GTP or its derivative, ribonucleoside 5 triphosphate, and the nucleic acid transcription initiator is guanosine, guanosine 5 monophosphate (GMP), guanosine 5 diphosphate. Fate (GD
P), general formula N ¹ (N) _n-1 G (where N ¹ is ribonucleoside, ribonucleoside 5 monophosphate, or ribonucleoside 5 diphosphate, and N is ribonucleoside 5 monophosphate) , N is an integer of 1 or more, and G is guanosine 5 monophosphate) and an oligoribonucleotide represented by the general formula N
² (N) _n-1 G (wherein N ² is a group represented by the following formula (1), N is ribonucleoside 5 monophosphate, n is an integer of 1 or more, and G is guanosine 5. The method according to claim 1, wherein the method is one or more selected from the group consisting of oligoribonucleotides represented by (5) monophosphate. Embedded image 5. The method according to claim 5, wherein the compression-suppressing ribonucleotide derivative comprises 7-deaza GTP, N ² -deamino GTP, and N-deamino GTP.
5. The method according to claim 4, wherein the method is selected from ² -monomethyl substituted GTP. 6. A compression-suppressing ribonucleotide derivative is used as ATP or its derivative, ribonucleoside 5 triphosphate, and the nucleic acid transcription initiator is adenosine, adenosine 5 monophosphate (AMP), or adenosine 5 diphosphate. Fate (AD
P), general formula N ¹ (N) _n-1 A (where N ¹ is ribonucleoside, ribonucleoside 5 monophosphate or ribonucleoside 5 diphosphate, and N is ribonucleoside 5 monophosphate) , N is an integer of 1 or more, and A is adenosine 5 monophosphate), and an oligoribonucleotide represented by the general formula N ² (N) _n-1 A (wherein N ² represents the following formula (1) ), N is ribonucleoside 5 monophosphate, n is an integer of 1 or more, and A is adenosine 5
2. The method according to claim 1, which is at least one member selected from the group consisting of oligoribonucleotides represented by the formula: Embedded image 7. The compression-suppressing ribonucleotide derivative comprises 7-deaza ATP, N ⁶ -deamino ATP, and N-deamino ATP.
The method according to claim 6, wherein the method is selected from ⁶ -monomethyl-substituted ATP. 8. A compression-suppressing ribonucleotide derivative is used as the ribonucleoside 5 triphosphates which are CTP or its derivative, and the nucleic acid transcription initiator is cytidine or cytidine 5 monophosphate (C
MP), cytidine 5 diphosphate (CDP), general formula N ¹ (N) _n-1 C (where N ¹ is ribonucleoside, ribonucleoside 5 monophosphate, or ribonucleoside 5 diphosphate; Is ribonucleoside 5 monophosphate, n is an integer of 1 or more, and C is cytidine 5 monophosphate), and an oligoribonucleotide represented by the general formula N ² (N)
_n-1 C (wherein, N ² is a group represented by the following formula (1),
N is ribonucleoside 5 monophosphate, n
Is an integer of 1 or more, and C is cytidine 5 monophosphate), or one or more selected from the group consisting of oligoribonucleotides represented by the formula:
The described method. Embedded image 9. The method of claim 9, wherein the compression-suppressing ribonucleotide derivative is 3-deaza CTP, N ⁴ -deamino CTP, and N-deamino CTP.
9. The method according to claim 8, wherein the method is selected from ⁴ -monomethyl substituted CTPs. 10. A compression-suppressing ribonucleotide derivative is used as UTP or its derivative, ribonucleoside 5 triphosphates, and the nucleic acid transcription initiator is uridine, uridine 5 monophosphate (UMP), uridine 5 diphosphate. Fate (UDP),
General formula N ¹ (N) _n-1 U (where N ¹ is ribonucleoside, ribonucleoside 5 monophosphate, or ribonucleoside 5 diphosphate, N is ribonucleoside 5 monophosphate, and n is An integer of 1 or more, and U is uridine 5 monophosphate) and an oligoribonucleotide represented by the general formula N
² (N) _n-1 U (wherein N ² is a group represented by the following formula (1), N is ribonucleoside 5 monophosphate, n is an integer of 1 or more, and U is uridine 5. The method according to claim 1, wherein the method is one or more selected from the group consisting of oligoribonucleotides represented by (5) monophosphate. 11. The method according to claim 10, wherein the compression-suppressing ribonucleotide derivative is 3-deaza-UTP.