JP2004329218A - Dna base-sequencing method by using compound having energy transfer function - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a compound capable of using the highly sensitive energy transfer theory, and to provide a DNA base-sequencing method capable of detecting a labeled DNA fragment at a high sensitivity by using the chain terminator method using the compound as the terminator. <P>SOLUTION: The method for determining a base sequence of DNA by the chain terminator method comprises performing a chain termination reaction by using 3'-deoxyribonucleotide residue, a terminator having two kinds of reporters capable of serving as a donor and a receptor in energy transfer and RNA polymerase. The two kinds of reporters are located at a sufficient interval for inducing energy transfer from the donor to the acceptor. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for determining a nucleotide sequence of DNA using a compound having an energy transfer function as a terminator, a primer or an initiator.

There is known a method of preparing a chain termination reaction product using DNA polymerase or RNA polymerase and separating and fractionating the product to determine a base sequence. In addition, a project for elucidating the gene sequence of a higher animal is in progress, and among them, development of a higher-speed nucleotide sequencing method is being attempted. For that purpose, the sequencing reaction product obtained using a fluorescently labeled terminator is separated and fractionated by electrophoresis, the resulting DNA fragment is excited with a laser, and the base sequence is determined by detecting the fluorescence, A DNA fluorescence sequencer has been used.
It is also known that the quantum yield of a fluorescent dye can be increased by using two fluorescent dyes (donor dye and acceptor dye) and using the principle of energy transfer. In recent years, energy transfer primers have been developed in which two fluorescent dyes are covalently bonded to oligonucleotides by introducing this energy transfer principle and used as primers for sequencing reactions (for example, Nature Medicine, 2 , 246-249 ( 1996), WO95 / 21266, JP-A-10-88124].

To speed up DNA sequencing, systems that can analyze many samples at once have been developed. In particular, the capillary fluorescence sequencer has an ideal structure that facilitates automatic loading of a sample and is easy to multiplex with no lane crossing. Two types of optical systems are used to promote the multiplexing of the fluorescence sequencer.
One is a scanning method and the other is an image method. In any case, the emergence of a highly sensitive detector capable of detecting even a small amount of DNA is a necessary condition in order to promote multiplexing. In the scanning method, in order to end the sequencing within a fixed time regardless of the number of capillaries, the more the number of capillaries increases, the shorter the time allocated to one capillary. Therefore, a detector with higher sensitivity is required. Also in the image method, the area of the visual field covered by the detector is constant regardless of the number of capillaries. In order to increase the number of capillaries, it is necessary to increase the number of pixels of the optical element and reduce the diameter of one capillary. Therefore, a detector with higher sensitivity is required.

On the other hand, in order to increase the DNA detection sensitivity, as described above, a technique using a fluorescent primer based on the principle of energy transfer for DNA sequencing has been developed. However, since this method is a fluorescent primer, it requires a very complicated process of performing a termination reaction on each of the four types of bases of AGCT and further subjecting the resulting mixture to electrophoresis. In addition, since no primer is used in a sequencing reaction by a transcription reaction using a promoter-dependent RNA polymerase, this energy transfer primer cannot be used.
In order to speed up DNA sequencing, a highly sensitive detection system with a high quantum yield, such as energy transfer, is required in a multiple-capillary sequencer. Nevertheless, systems using energy transfer primers have technical limitations, such as the complexity of pretreatment (DNA polymerase system) and the elimination of primers (RNA polymerase system).

Therefore, an object of the present invention is to eliminate the drawbacks in a system using an energy transfer primer, that is, it can be applied to a transcription reaction system using an RNA polymerase, and furthermore, the AGCT4 reaction products obtained separately as a pretreatment are mixed. An object of the present invention is to provide means that enables a sequencing method capable of high-sensitivity detection using energy transfer without going through complicated steps.
In particular, an object of the present invention is to provide a compound which can utilize the principle of energy transfer with high sensitivity, using this compound as a terminator, and using a chain terminator method to detect a labeled DNA fragment with high sensitivity. It is to provide a sequencing method.

  By the way, in the energy transfer primer disclosed in WO95 / 21266, two reporters that cause energy transfer are linked using a part of the oligonucleotide constituting the primer as a linker. However, in such a primer, when the sequence of the oligonucleotide serving as the primer is different, it is necessary to synthesize a different primer each time, which is practically disadvantageous. Further, in the energy transfer primer described in JP-A-10-88124, two reporters are linked by using an aliphatic or aromatic residue as a linker. Therefore, there is no problem as in the primer of WO95 / 21266, but even if the length of the linker is increased for the purpose of increasing the distance between the two reporters, even if the linker becomes a long chain, the two reporters will not be linked because of the binding mode of the linker. There is a problem that it is difficult to set the distance between them to a desired value.

Therefore, another object of the present invention is to solve the above problem, that is, without using a part of the primer sequence as a linker, and easily control the distance between two reporters, useful as an energy transfer primer It is to provide a compound.
It is a further object of the present invention to provide a method for determining the nucleotide sequence of DNA using the above compound as an energy transfer primer.
In the nucleotide sequencing method using a terminator, for example, 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 sequence of the template and 3 ′ deoxyribonucleotide are sequentially incorporated into the ribonucleotide sequence to synthesize a polyribonucleotide. Further, the resulting polyribonucleotide (nucleic acid transcription product) is separated, and the nucleotide sequence of the DNA is determined by reading the sequence of the nucleic acid from the obtained separated fraction. As a terminator for nucleic acid transcription, for example, a fluorescently labeled 3 ′ dNTP derivative is used, and the base sequence is determined by reading the label of the terminator.

  However, a terminator in which 3'dNTP is labeled with various labels may not be easily read into the nucleic acid sequence depending on the type of label and the binding mode of the label. In particular, the longer the chain length, the greater the tendency. Therefore, the terminator can be an unlabeled compound, and the nucleotide sequence can be determined using a labeled initiator. Further, at that time, the sensitivity of the label becomes a problem similarly to the case of the labeled terminator described above.

Therefore, a further object of the present invention is a method for determining a nucleotide sequence of DNA using RNA polymerase, which can be used as an initiator (transcription initiator) in a method for determining a nucleotide sequence without using a labeled terminator. And a compound having an energy transfer function.
It is a further object of the present invention to provide a method for determining the nucleotide sequence of DNA using the above compound as an energy transfer initiator.

   The present invention relates to a compound represented by the following general formula (1).

(Wherein Q represents a mono- or oligonucleotide residue, and V is -C≡C- (CH ₂ ) _n1 -NH- or -CH = CH- (CH ₂ ) _n2 -NH- (where n1 and n2 Represents an integer of 1 or more), R ¹ represents a trivalent group, R ² and R ³ each independently represent a hydrogen atom or a hydrocarbon group, or R ² and R ³ are bonded to each other And CH and NH adjacent to each other may form a ring, W ¹ and W ² each independently represent a fluorescent group, and m represents an integer of 1 or more.)

In the above compound, R ² , R ³ and m are preferably selected such that the distance between W ¹ and W ² is in the range of 10 to 100 °.
Further, in the above compound, Q is preferably a 2 ', 3'-dideoxyribonucleotide residue or a 3'-deoxyribonucleotide residue. This compound can be used as a terminator in a method for determining the base sequence of DNA by the chain terminator method.

The present invention relates to a method for determining the base sequence of DNA by the chain terminator method, wherein Q is a 2 ′, 3′-dideoxyribonucleotide residue or a 3′-deoxyribonucleotide residue. A chain termination reaction using the compound to be terminated as a terminator.
This method uses a compound represented by the general formula (1) in which Q is a 2 ', 3'-dideoxyribonucleotide residue as a terminator and uses a DNA polymerase, and Q uses a 3'-deoxyribonucleotide residue. The method includes a method using a compound represented by the general formula (1) as a terminator and using an RNA polymerase.

Further, in this method, four kinds of compounds corresponding to four kinds of bases selected from the compounds represented by the general formula (1) (provided that each compound has four different kinds of at least one of W ¹ and W ² ) ) As the terminator, and the chain termination reaction with the above four compounds can be performed in the same reaction system.

  The present invention relates to a compound represented by the general formula (1), wherein Q is an oligonucleotide residue having a 2'-deoxyribonucleotide residue at a terminal. This compound can be used as a primer in a method for determining the nucleotide sequence of DNA by the primer method.

  Furthermore, the present invention relates to a method for determining a nucleotide sequence of DNA by a primer method, wherein the above compound is used as a primer. In this method, an unlabeled terminator can be used.

  The present invention relates to a compound represented by the general formula (1), wherein Q is a mono- or oligonucleotide residue having no phosphate group at the 5 ′ end or having a mono- or diphosphate group. This compound can be used as an initiator in a method for determining the base sequence of DNA by the chain terminator method.

  Furthermore, the present invention is a method for determining the base sequence of DNA by the chain terminator method, wherein the 5 'end does not have a phosphate group, or a mono or oligonucleotide residue having a mono or diphosphate group, and The present invention relates to a method comprising performing a chain termination reaction using an initiator having two types of reporters, which can be a donor and an acceptor of energy transfer, and an RNA polymerase.

  In this method, a compound represented by the general formula (1) in which Q has no phosphate group at the 5 ′ end or is a mono or oligonucleotide residue having a mono or diphosphate group is used as an initiator. be able to. In addition, an unlabeled terminator can be used as the terminator.

Further, the present invention is a method for determining the nucleotide sequence of DNA by the chain terminator method, comprising a 3′-deoxyribonucleotide residue, a terminator having two types of reporters that can be donors and acceptors of energy transfer, and an RNA polymerase. And performing a chain termination reaction using the method.
In this method, the two reporters on the terminator are preferably located at a distance sufficient to cause energy transfer from the donor to the acceptor. Further, the distance sufficient for energy transfer from the donor to the acceptor to occur, for example, is in the range of 10-100 °. The reporter is selected from, for example, a group consisting of a group having a fluorescent property, a group emitting phosphorescence, a group labeled with spin, and a group having a high electron density.

  In this method, the donor of the terminator is, for example, a kind selected from the group consisting of a fluorescein dye, a rhodamine dye, and a 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene dye. And the acceptor is a member selected from the group consisting of a fluorescein dye, a rhodamine dye, and a 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene dye.

In this method, four types of terminators corresponding to four types of bases (each terminator has four different types of reporters as acceptors, respectively), and the chain termination reaction by the four types of terminators is the same reaction. It can be performed in the system.
In the method for determining a base sequence of the present invention, the chain termination reaction is preferably performed in the presence of inorganic pyrophosphatase.

Compound of the Present Invention In the compound of the present invention represented by the general formula (1), Q represents a mono- or oligonucleotide residue. More specifically, Q is, for example, a ribonucleotide residue, 2 ′, 3′-dideoxyribonucleotide residue, 2′-deoxyribonucleotide residue, 3′-deoxyribonucleotide residue, 5′-deoxyribonucleotide residue. It can be a nucleotide residue such as a group. A compound in which Q is a 2 ′, 3′-dideoxyribonucleotide residue or a 3′-deoxyribonucleotide residue can be used as a terminator in a method for determining a DNA base sequence by a chain terminator method. In particular, a compound in which Q is a 2 ', 3'-dideoxyribonucleotide residue is used as a terminator in a method using a DNA polymerase. A compound in which Q is a 3'-deoxyribonucleotide residue is used as a terminator in a method using RNA polymerase. 2 ′, 3′-dideoxyribonucleotide residue and 3′-deoxyribonucleotide residue include purine nucleotide residues represented by the following general formulas (2) and (3), and the following general formulas (4) and ( Pyrimidine nucleotide residues represented by 5) can be mentioned.

In the above formula, R ¹¹ and R ¹² may each be a hydrogen atom, or R ¹¹ may be a hydroxyl group and R ¹² may be a hydrogen atom. R ¹³ can _{-PO 3 H 2, -P 2 O} 6 H 3, which is -P ₃ O ₉ H ₄ or a salt thereof. Salts include alkali metal salts (e.g., sodium salts, potassium salts, lithium salts), alkaline earth metal salts (e.g., barium salts), ammonium salts, organic amine salts (e.g., triethylammonium salts, pyridine salts) and the like. Can be mentioned.

  Q can also be an oligonucleotide residue. Compounds in which Q is an oligonucleotide residue, in particular, a compound in which an oligonucleotide residue has a 2'-deoxyribonucleotide at the end, can be used as a primer in a method for determining a DNA base sequence by a primer method. The base sequence and length of the oligonucleotide residue can be appropriately determined in consideration of the function as a primer, and the length is, for example, in the range of 5 to 30 bases, and preferably in the range of 10 to 30 bases.

Further, Q can be a mono or oligonucleotide residue having no phosphate group at the 5 'end or having a mono or diphosphate group. Such a compound can be used as an initiator in a method for determining the base sequence of DNA by the chain terminator method. Mono or oligonucleotide residues having no phosphate group at the 5 'end or having a mono or diphosphate group include, for example, ribonucleoside residues, ribonucleoside 5' monophosphate residues, and ribonucleoside 5 'diphosphate residues. A phosphate residue, general formula N ¹ (N) _n , wherein N ¹ is a ribonucleoside, a ribonucleoside 5 ′ monophosphate, or a ribonucleoside 5 ′ diphosphate, and N is a ribonucleoside 5 ′ monophosphate An oligoribonucleotide residue represented by the formula: N ² (N) _n (wherein N ² is a group represented by the following formula (6)) , N is a ribonucleoside 5 ′ monophosphate, and n is an integer of 1 or more). It is it is possible.

More specifically, the mono- or oligonucleotide residues include, for example, guanosine, guanosine 5 ′ monophosphate (GMP), guanosine 5 ′ diphosphate (GDP), and the general formula N ¹ (N) _n-1 G (where N ¹ is ribonucleoside, ribonucleoside 5 ′ monophosphate, or ribonucleoside 5 ′ diphosphate, N is ribonucleoside 5 ′ monophosphate, n is an integer of 1 or more, G is guanosine 5 ′ monophosphate), and an oligoribonucleotide represented by the general formula N ² (N) _n-1 G (where N ² is a group represented by the above formula (6); Is a ribonucleoside 5 ′ monophosphate, n is an integer of 1 or more, and G is guanosine 5 ′ monophosphate. Mention may be made of each residue of the kind.
More specifically, as the above mono- or oligonucleotide residues, more specifically, adenosine, adenosine 5 ′ monophosphate (AMP), adenosine 5 ′ diphosphate (ADP), and the general formula N ¹ (N) _n-1 A (However, N ¹ is ribonucleoside, ribonucleoside 5 ′ monophosphate, or ribonucleoside 5 ′ diphosphate, N is ribonucleoside 5 ′ monophosphate, n is an integer of 1 or more, and A Is an adenosine 5 ′ monophosphate) and an oligoribonucleotide represented by the general formula N ² (N) _n-1 A (wherein N ² is a group represented by the above formula (6), and N is Ribonucleoside 5 ′ monophosphate, n is an integer of 1 or more, and A is adenosine 5 ′ monophosphate) Each residue of tides can be mentioned.
Further, specific examples of the above mono- or oligonucleotide residues include cytidine, cytidine 5 ′ monophosphate (CMP), cytidine 5 ′ diphosphate (CDP), and the general formula N ¹ (N) _n-1 C ( Wherein N ¹ is ribonucleoside, ribonucleoside 5 ′ monophosphate, or ribonucleoside 5 ′ diphosphate, N is ribonucleoside 5 ′ monophosphate, n is an integer of 1 or more, and C is Oligoribonucleotides represented by cytidine 5 ′ monophosphate) and a general formula N ² (N) _n-1 C (wherein N ² is a group represented by the above formula (6), and N is Nucleoside 5 ′ monophosphate, n is an integer of 1 or more, and C is cytidine 5 ′ monophosphate). Can be mentioned.

Further, specific examples of the above mono- or oligonucleotide residues include uridine, uridine 5 ′ monophosphate (UMP), uridine 5 ′ diphosphate (UDP), and the general formula N ¹ (N) _n-1 U ( Wherein N ¹ is ribonucleoside, ribonucleoside 5 ′ monophosphate, or ribonucleoside 5 ′ diphosphate, N is ribonucleoside 5 ′ monophosphate, n is an integer of 1 or more, and U is Oligoribonucleotides represented by uridine 5 ′ monophosphate) and a general formula N ² (N) _n-1 U (wherein N ² is a group represented by the above formula (6), and N is Nucleoside 5 ′ monophosphate, n is an integer of 1 or more, and U is uridine 5 ′ monophosphate) Can be mentioned.

The above-mentioned general formula _{^{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, in _{^{n 2 (n) n-1}} U, The bases of ribonucleoside, ribonucleoside 5 ′ monophosphate, and ribonucleoside 5 ′ diphosphate represented by N ¹ are not particularly limited, and can be appropriately selected from guanine, adenine, cytosine, and uridine, and are preferably guanine. Further, there is no particular limitation on the type of base of the 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. However, in practice, n is at most about 10 or less, preferably 5 or less, and more preferably 1 or 2.

In the compound represented by the general formula (1) of the present invention, V is -C≡C- (CH _{2) n1} -NH- or -CH = CH- (CH ₂₎ illustrating the _n2 -NH-. n1 and n2 represent an integer of 1 or more. Here, one end of a carbon atom of -C≡C- or -CH = CH- in V is a mono- or oligonucleotide residue represented by Q as described above, and at the 5-position of a pyrimidine nucleotide residue, In addition, the purine nucleotide residue binds to the 7th position.

Examples of the methylene chain represented _by- (CH ₂ ) _n1-and- (CH ₂ ) _n2- include a methylene chain in which n 1 and n 2 are 1 to 15, and specifically, a methylene group, ethylene Groups, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, heptamethylene group, octamethylene group, nonamethylene group, decamethylene group and the like.

However, when used as a terminator for a sequence reaction by RNA polymerase, from the viewpoint of high incorporation efficiency by RNA polymerase, n1 and n2 are preferably 4 or more, more preferably n1 and n2 are 4 to 10, and further preferably n1 and n2. And n2 is 4-8. When used as a terminator for a sequence reaction by a DNA polymerase, n1 and n2 are preferably 3 or more, more preferably n1 and n2 are 3 to 10, from the viewpoint of high incorporation efficiency by the DNA polymerase. When used as a primer or an initiator, n1 and n2 are appropriately determined in consideration of the activity of DNA polymerase or RNA polymerase used in the sequencing reaction.
R ¹ represents a trivalent group, for example,

（但し、R²¹〜R²³はそれぞれ独立して結合手または2価の炭化水素基を表す)等を挙げることができる。

(However, R ^{21 to} R ²³ each independently represent a bond or a divalent hydrocarbon group).

  The divalent hydrocarbon group may be aliphatic, aromatic, or a mixture of these. The divalent aliphatic hydrocarbon group may be linear, branched, or cyclic. Examples of the linear or branched divalent aliphatic hydrocarbon group include a divalent aliphatic hydrocarbon group having 1 to 6 carbon atoms, and specifically, a methylene group, an ethylidene group, 2-ethanediyl, propylidene, 1,2-propanediyl, 1,3-propanediyl, isopropylidene, butylidene, 1,2-butanediyl, 1,3-butanediyl, 1,4-butanediyl Group, 2-methyl-1,2-propanediyl group, 2-methyl-1,3-propanediyl group, pentylidene group, 1,2-pentanediyl group, 1,3-pentanediyl group, 1,4-pentanediyl group, 1,5-pentanediyl group, 2,3-pentanediyl group, 2,4-pentanediyl group, 2-methyl-1,2-butanediyl group, 2-methyl-1,3-butanediyl group, 2-methyl-1,4 -Butanediyl group, 2-methyl-1,5-butanediyl group, 2-methyl-2,3-butanediyl group, 2-methyl-2,4-butanediyl group, 2,2- Methyl-1,3-propanediyl, hexylidene, 1,2-hexanediyl, 1,3-hexanediyl, 1,4-hexanediyl, 1,5-hexanediyl, 1,6-hexane Examples thereof include a diyl group, a 2,3-hexanediyl group, a 2,4-hexanediyl group, and a 3,4-hexanediyl group. Among them, a divalent aliphatic hydrocarbon group having 1 to 4 carbon atoms is preferable. Examples of the cyclic divalent aliphatic hydrocarbon group include divalent aliphatic hydrocarbon groups having 3 to 7 carbon atoms, and specific examples thereof include a 1,2-cyclopropanediyl group and a 1,2-cyclobutane. Diyl group, 1,3-cyclobutanediyl group, 1,2-cyclopentanediyl group, 1,3-cyclopentanediyl group, 1,2-cyclohexanediyl group, 1,3-cyclohexanediyl group, 1,4-cyclohexane Diyl group, 1,2-cycloheptanediyl group, 1,3-cycloheptanediyl group, 1,4-cycloheptanediyl group and the like, among which divalent aliphatic hydrocarbon groups having 5 to 7 carbon atoms Is preferred. Examples of the divalent aromatic hydrocarbon group include a phenylene group, a biphenylene group, and a triphenylene group.

R ² and R ³ each independently represent a hydrogen atom or a hydrocarbon group, or R ² and R ³ may be bonded to each other to form a ring together with adjacent CH and NH. As the hydrocarbon group, for example, an aliphatic hydrocarbon group having 1 to 6 carbon atoms (which may be linear, branched, or cyclic; for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group , Isobutyl, tert-butyl, sec-butyl, n-pentyl, isopentyl, tert-pentyl, 1-methylpentyl, n-hexyl, isohexyl, cyclopropyl, cyclopentyl, cyclohexyl Aralkyl group having 7 to 10 carbon atoms (eg, benzyl group, phenethyl group, phenylpropyl group, methylbenzyl group, etc.), aryl group (eg, phenyl group, tolyl group, xylyl group, naphthyl group, biphenyl group, etc.) ) And the like. Examples of the ring formed by R ² and R ³ bonded to each other and adjacent CH and NH include a 3- to 6-membered ring which may further contain N and O. Examples thereof include an aziridine ring, an azetidine ring, a pyrrolidine ring, a pyrroline ring, an imidazolidine ring, an imidazoline ring, a pyrazolidine ring, a pyrazoline ring, a piperidine ring, a piperazine ring, and a morpholine ring. The number of repetitions m is an integer of 1 or more.

In the compounds of the present invention, R ² , R ³ and m are preferably chosen such that the distance between W ¹ and W ² is in the range from 10 to 100 °. Signals such as fluorescence resulting from the energy transfer varies depending on the distance between W ¹ and W ^2. The distance between W ¹ and W ^{2 at which} the strongest signal is obtained varies depending on the type of W ¹ and W ² . Therefore, R ² , R ³ and m are appropriately selected in consideration of the types of W ¹ and W ² .

Suitably, the distance between the two fluorescent groups is in the range of 10-100 °, preferably 20-60 °, more preferably about 30-50 °. When the ring formed by R ² and R ³ is a pyrrolidine ring, that is, a proline residue, 20 to 60 ° corresponds to a proline stretch of about 5 to 16 residues, and 30 to 50 ° corresponds to 8 to 12 residues. . It is appropriate that the above m is such that all amino acid residues including the proline stretch have a number corresponding to 20 to 60 °, preferably a number corresponding to 30 to 50 °.
W ¹ and W ² each independently represent a fluorescent group. Here, the fluorescent group is a group having a property of emitting fluorescence.

  Preferred fluorescent groups include fluorescein dyes, rhodamine dyes, 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene dyes, cyanine dyes, phthalocyanine dyes, squalanine dyes, and the like. Among them, fluorescein dyes, rhodamine dyes and 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene dyes are preferred.

More specifically, for example, 5 (or 6) carboxyfluorescein (hereinafter abbreviated as FAM), fluorescein, isothiocyanate, 5 (or 6) carboxy 4 ', 5'-dichloro-2' , 7'-Dimethoxyfluorescein (hereinafter abbreviated as JOE), 5 (or 6) carboxy 2 ', 4', 5 ', 7'-tetrachlorofluorescein, 5 (or 6) carboxy 2' , 4 ', 5', 7'-Tetrabromofluorescein, 5 (or 6) carboxy 4,7-dichloro-2 ', 7'-dimethoxyfluorescein, 5 (or 6) carboxy 4,7,4 ', 5'-tetrachloro-2', 7'-dimethoxyfluorescein, 5 (or 6) carboxy 2 ', 7'-dimethoxyfluorescein, 5 (or 6) carboxy 4, A7-dichloro-1' , 2 ', 7', 8'-Dibenzofluore Sein, 5 (or 6) carboxy 4,7-dichloro-1 ′, 2 ′, 7 ′, 8′-dibenzofluorescein, 5 (or 6) carboxytetramethylrhodamine (hereinafter abbreviated as TMR), 5 (or 6) carboxyrhodamine X (hereinafter abbreviated as XR), 5 (or 6) carboxyrhodamine 6G (hereinafter abbreviated as R6G), 5 (or 6) carboxyrhodamine 110 (hereinafter abbreviated as R110) 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a, 4a-diaza-s-indacene-8-propionic acid (hereinafter abbreviated as BODIPY493 / 503), 2,6-dibromo-4,4-difluoro, 5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-8-propionic acid (hereinafter abbreviated as BODIPY FL Br2) 4,4-difluoro-5-phenyl-4-bora-3a, 4a-diaza-s-indacene-8-propionic acid (hereinafter abbreviated as BODIPY R6G), 4,4-difluoro-5,7-diphenyl 4-bora-3a, 4a-diaza-s-indacene-8-propionic acid (hereinafter abbreviated as BODIPY530 / 550), 6-((4,4-difluoro-1,3-dimethyl-5 (4- Methoxyphenyl) -4-bora-3a, 4a-diaza-s-indacene-2-propionyl) amino) hexanoic acid (hereinafter abbreviated as BODIPY TMR), Oregon Green 488 carboxylic acid, ethidium bromide, 2-methoxy- Examples include those derived from fluorescent dyes such as 6-chloro-9-aminoacridine and 4-trifluoromethyl-7-ω-bromopropylaminocoumarin.
When the compound of the present invention is used as a terminator, in the method of determining the base sequence of DNA by the chain terminator method, four kinds of compounds designed to realize a stronger signal intensity more efficiently than the conventional four-color fluorescent terminator are used. It is important to be an energy transfer terminator. In order to carry out a termination reaction simultaneously for four bases of AGCT, four sets (combinations of donor dyes and acceptor dyes, which may be collectively referred to as a reporter) are provided.

W ¹ and W ² In the embodiment of the present invention, as any one of the donor dye and the other acts as an acceptor dye, as a set of W ¹ and W ^2, specifically, for example, the donor dye A (FAM), acceptor dye B (FAM, JOE, TMR, XR), donor dye A (FAM), acceptor dye B (FAM, R6G, TMR, XR), donor dye A (FAM), acceptor dye B (R6G , TMR, XR, R110), donor dye A (BODIPY 493/503), acceptor dye B (BODIPY FLBr2, BODIPY R6G, BODIPY TMR, BODIPY 530/550), donor dye A (FAM), acceptor dye B (BODIPY FLBr2) BODIPY R6G, BODIPY TMR, BODIPY 530/550), donor dye A Oregon Green 488), an acceptor dye B (BODIPY FLBr2, BODIPY R6G, BODIPY TMR, BODIPY 530/550) is set, and the like preferably not be construed as being limited thereto.

  Since the compound of the present invention has two kinds of fluorescent dyes bound to one mono- or oligonucleotide, when these are used as, for example, a terminator, there is an advantage that RNA polymerase and DNA polymerase can be incorporated without steric hindrance. Have. Further, the compound of the present invention has a chemical structure that adopts a specific high-order structure at a high speed so that it can be detected with high sensitivity based on the principle of energy transfer, and is useful as a terminator, a primer and an initiator.

  The compound of the present invention represented by the general formula (1) is, for example, a compound represented by the following general formula (7) and a compound represented by the following general formula (8) according to the following reaction formula (V). Can be synthesized by reacting

（式中、Ｑ及びＶは前記と同じ）

(Where Q and V are the same as above)

（式中、Ｓｕはコハク酸イミド基を示し、他は前記と同じ）

(In the formula, Su represents a succinimide group, and the others are the same as described above.)

  That is, as shown in the above reaction formula [V], a compound represented by the general formula (7) and a compound represented by the general formula (8) are mixed in a solvent such as DMF or DMF-water, If necessary, the compound of the general formula (1) can be obtained by reacting at 10 to 40 ° C. for one to several hours in the presence of a basic catalyst such as 4-dimethylaminopyridine.

  In addition, the compound represented by the general formula (7) can be obtained, for example, by the method described in JP-A No. 10-158293, columns 9 to 18, [0056] to [0067], specifically, the method described in [Example]. It can be synthesized by the method described in the section.

The compound represented by the general formula (8) can be synthesized according to (I) to (IV) in the above reaction scheme. Incidentally, Poly in the reaction scheme indicates a resin (solid phase). That is, for example, synthesized by a conventional method such as a solid phase method, for example, (Pro) m-εBoc-Lys (wherein, Pro represents a proline residue, Lys represents a lysine residue, and Boc is a t-butyloxycarbonyl group. And m represents a positive integer.) W ¹ is introduced into the N-terminus of [Reaction formula (I)], and then the Boc group is deprotected [Reaction formula (II)]. ² can be easily obtained by introducing [Reaction formula (III)] and further introducing a succinimide group (Su) [Reaction formula (IV)].

Base sequence determination method
(1) Method using a terminator having an energy transfer function The base sequence determination method of the present invention using a terminator having an energy transfer function includes the following methods.
A method for determining the nucleotide sequence of DNA by the chain terminator method, comprising using a terminator having a 3'-deoxyribonucleotide residue, and two types of reporters that can serve as energy transfer donors and acceptors, and an RNA polymerase. A method of performing a reaction (hereinafter, this method is referred to as a first method).

  Further, a method for determining the base sequence of DNA by the chain terminator method, comprising performing a chain termination reaction using the compound of the present invention represented by the general formula (1) as a terminator (hereinafter, this method is referred to as a second method). Method). This method includes a method using a compound represented by the general formula (1) in which Q is a 2 ′, 3′-dideoxyribonucleotide residue as a terminator and using a DNA polymerase, and a method using Q as a 3′-deoxyribonucleotide residue. There is a method using a compound represented by the general formula (1) as a terminator and using an RNA polymerase. The method using RNA polymerase overlaps with the first method.

  In the first method, a terminator having a 3'-deoxyribonucleotide residue and two types of reporters that can serve as donors and acceptors for energy transfer is used. The reporter can be selected, for example, from the group consisting of, for example, phosphorescent groups, spin-labeled groups, and electron-dense groups, as well as fluorescent groups. The two reporters on the terminator are preferably located at a distance sufficient to cause energy transfer from the donor to the acceptor, and a distance sufficient to cause energy transfer from the donor to the acceptor is, for example, 10 It is in the range of Å100 °.

When the reporter is a fluorescent group, the fluorescent group can be appropriately selected in consideration of fluorescence intensity, fluorescence wavelength, ease of incorporation by RNA polymerase, and the like. However, a fluorescent group is a fluorescent group that produces a detectable luminescent emission following stimulation by absorption of energy from a suitable source, such as an argon laser, wherein the fluorescent group has one of the luminescent emission wavelengths. It is preferable that the set is selected from a group that resonates with the other excitation wavelength.
As the preferred fluorescent group, and the like can be exemplified those mentioned for W ¹ and W ² above.

  When the reporter is a fluorescent group, the distance between two fluorescent dyes (donor and acceptor) is designed to be constant. As a structure for keeping the intermolecular distance constant, for example, a proline stretch can be mentioned as described above. Suitably, the distance between the two fluorescent dyes is in the range of 10 to 100 °, preferably 20 to 60 °, more preferably about 30 to 50 °. In the case of a proline stretch, 20-60 ° corresponds to about 5-16 residues and 30-50 ° corresponds to 8-12 residues.

  When the reporter is a fluorescent group, in the method of determining the base sequence of DNA by the chain terminator method, four types of energy transfer designed to realize a stronger signal intensity more efficiently than the conventional four-color fluorescent terminator It is preferred to select a terminator. In order to carry out a termination reaction simultaneously for four bases of AGCT, four sets (combination of a donor dye and an acceptor dye) are used. As such a set, specifically, for example, donor dye A (FAM), acceptor dye B (FAM, JOE, TMR, XR), donor dye A (FAM), acceptor dye B (FAM, R6G, TMR, XR), donor dye A (FAM), acceptor dye B (R6G, TMR, XR, R110), donor dye A (BODIPY 493/503), acceptor dye B (BODIPY FLBr2, BODIPY R6G, BODIPY TMR, BODIPY 530/550) ), Donor dye A (FAM), acceptor dye B (BODIPY FLBr2, BODIPY R6G, BODIPY TMR, BODIPY 530/550), donor dye A (Oregon green 488), acceptor dye B (BODIPY FLBr2, BODIPY R6G, BOD) PY TMR, BODIPY 530/550) is set, and the like preferably not be construed as being limited thereto.

Specific examples of such a terminator in which the reporter is a fluorescent group include the compound of the present invention represented by the general formula (1).
The second method for determining a base sequence of the present invention is a method based on the chain terminator method, wherein the chain termination reaction is performed using the compound of the present invention represented by the general formula (1) as a terminator. However, the second method of the present invention also includes a method using a DNA polymerase as a polymerase in addition to a method using RNA polymerase as a polymerase.

  In the first method and the second method of the present invention, a known method can be used as it is, except that the compound is used as a terminator. Methods using a DNA polymerase as a polymerase are disclosed in, for example, JP-A-1-180455, JP-A-5-502371, and JP-A-6-510433. In addition, the DNA polymerase can be a polymerase having improved heat resistance or a mutant polymerase having improved incorporation rate difference depending on the type of base.

  When a DNA polymerase is used as the polymerase and the compound represented by the general formula (1) is used as the terminator, the compound has a 2 ′, 3′-dideoxyribonucleotide residue and n1 or n2 is 3 or more. And more preferably an integer of 3 to 10 from the viewpoint that the incorporation by DNA polymerase is good and sequence data with high accuracy can be obtained.

When RNA polymerase is used as the polymerase, the terminator of the present invention having a 3′-deoxyribonucleotide residue or the compound of the present invention having a 3′-deoxyribonucleotide residue represented by the general formula (1) is used. As a method for determining a nucleotide sequence using RNA polymerase as a polymerase, for example, the method described in WO96 / 14434 can be used except for using the terminator or the compound of the present invention.
That is, in the presence of RNA polymerase and a DNA fragment containing a promoter sequence for the RNA polymerase, a ribonucleoside 5′-triphosphate composed of ATP, GTP, CTP and UTP or a derivative thereof and a terminator according to the present invention are provided. 3 ′ dATP selected from terminators, 3 ′ dGTP, 3 ′ dCTP, one or more of derivatives of 3 ′ dUTP, preferably four types of 3′-deoxyribonucleoside 5′-triphosphate (3′- (dNTP derivative) is reacted to obtain a nucleic acid transcription product, the obtained nucleic acid transcription product is separated, and the nucleic acid sequence is read from the obtained separated fraction, whereby the DNA base sequence can be determined.

  Further, the RNA polymerase can be a polymerase having improved heat resistance or a mutant polymerase having improved incorporation rate difference depending on the type of base. The DNA fragment used as a template is not limited except that it contains a promoter sequence for RNA polymerase. For example, it can be a DNA product obtained by amplifying a DNA fragment containing a promoter sequence by a polymerase chain reaction (PCR method). Further, without removing the primers and / or 2 ′ deoxyribonucleoside 5 ′ triphosphates and / or derivatives thereof used in the polymerase chain reaction from the amplified DNA product, the nucleic acid transcription production reaction in the method of the present invention can be performed. It can be carried out. Further, the DNA fragment containing the promoter sequence may be a DNA fragment obtained by ligating the promoter sequence and the DNA fragment to be amplified and then cloning using an appropriate host.

  When RNA polymerase is used as the polymerase and the compound represented by the general formula (1) is used as the terminator, the compound has a 3′-deoxyribonucleotide residue, and n1 or n2 is 4 or more, more preferably An integer of 4 to 10 is preferable from the viewpoint that the uptake by RNA polymerase is good and sequence data with high accuracy can be obtained.

Furthermore, in the method for determining the nucleotide sequence of a DNA of the present invention, four types of terminators corresponding to four types of bases selected from the terminator of the present invention or the compound represented by the general formula (1) (however, The terminator has four different types of reporters as acceptors, respectively), and the chain termination reaction can be performed in the same reaction system. When a compound represented by the general formula (1) is used as a terminator, ^one of W ¹ and W ² is an acceptor, and each compound has four different types of acceptors. In this method, the base sequence can be determined more efficiently with fewer operations by performing the incorporation reaction of the terminators corresponding to the four AGCTs in one tube.

  Furthermore, in the method of the present invention, the chain termination reaction is carried out in the presence of an inorganic pyrophosphatase as described later, by reducing the difference in peak height obtained for each ribonucleotide and reading the sequence. This is preferable from the viewpoint that the precision of the slab can be improved.

(2) Method Using a Primer Having an Energy Transfer Function The third method for determining the base sequence of DNA of the present invention is a method for determining the base sequence of DNA by a primer method, wherein Q is an oligonucleotide residue. A method comprising using, as a primer, a compound of the present invention represented by the formula (1), in which Q is an oligonucleotide residue having a 2'-deoxyribonucleotide residue at its terminal. .
In the third method of the present invention, a known dideoxy method using a DNA polymerase can be used except that the above compound is used as a primer. As a known method, for example, the method disclosed in WO95 / 21266 and JP-A-10-88124 can be used.

(3) Method Using an Initiator Having an Energy Transfer Function The fourth method for determining a DNA base sequence of the present invention is a method for determining a base sequence of DNA by a chain terminator method, wherein a phosphate group is added to the 5 ′ end. A chain termination reaction is carried out using an RNA polymerase and a mono- or oligonucleotide residue having no or a mono- or diphosphate group, and an initiator having two types of reporters that can be energy transfer donors and acceptors. A method characterized by the following. As the above-mentioned initiator, the compound of the present invention represented by the general formula (1) in which Q is a mono- or oligonucleotide residue having no phosphate group at the 5 'end or having a mono- or di-phosphate group is used. be able to.

  More specifically, this method is a method for preparing a ribonucleoside 5 ′ triphosphate comprising ATP, GTP, CTP and UTP or a derivative thereof in the presence of an RNA polymerase and a DNA fragment containing a promoter sequence for the RNA polymerase. And 3 ′ dATP, 3 ′ dGTP, 3 ′ dCTP, 3 ′ dUTP and one type of 3 ′ deoxyribonucleoside 5 ′ triphosphate (hereinafter referred to as 3 ′ dNTP derivative) selected from the group consisting of derivatives thereof. A method for determining a nucleotide sequence of DNA, comprising obtaining a nucleic acid transcription product, separating the obtained nucleic acid transcription product, and reading the sequence of the nucleic acid from the obtained separated fraction, wherein the initiator of the nucleic acid transcription reaction is: The above initiator is used. An unlabeled compound can be used as the 3'dNTP derivative as a terminator.

  Using a DNA fragment containing a promoter sequence for RNA polymerase as a template, a method for enzymatically synthesizing a nucleic acid transcription product using RNA polymerase, a method for separating a nucleic acid transcription product, and a method for separating nucleic acids from separated fractions. Any method of reading the sequence is a known method in principle. Accordingly, any of the known methods, conditions, devices, and the like can be appropriately used in these respects. For example, the description regarding the terminator method described in WO96 / 14434 can be appropriately referred to.

  The DNA fragment used as a template is not limited except that it contains a promoter sequence for RNA polymerase. 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, the nucleic acid transcription production reaction in the method of the present invention is carried out without removing the primer and / or 2 ′ deoxyribonucleoside 5 ′ triphosphate and / or a derivative thereof used in the PCR method from the amplified DNA product. be able to. Further, the DNA fragment containing the promoter sequence may be a DNA fragment obtained by ligating the promoter sequence and the DNA fragment to be amplified and then cloning using an appropriate host. That is, in the present invention, there are no particular restrictions on the DNA sequence to be amplified, primers, amplification conditions, 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, and 200 μM of each 2 ′ deoxyribonucleoside 5 ′ trifoam. For example, Taq polymerase or the like can be used as a DNA polymerase in a volume of 20 μl containing sulfate (dATP, dGTP, dCTP, dTTP).

However, it is necessary that either one of the primers for the polymerase chain reaction or the amplified insert DNA contains a promoter sequence for an RNA polymerase described later. In the direct transcription sequencing method, in the PCR method, a primer having a phage promoter sequence is used as one of two types of primers, or a phage promoter sequence is provided in the amplified inserted DNA. The resulting PCR product can be subjected to in vitro transcription using an RNA polymerase driven by the promoter.
The promoter sequence for RNA polymerase can be appropriately selected depending on the type of RNA polymerase to be used.

  In the fourth method of the present invention, a transcript such as RNA is synthesized from a DNA fragment containing a promoter sequence. Since the DNA 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.

  Synthesis of transcripts such as RNA is carried out in the presence of the above-mentioned nucleic acid transcription initiator and RNA polymerase by using ribonucleoside 5 ′ triphosphates (NTPs) composed of ATP, GTP, CTP and UTP or derivatives thereof and one type of 3TP. 'React the dNTP derivative. The 3 'dNTP derivative is used herein as a generic term for 3' dATP, 3 'dGTP, 3' dCTP, 3 'dUTP and derivatives thereof. As the ribonucleoside 5 'triphosphates (NTPs), at least four types of compounds having different bases are necessary for the synthesis of a transcript, including the case where some of them are derivatives such as NTP.

  By incorporating a 3 'dNTP derivative into the 3' end of the RNA or nucleic acid that is a transcription product, the 3 'hydroxy group is lost and the 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 ribonucleoside analogs, they can be used for determination of RNA or nucleic acid sequences [Vladimir D. Axelreder al. (1985) Biochemistry Vol. 24, 5716-5723].

  As for the 3 ′ dNTP derivative, one type is used for one nucleic acid transcription reaction, the nucleic acid transcription reaction is performed four times using four different types of 3 ′ dNTP derivatives, and the 3 ′ dNTP derivative at the 3 ′ end has a different base. Four transcripts are obtained. A single nucleic acid transcription reaction yields a transcription product that is a mixture of various RNA or nucleic acid fragments having the same 3 'dNTP derivative at the 3' end and different molecular weights. The resulting four transcription products can be independently subjected to separation and sequence reading described below. Alternatively, two or more of the four types of transcription products can be mixed, and this mixture can be subjected to separation and sequence reading.

  The RNA polymerase used in the fourth method of the present invention may be either a wild-type RNA polymerase or a mutant RNA polymerase. For the RNA polymerase, the description relating to the first or second method can be referred to.

Inorganic pyrophosphatase In the first to fourth methods 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 making it possible to obtain more accurate sequence data.
Pyrophosphorolysis occurs by increasing the amount of pyrophosphate produced by DNA synthesis, and serves to promote the reaction in the direction in which the synthesized DNA product is degraded. As a result, pyrophosphorolysis will inhibit sequencing in the dideoxy sequencing method using DNA polymerase. On the other hand, it is known that when inorganic pyrophosphatase is used in the dideoxy sequencing method using DNA polymerase, pyrophosphorolysis is inhibited and stable sequence data can be obtained [Japanese Patent Laid-Open No. 4-506002]. ].

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, commercially available from Sigma as INORGANIC PYROPHOSPHATASE and from Boehringer as pyrophosphatase. The amount of the inorganic pyrophosphatase used depends on the degree of the activity of the inorganic pyrophosphatase and the RNA polymerase. For example, the amount is preferably in the range of 10 ^-6 to 10 ^-2 units per 1 unit of the RNA polymerase. is there.

  According to the present invention, the sensitivity of the corresponding signal is 5 to 20 times higher than that of a normal fluorescent label terminator having only one reporter such as a fluorescent dye with the same number of molecules. In particular, an effect is particularly exhibited in the case of electrophoresis using an ultra-fine lithography channel or the like by using a multiplexed capillary sequencer or a lithography channel. In particular, the tendency of multiplexing is that the cross-sectional area of one electrophoretic lane tends to decrease, and it is considered that the amount of DNA that can be loaded is almost proportional to the cross-sectional area.

  Therefore, the amount of DNA that can be loaded is considered to be inversely proportional to the square of the diameter of the electrophoresis lane. Therefore, for multiplication, the width of the ultrafine electrophoresis channel by capillary array or lithography is set to the diameter of one capillary. If the width of the entire capillary array is constant, the sensitivity is required to be proportional to the square of the number.

  The present invention is considered to be extremely effective in diversifying the sequencing lanes. In addition, in the present invention, since a structure having a very large molecular weight is bound to one terminator nucleotide, the mobility of the electrophoresis of the finally formed sequencing reaction product tends to be slower than when a normal terminator is used. is there. Therefore, it is appropriate to newly program computer software for base call of the sequencer.

Hereinafter, the present invention will be described in more detail with reference to Examples.
The abbreviations used in the following examples are as follows.
Fmoc: 9-fluorenylmethyloxycarbonyl group
Boc: t-butyloxycarbonyl group
Lys: Lysine Pro: Proline Ac: Acetyl group

Synthesis Example 1 Synthesis of FAM- (Pro) 8-Lys (εTMR) (1) Synthesis of FAM- (Pro) 8-Lys αFmoc-εBoc-Lys-Alko resin (100 to 200 mesh, manufactured by Watanabe Chemical Industry Co., Ltd.) ) 5.0 g (2.4 mmol as αFmoc-εBoc-Lys) was used as a starting material, and was synthesized by a solid phase method according to the method described in the literature (Basic and Experimental Peptide Synthesis (Maruzen Publishing), p218).
That is, first, the αFmoc-εBoc-Lys-Alko resin was treated with piperidine to remove the Fmoc group. A 1-methyl-2-pyrrolidone solution containing 3 equivalents of Fmoc-Pro (manufactured by Wako Pure Chemical Industries, Ltd.), 3 equivalents of HOBt, and 3 equivalents of Lys on the resin N, N′-diisopropylcarbodiimide was added, and amino acids were successively introduced by performing a condensation reaction at room temperature for 5 hours. After introducing all amino acids, 3-fold equivalent of 5-carboxyfluorescein succinimide ester (Molecular Probes) was condensed. After completion of the reaction, the resin was washed with MeOH, 100 ml of a mixed solution of 55% TFA-dichloromethane and anisole (1 ml) was added, and the mixture was stirred and reacted at room temperature for 1 hour to simultaneously cut the target FAM-labeled polypeptide from the resin and simultaneously form a Boc group (Lys Was cleaved. After completion of the reaction, the resin was removed by filtration, the filtrate was concentrated under reduced pressure, and ether was added to precipitate a reaction product. The precipitate was collected and dried in a desiccator to obtain 2.2 g of FAM- (Pro) 8-Lys.

(2) Synthesis of FAM- (Pro) 8-Lys (εTMR) 500 mg of FAM- (Pro) 8-Lys synthesized in Synthesis Example 1- (1) was dissolved in 1 ml of DMF, and 3 equivalents of triethylamine and 2.5 equivalents were used. Of 5-carboxytetramethylrhodamine succinimide ester (manufactured by Molecular Probes) was added and reacted at room temperature for 19 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the obtained residue was purified using a gel filtration column (Sephadex LH20, manufactured by Pharmacia) to obtain 420 mg of FAM- (Pro) 8-Lys (εTMR).

Synthesis Example 2 Synthesis of FAM- (Pro) 8-Lys (εXR) 500 mg of FAM- (Pro) 8-Lys obtained in Synthesis Example 1- (1) and 2.5 equivalents of 5-carboxy-X-rhodamine succinimide ester (Manufactured by Molecular Probes Co., Ltd.) and 380 mg of FAM- (Pro) 8-Lys (εXR) were obtained by using the same reagents as in Synthesis Example 1- (2) and performing the same operation.

Synthesis Example 3
Synthesis of FAM- (Pro) 8-Lys (εR6G) 500 mg of FAM- (Pro) 8-Lys obtained in Synthesis Example 1- (1) and 2.5 equivalents of 5-carboxyrhodamine 6G succinimide ester (Molecular Probes, Inc.) Using the same reagents as in Synthesis Example 1- (2), the same operation was performed to obtain 400 mg of FAM- (Pro) 8-Lys (εR6G).

Synthesis Example 4 Synthesis of FAM- (Pro) 8-Lys (εR110) 500 mg of FAM- (Pro) 8-Lys obtained in Synthesis Example 1- (1) and 3.5 equivalents of 5-carboxyrhodamine 110 bis-trifluoroacetate Using succinimide ester (manufactured by Molecular Probes) and the same reagent as in Synthesis Example 1- (2), after completion of the reaction, the same operation was performed to obtain 300 mg of FAM- (Pro) 8-Lys (εR110). Was.

Synthesis Example 5 Synthesis of FAM- (Pro) 10-Lys (εTMR) (1) Synthesis of FAM- (Pro) 10-Lys αFmoc-εBoc-Lys-Alko resin (100 to 200 mesh, manufactured by Watanabe Chemical Industry Co., Ltd.) Using 5.0 g (2.4 mmol as αFmoc-εBoc-Lys) as a starting material, H-Pro10-Lys (Boc) -Alko resin was synthesized by the same operation as in Synthesis Example 1 (yield 6.8 g). 5-Carboxyfluorescein succinic acid prepared from 5-carboxyfluorescein (manufactured by Molecular Probes) (0.6 g), N-hydroxysuccinimide (0.37 g) and diisopropylcarbodiimide (500 μl) per 1 g of the dried resin The imide ester was condensed.
After the completion of the reaction, the resin was washed with DMF and MeOH, a mixed solution of 95% TFA-anisole (8 ml) was added, and the mixture was stirred and reacted at room temperature for 1 hour to cleave the target FAM-labeled polypeptide from the resin. After completion of the reaction, the resin was removed by filtration, the filtrate was concentrated under reduced pressure, and ether was added to precipitate a reaction product. The precipitate was collected and dried in a desiccator to obtain FAM- (Pro) 10-Lys (0.63 g).

(2) Synthesis of FAM- (Pro) 10-Lys (εTMR) FAM- (Pro) 10-Lys (13.7 mg) synthesized in Synthesis Example 5- (1) was dissolved in DMF (1 ml), and triethylamine (20 μl) was dissolved. ) And 6-carboxytetramethylrhodamine succinimide ester (manufactured by Molecular Probes) (4.88 mg) were added and reacted at room temperature for 19 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the obtained residue was purified using a gel filtration column (Sephadex LH20, manufactured by Pharmacia) to obtain FAM- (Pro) 10-Lys (εTMR) (10.9 mg). .

Synthesis Example 6 Synthesis of FAM- (Pro) 10-Lys (εXR) FAM- (Pro) 10-Lys (9.63 mg) obtained in Synthesis Example 5- (1) was dissolved in DMF (1 ml), and triethylamine (20 μl) was dissolved. ) And 6-carboxy-X-rhodamine succinimide ester (manufactured by Molecular Probes, Inc.) (3.78 mg) were condensed by the same operation as in Synthesis Example 5- (2), purified, and purified by FAM- (Pro ) 10-Lys (εXR) (6.53mg) was obtained.

Synthesis Example 7 Synthesis of FAM- (Pro) 10-Lys (εR6G) FAM- (Pro) 10-Lys (3.18 mg) obtained in Synthesis Example 5- (1) was dissolved in DMF (0.5 ml), and triethylamine ( 10 μl) and 6-carboxyrhodamine 6G succinimide ester (manufactured by Molecular Probes, Inc.) (1.11 mg) were condensed and purified by the same operation as in Synthesis Example 5- (2), followed by purification of FAM- (Pro). 10-Lys (εR6G) (2.66 mg) was obtained.

Synthesis Example 8 Synthesis of FAM- (Pro) 10-Lys (εR110) FAM- (Pro) 10-Lys (5.00 mg) obtained in Synthesis Example 5- (1) was dissolved in DMF (0.5 ml), and triethylamine ( 10 μl) and 5-carboxyrhodamine 110 bis-trifluoroacetate succinimide ester (manufactured by Molecular Probes) (2.07 mg) were condensed and purified by performing the same operation as in Synthesis Example 5- (2). FAM- (Pro) 10-Lys (εR110) (3.00 mg) was obtained.

Synthesis Example 9 Synthesis of FAM- (Pro) 12-Lys (εTMR) (1) Synthesis of FAM- (Pro) 12-Lys αFmoc-εBoc-Lys-Alko resin (100 to 200 mesh, manufactured by Watanabe Chemical Industry Co., Ltd.) H-Pro12-Lys (Boc) -Alko resin was synthesized by performing the same operation as in Synthesis Example 1 using 5.0 g (2.4 mmol as αFmoc-εBoc-Lys) as a starting material (yield 7.2 g). 5-carboxyfluorescein succinic acid prepared from 5-carboxyfluorescein (manufactured by Molecular Probes) (0.47 g), N-hydroxysuccinimide (0.27 g) and diisopropylcarbodiimide (330 μl) per 1 g of the dried resin The imide ester was condensed.
After the completion of the reaction, the resin was washed with DMF and MeOH, a mixed solution of 95% TFA-anisole (8 ml) was added, and the mixture was stirred and reacted at room temperature for 1 hour to cleave the target FAM-labeled polypeptide from the resin. After completion of the reaction, the resin was removed by filtration, the filtrate was concentrated under reduced pressure, and ether was added to precipitate a reaction product. The precipitate was collected and dried in a desiccator to obtain FAM- (Pro) 12-Lys (0.58 g).

(2) Synthesis of FAM- (Pro) 12-Lys (εTMR) FAM- (Pro) 12-Lys (14.7 mg) synthesized in Synthesis Example 9- (1) was dissolved in DMF (1 ml), and triethylamine (20 μl) was dissolved. ) And 6-carboxytetramethylrhodamine succinimide ester (manufactured by Molecular Probes) (4.17 mg) were added, and the mixture was reacted at room temperature for 19 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the obtained residue was purified using a gel filtration column (Sephadex LH20, manufactured by Pharmacia) to obtain FAM- (Pro) 12-Lys (εTMR) (7.6 mg). .

Synthesis Example 10 Synthesis of FAM- (Pro) 12-Lys (εXR) FAM- (Pro) 12-Lys (3.76 mg) obtained in Synthesis Example 9- (1) was dissolved in DMF (0.5 ml), and triethylamine ( 10 μl) and 6-carboxy-X-rhodamine succinimide ester (manufactured by Molecular Probes, Inc.) (1.33 mg) were condensed and purified by the same operation as in Synthesis Example 9- (2), followed by purification of FAM- ( Pro) 12-Lys (εXR) (6.53 mg) was obtained.

Synthesis Example 11 Synthesis of FAM- (Pro) 12-Lys (εR6G) FAM- (Pro) 12-Lys (3.18 mg) obtained in Synthesis Example 9- (1) was dissolved in DMF (0.5 ml), and triethylamine ( 10 μl) and 6-carboxyrhodamine 6G succinimide ester (manufactured by Molecular Probes, Inc.) (1.11 mg) were condensed and purified by the same operation as in Synthesis Example 9- (2), followed by purification of FAM- (Pro). 12-Lys (εR6G) (2.75 mg) was obtained.

Synthesis Example 12 Synthesis of FAM- (Pro) 12-Lys (εR110) FAM- (Pro) 12-Lys (5.00 mg) obtained in Synthesis Example 9- (1) was dissolved in DMF (0.5 ml), and triethylamine ( 10 μl) and 5-carboxyrhodamine 110 bis-trifluoroacetate succinimide ester (manufactured by Molecular Probes) (0.86 mg) were condensed and purified by performing the same operation as in Synthesis Example 9- (2). FAM- (Pro) 12-Lys (εR110) (1.2 mg) was obtained.

Synthesis Example 13 Synthesis of FAM- (Pro) 4-Lys (εTMR) (1) Synthesis of FAM- (Pro) 4-Lys αFmoc-εBoc-Lys-Alko resin (100 to 200 mesh, manufactured by Watanabe Chemical Industry Co., Ltd.) H-Pro4-Lys (Boc) -Alko resin was synthesized by performing the same operation as in Synthesis Example 1 using 5.0 g (2.4 mmol as αFmoc-εBoc-Lys) as a starting material (yield 5.4 g). 5-carboxyfluorescein (manufactured by Molecular Probes) (256 mg), 400 mg of dried resin, 5-carboxyfluorescein succinimide ester prepared from N-hydroxysuccinimide (72 g) and diisopropylcarbodiimide (90 μl) were used. Condensed. After completion of the reaction, the resin was washed with DMF and MeOH, a mixed solution of 95% TFA-anisole (8 m) was added, and the mixture was stirred and reacted at room temperature for 1 hour to cleave the desired FAM-labeled polypeptide from the resin. After completion of the reaction, the resin was removed by filtration, the filtrate was concentrated under reduced pressure, and ether was added to precipitate a reaction product. The precipitate was collected and dried in a desiccator to obtain FAM- (Pro) 4-Lys (66.8 mg).

(2) Synthesis of FAM- (Pro) 4-Lys (εTMR) FAM- (Pro) 4-Lys (10 mg) synthesized in (1) was dissolved in DMF (1 ml), and triethylamine (20 μl) and 6-carboxy Tetramethylrhodamine succinimide ester (manufactured by Molecular Probes) (5.28 mg) was added and reacted at room temperature for 19 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the obtained residue was purified using a gel filtration column (Sephadex LH20, manufactured by Pharmacia) to obtain FAM- (Pro) 4-Lys (εTMR) (9.8 mg). .

Synthesis Example 14 Synthesis of FAM- (Pro) 6-Lys (εTMR) (1) Synthesis of FAM- (Pro) 6-Lys αFmoc-εBoc-Lys-Alko resin (100 to 200 mesh, manufactured by Watanabe Chemical Industry Co., Ltd.) H-Pro4-Lys (Boc) -Alko resin was synthesized by performing the same operation as in Synthesis Example 1 using 5.0 g (2.4 mmol as αFmoc-εBoc-Lys) as a starting material (yield: 5.8 g). 5-Carboxyfluorescein succinimide ester prepared from 5-carboxyfluorescein (manufactured by Molecular Probes) (128 mg), N-hydroxysuccinimide (73.7 mg) and diisopropylcarbodiimide (90 μl) relative to 218 mg of the dried resin Was condensed. After completion of the reaction, the resin was washed with DMF and MeOH, a mixed solution of 95% TFA-anisole (8 m) was added, and the mixture was stirred and reacted at room temperature for 1 hour to cleave the desired FAM-labeled polypeptide from the resin. After completion of the reaction, the resin was removed by filtration, the filtrate was concentrated under reduced pressure, and ether was added to precipitate a reaction product. The precipitate was collected and dried in a desiccator to obtain FAM- (Pro) 6-Lys (20.6 mg).

(2) Synthesis of FAM- (Pro) 6-Lys (εTMR) FAM- (Pro) 6-Lys (12 mg) synthesized in (1) was dissolved in DMF (1 ml), and triethylamine (20 μl) and 6-carboxy were added. Tetramethylrhodamine succinimide ester (manufactured by Molecular Probes) (5.5 mg) was added and reacted at room temperature for 19 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the obtained residue was purified using a gel filtration column (Sephadex LH20, manufactured by Pharmacia) to obtain FAM- (Pro) 6-Lys (εTMR) (9.29 mg). .

Reference Example 1 Synthesis of Ac- (Pro) 10-Lys (εTMR) (1) Synthesis of Ac- (Pro) 10-Lys H-Pro10-Lys (Boc) -Alko resin obtained in Synthesis Example 5- (1) (1.5 g) was swollen in DMF (35 ml), acetic anhydride (230 μl) and triethylamine (336 μl) were added, and the mixture was reacted for 20 hours. After the completion of the reaction, the resin was washed with DMF and MeOH, a mixed solution of 95% TFA-anisole (8 ml) was added, and the mixture was stirred and reacted at room temperature for 1 hour to cleave the target FAM-labeled polypeptide from the resin. After completion of the reaction, the resin was removed by filtration, the filtrate was concentrated under reduced pressure, and ether was added to precipitate a reaction product. The precipitate was collected and dried in a desiccator to obtain Ac- (Pro) 10-Lys (0.45 g).
(2) Synthesis of Ac- (Pro) 10-Lys (εTMR) Ac- (Pro) 10-Lys (11.8 mg) synthesized in Reference Example 1- (1) was dissolved in DMF (1 ml), and triethylamine (20 μl) was dissolved. ) And 6-carboxytetramethylrhodamine succinimide ester (manufactured by Molecular Probes) (5.7 mg) were added and reacted at room temperature for 19 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the obtained residue was purified using a gel filtration column (Sephadex LH20, manufactured by Pharmacia) to obtain Ac- (Pro) 10-Lys (εTMR) (6.0 mg). .

Reference Example 2 Synthesis of Ac- (Pro) 10-Lys (εXR) Ac- (Pro) 10-Lys (10 mg) obtained in Reference Example 1- (1) was dissolved in DMF (1 ml), and triethylamine (20 μl) was obtained. And 6-carboxy-X-rhodamine succinimide ester (manufactured by Molecular Probes, Inc.) (6.5 mg) were condensed and purified by the same operation as in Reference Example 1- (2) to obtain Ac- (Pro). 10-Lys (εXR) (4.2 mg) was obtained.

Reference Example 3 Synthesis of Ac- (Pro) 10-Lys (εR6G) Ac- (Pro) 10-Lys (10 mg) obtained in Reference Example 1- (1) was dissolved in DMF (1 ml), and triethylamine (20 μl) was obtained. And 6-carboxyrhodamine 6G succinimide ester (manufactured by Molecular Probes, Inc.) (5.3 mg) were condensed by the same operation as in Reference Example 1- (2), purified, and purified with Ac- (Pro) 10- Lys (εR6G) (2.66 mg) was obtained.

Reference Example 4 Synthesis of Ac- (Pro) 10-Lys (εR110) AC- (Pro) 10-Lys (10.4 mg) obtained in Reference Example 1- (1) was dissolved in DMF (1 ml), and triethylamine (20 μl) was dissolved. ) And 5-carboxyrhodamine 110 bis-trifluoroacetate succinimide ester (manufactured by Molecular Probes, Inc.) (8.3 mg) were condensed and purified by the same operation as in Reference Example 1- (2) to obtain Ac. -(Pro) 10-Lys (εR110) (4.8 mg) was obtained.

Experimental example 1
Energy transfer dye of the compound of proline residue 10 (FAM- (Pro) 10-Lys (εTMR), FAM- (Pro) 10-Lys (εXR), FAM- (Pro) 10-Lys (εR6G), FAM- ( Pro) 10-Lys (εR110)] and a single dye (Ac- (Pro) 10-Lys (εTMR), Ac- (Pro) 10-Lys (εXR), Ac- (Pro) 10-Lys (εR6G), Ac- (Pro) 10-Lys (εR110)] in 40 mM Tris-HCl buffer (pH 8.0) was compared for its fluorescence emission intensity.
Each dye solution was excited at an excitation wavelength of 488 nm using a Hitachi F-4010 spectrofluorometer, and the fluorescence wavelengths were measured by TMR (580 nm), XR (610 nm), R6G (560 nm), and R110 (530 nm), respectively. . FIG. 6 shows the relative intensities of the obtained dyes as bar graphs.
As can be seen from FIG. 6, it can be seen that the energy transfer dye exhibits much stronger fluorescence than the acceptor dye itself.

Example 1
Synthesis of FAM and TMR-labeled 3'-deoxyuridine-5'-triphosphate (FAM, TMR-labeled 3'-dUTP) (Compound 10) FAM- (Pro) 8-Lys (εTMR) obtained in Synthesis Example 1 To a solution of (54.1 mg) in DMF (1 ml) were added N, N'-disuccinimidyl carbonate (8.2 mg) and 4-dimethylaminopyridine (3.9 mg) under a nitrogen stream, and the mixture was reacted at room temperature for 2 hours. The reaction solution was added to a DMF-water mixture containing 5- (6 "-amino-1" -hexynyl) -3'-deoxyuridine-5'-triphosphate (8 µmol), and further stirred at room temperature overnight. Water (30 ml) was added to the reaction solution to dilute it, followed by DEAE-Toyopearl ion exchange column chromatography (1.7 × 15 cm; eluent: triethylammonium hydrogen carbonate buffer containing 40% acetonitrile (pH 7.5) 0.1 M → 0.7 M linear) Purification was performed using a concentration gradient (total volume: 2 L).

Example 2
Synthesis of FAM and XR-labeled 3′-deoxycytidine-5′-triphosphate (FAM, XR-labeled 3′-dCTP) (Compound 11) FAM- (Pro) 8-Lys (εXR) obtained in Synthesis Example 2 To a solution of (71.9 mg) in DMF (1 ml) were added N, N'-disuccinimidyl carbonate (10.2 mg) and 4-dimethylaminopyridine (4.9 mg) under a nitrogen stream, and reacted at room temperature for 2 hours. The reaction solution was added to a DMF-water mixture containing 5- (6 "-amino-1" -hexynyl) -3'-deoxycytidine-5'-triphosphate (10 µmol), and further stirred at room temperature overnight. The product was purified as in Example 1.

Example 3
Synthesis of FAM and R6G-labeled 3′-deoxyadenosine-5′-triphosphate (FAM, R6G-labeled 3′-dATP) (Compound 12) FAM- (Pro) 8-Lys (εR6G) obtained in Synthesis Example 3 To a solution of (55.1 mg) in DMF (1 ml) were added N, N'-disuccinimidyl carbonate (8.2 mg) and 4-dimethylaminopyridine (3.9 mg) under a nitrogen stream, and reacted at room temperature for 2 hours. The reaction solution was added to a DMF-water mixture containing 5- (6 "-amino-1" -hexynyl) -3'-deoxyadenosine-5'-triphosphate (8 µmol), and further stirred at room temperature overnight. The product was purified as in Example 1.

Example 4
Synthesis of FAM and R110-labeled 3′-deoxyguanosine-5′-triphosphate (FAM, R110-labeled 3′-dGTP) (Compound 13) FAM- (Pro) 8-Lys (εR110) obtained in Synthesis Example 4 To a solution of (52.4 mg) in DMF (1 ml) were added N, N'-disuccinimidyl carbonate (8.2 mg) and 4-dimethylaminopyridine (3.9 mg) under a nitrogen stream, and the mixture was reacted at room temperature for 2 hours. The reaction solution was added to a DMF-water mixture containing 5- (6 "-amino-1" -hexynyl) -3'-deoxyguanosine-5'-triphosphate (8 µmol), and further stirred at room temperature overnight. The product was purified as in Example 1.
The structural formulas of Compounds 10 to 13 obtained in Examples 1 to 4 are shown below.

Reference Example 5
Cloning of Wild Type T7 RNA Polymerase Gene and Construction of Expression Plasmid T7 phage using Escherichia coli as a host was purified as follows. Escherichia coli C600 was inoculated into 200 ml of an LB medium (a medium in which 10 g of Bacto tryptone, 5 g of Bacto yeast extract, and 5 g of NaCl were dissolved in 1 liter of water, adjusted to pH 7.5, and then sterilized in an autoclave). (600 nm) = 1.0, infection was carried out at a multiplicity of infection of about 2, then OD was measured over time, and when OD dropped sharply, the bacterial residue was removed by centrifugation to remove NaCl. And polyethylene glycol 6000 were added to the final concentrations of 0.5 M and 10%, respectively, and after stirring well, the mixture was allowed to stand at 4 ° C. overnight to form a precipitate. The precipitate was collected by centrifugation, and suspended in SM buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgSO ₄ , 50 mM NaCl, 0.01% gelatin). This concentrated solution of T7 phage was then placed on a CsCl solution of different density carefully layered on a centrifuge tube (from the bottom layer, a solution with a CsCl concentration of 1.267 g / ml, 0.817 g / ml, 0.705 g / ml). The layers were overlaid and centrifuged at 22,000 rpm for 2 hours to form a phage layer. The white band of the phage was carefully separated and dialyzed against TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). , CsCl components were removed. The phage solution was further modified with phenol to denature the phage protein, and the genomic DNA of T7 phage was purified.

  The T7 RNA polymerase gene is encoded at positions 3171 to 5822 out of this genomic DNA, 39,937 base pairs. (The entire nucleotide sequence of the T7 genomic gene has already been reported by Dunn et al. (1983, J. Mol. Biol., 166 (4): 477-535). However, there are some corrections (see the T7 phage DNA sequence in GeneBank, Accession No. V01148 J02518 X00411)]. This genomic DNA was used as a template to amplify using PCR, and cloned into an expression vector as follows (see FIG. 2). That is, a primer specific to the N-terminal amino acid region upstream of the T7 RNA polymerase gene (T7Rpol-N 5'-ATA TTT TAG CCA TGG AGG ATT GAT ATA TGA ACA CGA TTA ACA TCG CTA AG -3 '), and a primer specific to the C-terminal amino acid region downstream (T7Rpol-C 5'-ATA TTT TAG CCA TGG TAT AGT GAG TCG TAT TGA TTT GCG -3') The enzyme gene was amplified by the PCR method. This DNA fragment was digested with NcoI, subjected to 1% agarose electrophoresis, the target DNA fragment was excised from the agarose, and purified using Gene Pure Kit (Nippon Gene). This was ligated with an expression vector pTrc99a (Pharmacia Biotech) digested with NcoI and dephosphorylated to construct pT7R which overexpresses T7 RNA polymerase. Plasmid pT7R expressing wild-type T7 RNA polymerase is transformed into Escherichia coli DH5α, Escherichia coli showing resistance to the antibiotic ampicillin is cultured, IPTG is added to the culture solution, and the Trc promoter contained in the expression vector pT7R is activated. Let it go. Two hours after IPTG addition, Escherichia coli was recovered, and the whole protein was analyzed by SDS-polyacrylamide gel electrophoresis.A protein band was detected only when IPTG was added, near the molecular weight of T7 RNA polymerase of 99 kDa. . This protein was further purified by partially improving the method already described in Zawadzki, V. et al., 1991, Nucl. Acids Res. The purification was carried out by almost the same method as the polymerase purification method). As a result, it had the activity of an RNA polymerase that acts specifically on the T7 promoter.

Reference Example 6
Construction of expression plasmid for producing mutant T7 RNA polymerase
(1) Construction of an expression plasmid for producing mutant T7 RNA polymerase F644Y (see FIG. 3)
Using pT7R, into which the wild-type T7 RNA polymerase gene has been inserted, as a template, a mutation was introduced by PCR using the PCR method in the region flanked by the restriction enzymes HpaI and NcoI corresponding to the C-terminal side of the T7 RNA polymerase gene. did. To illustrate in more detail, the primer to be mutated is divided into left and right with the base to be mutated as a boundary, and the mutated primer F646Y (+) (5′-GTT GAC GGA AGC CGT ACT CTT TGG AC-3 ′), F646Y (− ) (5'-GTC CAA AGA GTA CGG CTT CCG TCA AC-3 ') and primer T7RNAP-HpaI-N (5'-CGC GCG GTT AAC TTG CTT CCT AG- 3 ′), each DNA fragment was amplified by PCR using pTrc99a-PstI-C (5′-GCA TGC CTG CAG GTC GAC TCT AG-3 ′). These DNA fragments have complementary parts, and by repeating denaturation, annealing, and extension reactions, DNA fragments into which the desired mutation was introduced were prepared. This DNA fragment was purified by agarose gel electrophoresis by cutting out only the DNA fragment of the desired size, and using this as a template, reamplified using primers T7RNAP-HpaI-N and pTrc99a-PstI-C, It was cut with Hpa I and Pst I. This DNA was subjected to 1% agarose electrophoresis, separated, and then a target DNA fragment was cut out and purified. Mutation is introduced by replacing this DNA fragment with the HpaI and PstI DNA fragments of pT7R, transformed into Escherichia coli DH5α, and a plasmid having the mutation introduced is selected, and finally the nucleotide sequence is confirmed. It was confirmed whether the mutation was introduced at the target position. Then, an expression plasmid pT7RF644Y for producing a mutant T7 RNA polymerase F644Y was obtained. Production of mutant T7 RNA polymerase F644Y from this plasmid could be induced by culturing Escherichia coli containing this plasmid and adding IPTG, as in the production of wild-type T7 RNA polymerase.

(2) Construction of an expression plasmid for producing mutant T7 RNA polymerase L665P / F677Y (see FIGS. 4 and 5)
The construction of mutant T7 RNA polymerase L665P / F667Y was carried out in the following manner based on the PCR method, as in the construction of F644Y.
First, a restriction enzyme XhoI (CTC GAG) was introduced into a T7 RNA polymerase gene region in an expression vector pT7R having a wild-type T7 RNA polymerase gene to facilitate a mutation introduction operation. More specifically, the combination of primer ApaF1 (5′-CAT CTG GTC GCA TTG GGT CAC-3 ′) and primer Xho-R (5′-CCAGT GTT CTC GAG TGG AGA-3 ′), -F (5'-CTA AGT CTC CAC TCG AGA ACA CTT GG-3 ') and primer AflII-R (5'-CAG CCA GCA GCT TAG CAG CAG-3'), each with the expression vector pT7R as a template Was used to perform PCR. The amplified former DNA fragment was reacted with restriction enzymes ApaI and XhoI, and the latter amplified DNA fragment was reacted with restriction enzymes AflII and XhoI, respectively.The expression vector pT7R was previously treated with ApaI and AflII, and all were treated with T4 DNA ligase. The binding was performed using This reaction product was transformed into Escherichia coli DH5α, and a plurality of colonies growing on agar plates containing the antibiotic ampicillin were obtained. Some of these colonies were selected, cultured and plasmid DNA was extracted to obtain a plasmid pT7R-Xho having a restriction enzyme XhoI site in the T7 RNA polymerase gene region (see FIG. 4). The XhoI site can be cleaved by treatment with the restriction enzyme XhoI, and the nucleotide sequence of DNA can be determined to confirm its presence. Using this plasmid pT7R-Xho as a template, a combination of primer Xho-R and primer 667R (5′-GCT GAG TGT ACA TCG GAC CCT-3 ′) and primer 667F (5′-GCT GAG TGT ACA TCG GAC CCT-3 ′) ) And the primer AflIIR were each subjected to PCR. Using this PCR product directly as a template, the nucleotide sequence of DNA was determined, and after confirming the sequences of primers 667R and 667F, each was subjected to 2% agarose electrophoresis (agarose used was Nippon Gene's agarose X) to obtain the desired product. A DNA fragment having a size was cut out, and the DNA fragment was purified using a Gene Pure Kit. The two purified DNAs are mixed, and PCR is performed using primers XhoF and AflIIR as templates. The amplified DNA fragment is subjected to restriction enzyme mapping and DNA base sequence analysis to confirm that the fragment is the target fragment. An enzyme reaction was carried out using XhoI and AflII, and this was ligated to a plasmid pT7R-Xho previously treated with restriction enzymes XhoI and AflII using T4 DNA ligase. This reaction product was transformed into Escherichia coli DH5α, and a plurality of colonies growing on agar plates containing the antibiotic ampicillin were obtained. Some of these colonies are selected, cultured, plasmid DNA is extracted, the DNA base sequence is determined whether the desired mutation has been introduced, and confirmed, and finally the desired mutant T7 RNA polymerase L665P / F644Y An expression plasmid pT7RL665P / F667Y for producing E. coli was constructed (see FIG. 5). The production of mutant T7 RNA polymerase L665P / F667Y from this plasmid could be induced by culturing Escherichia coli containing this plasmid and adding IPTG, as in the production of wild-type T7 RNA polymerase.

Reference Example 7
Purification of mutant T7 RNA polymerase The mutant T7 RNA polymerase protein introduced into E. coli was purified.
The wild type of this protein has already been described in Chamberlin, M et al. Nature, 228: 227-231 (1970), Davanloo et al., Proc. Natl. Acad. Sci. USA., 81: 2035-2039 (1984). )It is described in. Further, mass production is reported in Zawadzki, V et al., Nucl. Acids Res., 19: 1948 (1991).
All mutant T7 RNA polymerases can be purified by essentially the same method. Depending on the mutation site, the expression level and the behavior of column chromatography may be slightly different. Hereinafter, a method for purifying the mutant T7 RNA polymerase F644Y will be exemplified. The F644Y expression vector pT7RF644Y was introduced into Escherichia coli DH5α, and in an LB medium containing the antibiotic ampicillin, first, when the OD (600 nm) became 0.4 to 0.6 in a test tube culture, isopropyl-β-thiogalactopyrano Cid (IPTG) is added to a final concentration of 0.4 mM, and the cells are further cultured for 8 hours. At this time, Escherichia coli cells are collected by centrifugation, and typically 10 g of wet E. coli is obtained from 2 liters of the culture solution. When the E. coli cells are not used immediately, they can be stored in a freezer at -20 ° C or lower.
From this stage on, all steps of enzyme purification are carried out at a temperature below room temperature, preferably 0-5 ° C, unless otherwise specified. At this time, the E. coli was washed with a washing buffer (20 mM Tris-HCl, pH 8.1, 130 mM NaCl, 2 mM EDTANa2 at 25 ° C.) 10 times the cell weight, and centrifuged again (5,000 × g, 10 ° C. at 4 ° C.). For 10 minutes, and add 10 volumes of sonication buffer [50 mM Tris-HCl, pH 8.1, 100 mM NaCl, 0.1 mM EDTANa2, 5 mM dithiothreitol (DTT), 0.1 mM benzamidine, 30 μg / ml phenylmethylsulfonylfluoride (PMSF), 10 μg / ml, bacitracin], and sonicate at 80 W for 15 minutes using Sonifier 450 (Branson) to disrupt the cells and reduce the viscosity. Subsequently, the mixture was centrifuged at 12,000 × g and 4 ° C. for 10 minutes to remove cell debris. While stirring the resulting supernatant, 10% streptomycin sulfate was slowly added dropwise to a final concentration of 2.0%, and then stirring was continued for another 30 minutes. 12,000xg. Centrifuge at 4 ° C for 10 minutes to remove the precipitate, and stir while slowly adding ammonium sulfate powder to form a precipitate. In this case, the precipitate is first collected with 30% saturated ammonium sulfate (30% ammonium sulfate precipitation), and the supernatant is further added with stirring ammonium sulfate so as to become 60% saturated ammonium sulfate, and the precipitate is formed again (30-60% ammonium sulfate). Ammonium sulfate precipitation), and further, ammonium sulfate powder was added to the supernatant so as to become 90% saturated ammonium sulfate. A part of these three ammonium sulfate fractions were subjected to SDS-acrylamide gel electrophoresis and analyzed for protein. Most of the mutant T7 RNA polymerase of interest was present in the 30-60% ammonium sulfate fraction. Purification proceeded in minutes. The 30-60% ammonium sulfate fraction was suspended in a small amount of column buffer (20 mM KPO ₄ , pH 7.7, 100 mM NaCl, 1 mM DTT, 30 μg / ml PMSF) and dialyzed against the same buffer (500 ml) for 16 hours. , Desalted. The dialysate is added to a 5 ml column volume of heparin-Sepharose (Pharmacia Biotech). Then, the column is washed with the same buffer until no 280 nm ultraviolet absorbing substance is detected, and eluted with a linear concentration gradient of 0.1 M to 0.64 M NaCl in the same buffer, which is about 40 times the column volume. I do. The eluate is collected by fractionating an appropriate amount into a test tube, immediately performing SDS-acrylamide gel electrophoresis, analyzing the protein, and fractionating the protein in the vicinity of the molecular weight considered to be the target mutant T7 RNA polymerase. To inspect. A typical example would be found near 0.4M NaCl. The protein fractions were collected containing, about 1 liter of column buffer _{(20 mM KPO 4, pH7.7,} 100 mM NaCl, 1mM DTT, 30μg / ml PMSF) with respect dialyzed 16 hours, the desalting went. The dialyzed and desalted fraction is added to a 5 ml column volume of Q-sepharose (Pharmacia Biotech) pre-equilibrated with the same buffer, and the same buffer detects a 280 nm ultraviolet absorbing substance. And elute with a linear gradient of 0.1 M to 0.64 M NaCl in the same buffer at approximately 40 column volumes. The eluate is collected by fractionating an appropriate amount into a test tube, immediately performing SDS-acrylamide gel electrophoresis, analyzing the protein, and determining the fraction in which the protein exists near the molecular weight considered to be the target mutant T7 RNA polymerase. inspect. A typical example would be found near 0.24M NaCl. The fractions containing this protein are collected, dialyzed against 500 ml of storage buffer (50% glycerol, 20 mM KPO ₄ , pH 7.7, 100 mM NaCl, 1 mM DTT, 30 μg / ml PMSF) for 16 hours. Store at -20 ° C until In this state, the in vitro RNA synthesis activity or contaminating ribonuclease activity is tested. As an example of this method, the in vitro RNA synthesis activity is determined by using a plasmid containing a T7 promoter as a template, and using a commercially available wild-type T7 RNA polymerase (BRL Gibco) as a standard product by enzyme dilution. Then, an RNA synthesis reaction was performed, and an approximate titer was estimated by agarose electrophoresis of the synthesized RNA. At this time, the degree of degradation of the synthesized RNA is also observed, and at the same time, a simple assay for contaminating ribonuclease is possible. As a typical example, 2,500,000 units of mutant T7 RNA polymerase F644Y protein were purified from 1 liter of culture solution by a purification method based on the above steps, and this sample had almost no RNase contamination. Absent.

Reference Example 8
Purification of pyro-dried fatter without RNase activity contamination
Inorganic pyrophosphatase (PPase) containing no RNase was purified as follows, but is not limited to the following method.

  As starting materials for inorganic pyrophosphatase, 4 mg (680 units) of a crude yeast-derived product (Sigma I-1643, EC. 3.6.1.1) manufactured by Sigma was added to a buffer (20 mM Tris-HCl, 1 mM EDTA, The suspension was suspended in 1 ml of pH 7.9), dialyzed against the same buffer for 2 hours, desalted, and subjected to SP Sepharose column chromatography (Pharmacia Biotech) with a column volume of 1 ml. Specifically, the column buffer is sufficiently washed with about 20 times the volume of the same column buffer until no 280 nm ultraviolet absorbing substance is detected, and 0 to 0.1 M NaCl is used using about 20 times the column volume of the same buffer. Elute with a linear gradient. The eluate was fractionated and collected in an appropriate amount, immediately subjected to SDS-12.5% polyacrylamide electrophoresis, and a fraction containing a protein having a molecular weight of about 32 kDa was examined. In a typical example, the PPase fraction should be found in the unadsorbed part. The fractions containing this protein are collected, adsorbed on a 1 ml column volume of Q-Sepharose (Pharmacia Biotech), and a linear concentration of 0 to 1.0 M NaCl is used using about 20 times the column volume in the same buffer. Elute with a gradient. The eluate was fractionated and collected in an appropriate amount, immediately subjected to SDS-12.5% polyacrylamide electrophoresis, and a fraction containing a protein having a molecular weight of about 32 kDa was examined. In a typical example, it should be found around 0.35M NaCl. Collect fractions containing a protein with a molecular weight of 32 kDa, dialyze against 500 ml of a storage buffer (20 mM Tris-HCl, 1 mM EDTA, 50% glycerol, pH 7.9) for 16 hours and store at -20 ° C until use I do. In a typical example, a standard of 425 units of PPase protein and 0.425 units / μl can be obtained at a recovery of 62.5%.

In this state, the degree of RNase contamination was examined using 8 μg of rRNA (16S and 23S) of E. coli as a substrate. Specifically, E. coli rRNA was added to a buffer of 8 mM MgCl ₂ , 2 mM spermidine- (HCl) ₃ , 5 mM DTT, 40 mM Tris / HCl pH 8.0, added PPase, equivalent to 0.17 units, and reacted at 37 ° C. for 4 hours. I let it. This RNA was subjected to 1.0% agarose electrophoresis under denaturing conditions in the presence of formamide. When xylene cyanol, which was added together, reached about 1/3 of the agarose gel, electrophoresis was stopped and ultraviolet light (wavelength 254 nm) and photographed to examine the degree of RNA degradation. At this time, when the crude product was compared with the purified PPase, RNA degradation was observed in the crude product, but no significant RNA degradation activity was detected in the purified product.

Example 5
Comparison of sequencing examples using energy transfer terminator and conventional dye terminator in in vitro transcription reaction using mutant RNA polymerase
The effect of 3'dNTP on the sequencing of the energy transfer terminator was examined by an in vitro transcription reaction using mutant T7 RNA polymerase F644Y. The sequencing reaction used the method indicated by Melton, DA (1984, Nucleic Acids Res., 12: 7035-7056). More specifically, a plasmid vector pBluescriptKS (+) (Stratagene) having a T7 promoter was reacted with a restriction enzyme PvuII, and a linearized one was used as a template. As a 3 ′ dNTP energy transfer terminator derivative, a dye terminator having a proline spacer, which is a compound 10 to 13 synthesized in Examples 1 to 4, was used. Specifically, 4 μM FAM and R6G-labeled 3′-dATP, 4 μM FAM and R110-labeled 3′-dGTP, 80 μM FAM and XR-labeled 3′-dCTP and 20 μM FAM and TMR-labeled 3′-dUTP, 500 μM GTP, UTP and 250 μM ATP, CTP, 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 F644Y (25U) Then, yeast-derived inorganic pyrophosphatase (0.045U) was added to make a total reaction volume of 10 µl, and the reaction was carried out at 37 ° C for 1 hour.

The reaction using a dye terminator having one fluorescent dye for comparison was performed using 4 μM R6G-4x-3′dATP, 4 μM R110-4x-3′dGTP, 80 μM XR-4x-3′dCTP, and 20 μM TMR-. 4x-3'dUTP), 500 μM GTP, UTP and 250 μM ATP, CTP, 8 mM MgCl ₂ , 2 mM spermidine- (HCl) ₃ , 5 mM DTT, 40 mM Tris / HCl pH 8.0 (BRL, Gibco) Mutant T7 RNA polymerase F644Y (25 U) and inorganic pyrophosphatase derived from yeast (0.05 U) were added to make a total reaction volume of 10 μl and reacted at 37 ° C. for 1 hour. Next, in order to remove the unreacted terminator remaining in the reaction product, the transcript was purified by gel filtration using a Sephadex G-50 column (manufactured by Pharmacia Biotech), and the purified product was centrifugally evaporated. And evaporated to dryness.

  The dried reaction product was dissolved in 6 μl of formamide / EDTA / Blue dextrane loading buffer according to ABI PRISM 377 DNA Sequencing System instruction manual Ver.1.0 of ABI (Perkin Elmer Corporation, Applied Biosystems Division), and 2 μl of the solution was dissolved. Using a denaturing gel for sequence analysis containing 6M urea / 4% Long Ranger ™ acrylamide solution (FMC), analysis was performed by ABI 377 DNA Sequencer and an analysis program (Sequencing Analysis Ver.3.0).

As a result, when the energy transfer terminator was used, the strength of each sequence ladder was found to be high. The reaction product was diluted with formamide / EDTA / Blue dextrane loading buffer, and 2 μl of the diluted product was electrophoresed. As a result, it was found that a 10-fold dilution could be sequenced with almost the same peak intensity as when using a conventional die terminator. FIG. 1 shows a typical example of a sequence pattern obtained by diluting a reaction product using the energy transfer terminator by 10 times with a sequence pattern using a conventional die terminator. From these results, it was found that the use of the energy transfer terminator enables sequencing with a sensitivity 10 times that of the conventional die terminator.
As a result of performing sequencing using the mutant T7 RNA polymerase L665P / F667Y instead of the mutant T7 RNA polymerase F644Y, the same results as described above were obtained.

  Chemistry of R6G-4x-3'dATP (14), R110-4 x-3'dGTP (15), XR-4x-3'dCTP (16) and TMR-4x-3'dUTP (17) used above The structural formula is shown below.

FIG. 1 shows a sequence pattern obtained in Example 5. FIG. 2 is a construction diagram of a plasmid expressing wild-type T7 RNA polymerase, pT7R. FIG. 3 is a construction diagram of pT7RF644Y, a plasmid expressing the mutant T7 RNA polymerase F644Y. FIG. 4 is a construction diagram of pT7R-Xho, an improved pT7R plasmid having a restriction enzyme XhoI site in the T7 RNA polymerase gene. FIG. 5 is a construction diagram of pT7RL665P / F667Y, a plasmid expressing the mutant T7 RNA polymerase L665P / F667Y. FIG. 6 is a comparison diagram of the fluorescence intensity of the energy transfer dye obtained in Experimental Example 1.

Claims (7)

A method for determining the nucleotide sequence of DNA by the chain terminator method, comprising using a terminator having a 3'-deoxyribonucleotide residue, and two types of reporters that can serve as energy transfer donors and acceptors, and an RNA polymerase. A method comprising performing a reaction. The method of claim 1, wherein the two reporters on the terminator are located a distance sufficient to cause energy transfer from the donor to the acceptor. 3. The method of claim 2, wherein the distance sufficient to cause energy transfer from the donor to the acceptor is in the range of 10-100 °. The method according to any one of claims 1 to 3, wherein the reporter is selected from the group consisting of a fluorescent group, a phosphorescent group, a spin-labeled group, and a group having a high electron density. The donor is a kind selected from the group consisting of fluorescein dyes, rhodamine dyes and 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene dyes, and the acceptor is a fluorescein dye The method according to any one of claims 1 to 4, wherein the method is a kind selected from the group consisting of a rhodamine dye and a 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene dye. Using four types of terminators corresponding to the four types of bases (each terminator has four different types of reporters as acceptors, respectively), and performing a chain termination reaction by the above four types of terminators in the same reaction system The method according to claim 1. The method according to any one of claims 1 to 6, wherein the chain termination reaction is performed in the presence of an inorganic pyrophosphatase.

Images