JP2005143484A - Method for cutting dna and kit for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for regiospecifically and efficiently cutting a DNA. <P>SOLUTION: This method for cutting the DNA comprises hybridizing a target DNA with a complementary chain A complementary to a 5' terminal region of a target part of the target DNA together with a complementary chain B complementary to a 3' terminal region of the target part of the target DNA, so as to obtain a nucleic acid complex, and then contacting the nucleic acid complex with a cerium (IV) ion or a cerium (IV) complex, wherein a phosphoric acid group or a derivative containing the group is introduced into the 5' terminal of the complementary chain A and/or the 3' terminal of the complementary chain B. Further, a kit capable of being used in conducing the method is provided. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

Industrial field

  The present invention relates to a method and kit for specifically cleaving DNA, and more particularly to a method and kit for cleaving DNA specifically in an artificially created chemical structure.

  A simple method for cleaving a DNA strand at an arbitrary position is expected to play a major role in various fields dealing with genes such as medicine and molecular biology. Currently, restriction enzymes are widely used to cleave DNA. However, restriction enzymes have low base sequence specificity and cannot selectively cleave huge DNA at specific positions. Moreover, as a method for cleaving DNA without using a natural enzyme, attempts have been made so far to cleave DNA with metal ions or metal ion complexes. However, for example, cerium (IV) ions are known to have DNA-cleaving ability, but have little base sequence specificity. That is, a method for cutting a huge DNA at a specific position has not been developed yet.

Problems to be solved by the present invention

  An object of the present invention is to provide a method for efficiently cleaving DNA at a specific position. Another object of the present invention is to provide a kit that specifically and efficiently cleaves DNA.

Means for solving the problem

The subject of the present invention is
(1) A target DNA (target DNA) is a complementary strand A having a sequence complementary to the 5 ′ end region of the target portion of the target DNA and a sequence complementary to the 3 ′ end region of the target portion of the target DNA. In the method of cleaving a target DNA by hybridizing with a complementary strand B having a nucleic acid complex and then contacting the nucleic acid complex with a cerium (IV) ion or a cerium (IV) complex, the 5 ′ of the complementary strand A A method for cleaving DNA, wherein a phosphate group or a derivative containing the same is introduced into the terminal and / or the 3 ′ terminal of the complementary strand B.
(2) A gap exists between the 5 ′ end region of the target DNA to which the complementary strand A hybridizes and the 3 ′ end region of the target DNA to which the complementary strand B hybridizes, and the desired cleavage point is in the gap. (1) The method for cleaving DNA according to (1).
(3) The DNA cutting method according to (1) to (2), wherein the cerium (IV) complex is a complex of cerium (IV) and polyamine-N-polycarboxylic acid.
(4) The DNA cutting method according to any one of (1) to (3), wherein both or one of the complementary strand A and the complementary strand B is DNA.
(5) The DNA cutting method according to any one of (1) to (3), wherein both or one of the complementary strand A and the complementary strand B is a peptide nucleic acid (PNA).
(6) A target DNA (target DNA) is a complementary strand A having a sequence complementary to the 5 ′ end region of the target portion of the target DNA and a sequence complementary to the 3 ′ end region of the target portion of the target DNA. In the method of cleaving a target DNA by hybridizing with a complementary strand B having a nucleic acid complex and then contacting the nucleic acid complex with a cerium (IV) ion or a cerium (IV) complex, the 5 ′ of the complementary strand A This was achieved by a kit characterized in that a phosphate group or a derivative containing the same was introduced into the terminal and / or the 3 ′ terminal of the complementary strand B. The structure of the nucleic acid complex in item (1) is shown in FIGS.

FIG.

FIG.

Detailed Description of the Invention

  According to the first aspect of the present invention, the target DNA (target DNA) is converted into a complementary strand A having a sequence complementary to the 5 ′ end region of the target portion of the target DNA and 3 ′ of the target portion of the target DNA. A method of cleaving a target DNA comprising hybridizing with a complementary strand B complementary to a terminal region to obtain a nucleic acid complex, and then contacting the nucleic acid complex with a cerium (IV) ion or a cerium (IV) complex. Thus, a method is provided in which a phosphate group or a derivative containing the same is introduced into the 5 ′ end of the complementary strand A and / or the 3 ′ end of the complementary strand B.

  Here, the complementary strands A and B are DNA, peptide nucleic acid (PNA), RNA, and derivatives thereof, and are preferably DNA or peptide nucleic acid (PNA) because of their ease of synthesis and handling. Here, PNA is a polymer having an amide bond in the main chain and a nucleobase in the side chain. For example, PNA is described on page 66 of “New Central Dogma of Biochemistry” (published by Kagaku Dojin, 2002). Can be used.

  When a phosphate group or a derivative containing the same is introduced into the 5 'end of the complementary strand A and the 3' end of the complementary strand B, the structure of the introduced portion can be, for example, any of FIG.

FIG.

In FIG. 3, X represents an oxygen atom or a CH ₂ group. M is an integer of 1 to 20, preferably 6 to 12, n is an integer of 1 to 10, preferably 3 or 4, and q and r are integers of 0 to 5 and most preferably 0.

  Cerium (IV) ions include diammonium cerium (IV) sulfate aqueous solution, cerium sulfate (IV) aqueous solution, oxide of cerium (III) chloride aqueous solution, oxide of cerium (III) sulfate aqueous solution, cerium (III) perchlorate Any aqueous solution containing Ce (IV) ions, such as an aqueous oxide, may be introduced into the reaction system in any form, but diammonium cerium (IV) sulfate is preferred. The cerium complex having the ability to cleave DNA can be obtained, for example, by contacting cerium (IV) with polyamine-N-polycarboxylic acid in water. As the polyamine-N-polycarboxylic acid, those described in “Metal Chelate, I-IV” (Nanedo, issued in 1967) can be used. For example, ethylenediaminetetraacetic acid, 1,3-diaminopropane-N, N, N ′, N′-tetraacetic acid, 1,4-diaminobutane-N, N, N ′, N′-tetraacetic acid, diethylenetriaminepentaacetic acid, Examples include, but are not limited to, triethylenetetramine-N, N, N ′, N ″, N ′ ″, N ′ ″-hexaacetic acid.

  By contacting the nucleic acid complex with cerium (IV) ions or cerium (IV) complexes, the target DNA is cleaved. The obtained fragment can be purified by polyacrylamide gel electrophoresis, agarose gel electrophoresis, or HPLC, if necessary. Antisense oligonucleotides and peptide nucleic acids hybridized to the target DNA may be separated by conventional methods.

Detailed Description of the Invention

The preferred usage forms of the present invention are shown below.
(1) A target DNA is hybridized with a complementary strand A and a complementary strand B containing a phosphate group at the 5 ′ end to obtain a nucleic acid complex, and then the nucleic acid complex is converted into cerium (IV) ion or cerium (IV). A method of cleaving a target DNA comprising contacting with a complex.
(2) A target DNA is hybridized with a complementary strand A and a complementary strand B containing a phosphate group at the 3 ′ end to obtain a nucleic acid complex, and then the nucleic acid complex is converted into cerium (IV) ion or cerium (IV). A method of cleaving a target DNA comprising contacting with a complex.
(3) The target DNA is hybridized with a complementary strand A containing a phosphate group at the 5 ′ end and a complementary strand B containing a phosphate group at the 3 ′ end to obtain a nucleic acid complex, A method of cleaving a target DNA comprising contacting with cerium (IV) ions or a cerium (IV) complex.
(4) A gap exists between the 5 ′ end region of the target DNA to which the complementary strand A hybridizes and the 3 ′ end region of the target DNA to which the complementary strand B hybridizes, and the desired cleavage point is in the gap. A method characterized in that it exists.
(5) The DNA according to any one of (1) to (4), wherein the cerium (IV) is an oxide of a diammonium cerium (IV) sulfate aqueous solution or a cerium (III) chloride aqueous solution. Cutting method.
(6) The method for cleaving DNA according to any one of (1) to (4), wherein the cerium complex is a complex of cerium (IV) and polyamine-N-polycarboxylic acid.
(7) The distance between the 5 ′ end region of the target DNA to which the complementary strand A hybridizes and the 3 ′ end region of the target DNA to which the complementary strand B hybridizes, that is, the size of the gap is typically 0. A method for cleaving DNA, characterized in that it is 10 to 10 base pairs, preferably 2 to 5 base pairs, more preferably 2 and 3 base pairs.
(8) The method for cleaving DNA according to (1) to (7), wherein the complementary strands A and B are at least 6 base pairs, preferably at least 9 base pairs.

  Although the DNA cleavage test was carried out as follows, the present invention is not limited to the following examples.

Synthesis of modified DNA Synthesis of phosphoric acid-modified DNA Using a commercially available reagent (Glen Research), the DNA was synthesized by an automatic DNA synthesizer (Applied Biosystems). Phosphoric acid modification on the 5′-terminal side was performed using a chemically phosphorylated amidite monomer. In addition, the introduction of phosphoric acid to the 3′-terminal side was performed by using phosphorylated CPG as a fixed carrier. The unmodified DNA was outsourced to Sigma Genosys.

DNA cleavage reaction Two phosphate modifications in 1 μM solution (NaCl 100 mM, HEPES buffer 8.75 mM, pH 7) of substrate DNA labeled with ³² P at the 5′-end (DNA commissioned for synthesis by Sigma Genosys) The aqueous DNA solution was added to a final concentration of 2 μM. The mixture was heated at 90 ° C. for 1 minute and gradually cooled to room temperature to form a double strand. A Ce (IV) / EDTA aqueous solution was added thereto so as to have a final concentration of 500 μM, and the mixture was reacted at 37 ° C. for 2 to 95 hours. The analysis confirmed the cleavage of the substrate by 20% denaturing polyacrylamide gel electrophoresis.

Results The modified DNA sequence (20mer) and DNA sequence (50 to 43mer) used in the experiment were as shown in FIG.

FIG.

DNA cleavage test (1)
The following DNA was prepared and reacted with DNA (D50) according to the conditions described in “II DNA cleavage reaction” (however, the reaction time was 15 hours). The result was as shown in FIG.

FIG.

Lane 1: untreated, Lane 2: Ce (IV) · EDTA complex only, Lane 3, D20LP + D20RP in the absence of Ce (IV) · EDTA complex, Lane 4: Ce (IV) · EDTA complex and D20LP, Lane 5: Ce (IV) · EDTA complex and D20L + D20R, Lane 6: Ce (IV) · EDTA complex and D20LP + D20R, Lane 7: Ce (IV) · EDTA complex and D20L + D20RP, Lane 8: Ce (IV) · EDTA complex D20LP + D20RP.
When two phosphate-modified DNAs were used, they were efficiently cleaved at the gap portion (lane 8) (the lane M was a standard lane in which the 5′-end of the synthetic DNA was labeled with ³² P. And the gap portion corresponds to the portion between M20 and M30).
The cut band was read with an imaging analyzer FujixFLA-3000G manufactured by Fuji Film, and the density of the band was calculated with analysis software. As a result, the cutting efficiency at the gap was about 10%.
Even when one phosphate-modified DNA and one unmodified DNA are used (lane 6 and lane 7), the cleavage is almost as strong as when two phosphate-modified DNAs are used. Observed.
When unmodified DNA was used (lane 5), selective cleavage at the gap portion was observed, but the cleavage efficiency was very low.
As described above, by introducing phosphate at both ends of the gap of the substrate DNA using phosphate-modified DNA, the specificity of cleavage at the gap site is significantly improved.
Although not bound by the following theory, it is considered that cleavage at the gap site is realized in order to efficiently bind Ce (IV) / EDTA at the phosphate site.

DNA cleavage experiment (2)
The following DNA was prepared and reacted with DNA (D45) according to the conditions described in “II DNA cleavage reaction” (however, the reaction time was 95 hours). The result was as shown in FIG.

FIG.

Lane 1: untreated, Lane 2: Ce (IV) · EDTA complex only, Lane 3, D20L + D20R in the absence of Ce (IV) · EDTA complex, Lane 4: Ce (IV) · EDTA complex and D20LS3 + D20R, Lane 5: Ce (IV) · EDTA complex and D20L + D20RS3, Lane 6: Ce (IV) · EDTA complex and D20L S3 + D20R S3.
When a gap was formed using two phosphate-modified DNAs having a linker between the terminal phosphate and DNA, the gap was efficiently cleaved (lane 6).
The cut band was read with Fujifilm Imaging Analyzer FujiFLA-3000G, and the density of the band was calculated with analysis software. As a result, the cutting efficiency at the gap was about 25%.
In addition, even when one phosphate-modified DNA and one unmodified DNA were used (lanes 4 and 5), gap-selective cleavage was observed, and the cleavage efficiency was 15 to 20%. .
On the other hand, when unmodified DNA was used (lane 3), although selective cleavage at the gap portion was observed, the cleavage efficiency was low, and the efficiency was about 5%.
As described above, even when phosphoric acid is introduced before the linker, efficient cleavage is realized.

DNA cleavage experiment (3)
The following DNA was prepared and reacted with DNA (D45) according to the conditions described in “II DNA cleavage reaction” (however, the reaction time was 48 hours). The result was as shown in FIG.

FIG.

Lane 1: untreated, Lane 2: Ce (IV) · EDTA complex only, Lane 3, D20LP + D20RP in the absence of Ce (IV) · EDTA complex, Lane 4: Ce (IV) · EDTA complex and D20LP + D20RP, Lane 5: D20LS1 + D20R S1 in the absence of EDTA complex, Lane 6: Ce (IV) .EDTA complex and D20LS1 + D20R S1. Lane 7: D20L S2 + D20R S2 in the absence of EDTA complex, Lane 8: Ce (IV) .EDTA complex and D20L S2 + D20R S2. Lane 9: D20LS1 + D20R S1 in the absence of EDTA complex, Lane 10: Ce (IV) .EDTA complex and D20LS1 + D20R S1. Lane 11: D20LS3 + D20R S3 in the absence of EDTA complex, Lane 12: Ce (IV) .EDTA complex and D20LS3 + D20R S3. Lane 13: D20L S4 + D20R S4 in the absence of EDTA complex, Lane 14: Ce (IV) .EDTA complex and D20L S4 + D20R S4.
Even when various linkers were used, efficient cleavage at the gap site was realized. The cut band was read with an imaging analyzer Fujix FLA-3000G manufactured by Fuji Film Co., Ltd., and the density of the band was calculated with analysis software.
As a result, the cleavage efficiency at the gap was about 10 to 15%, which was comparable with any linker.
As described above, even when phosphoric acid is introduced into the tip of a linker having various lengths, efficient cleavage is realized.

2 shows a nucleic acid complex formed in the first embodiment of the present invention. The nucleic acid complex formed in the first embodiment of the present invention, wherein the portion from the 5 'end to the 3' end of the target DNA is the target portion. A form in which a phosphate group or a derivative thereof is bound to complementary strand A and complementary strand B. Reaction substrate DNA used in Examples, and structures of complementary strands A and B. The result of DNA cleavage test (1) is shown. Lane 1: untreated, Lane 2: Ce (IV) · EDTA complex only, Lane 3, D20LP + D20RP in the absence of Ce (IV) · EDTA complex, Lane 4: Ce (IV) · EDTA complex and D20LP, Lane 5: Ce (IV) · EDTA complex and D20L + D20R, Lane 6: Ce (IV) · EDTA complex and D20LP + D20R, Lane 7: Ce (IV) · EDTA complex and D20L + D20RP, Lane 8: Ce (IV) · EDTA complex D20LP + D20RP. The result of DNA cleavage test (2) is shown. Lane 1: untreated, Lane 2: Ce (IV) · EDTA complex only, Lane 3, D20L + D20R in the absence of Ce (IV) · EDTA complex, Lane 4: Ce (IV) · EDTA complex and D20LS3 + D20R, Lane 5: Ce (IV) · EDTA complex and D20L + D20RS3, Lane 6: Ce (IV) · EDTA complex and D20L S3 + D20R S3. The result of DNA cleavage test (3) is shown. Lane 1: untreated, Lane 2: Ce (IV) · EDTA complex only, Lane 3, D20LP + D20RP in the absence of Ce (IV) · EDTA complex, Lane 4: Ce (IV) · EDTA complex and D20LP + D20RP, Lane 5: D20LS1 + D20R S1 in the absence of EDTA complex, Lane 6: Ce (IV) .EDTA complex and D20LS1 + D20R S1. Lane 7: D20LS2 + D20R S2 in the absence of EDTA complex, Lane 8: Ce (IV) .EDTA complex and D20L S2 + D20R S2. Lane 9: D20LS1 + D20R S1 in the absence of EDTA complex, Lane 10: Ce (IV) .EDTA complex and D20LS1 + D20R S1. Lane 11: D20LS3 + D20R S3 in the absence of EDTA complex, Lane 12: Ce (IV) .EDTA complex and D20LS3 + D20R S3. Lane 13: D20L S4 + D20R S4 in the absence of EDTA complex, Lane 14: Ce (IV) .EDTA complex and D20L S4 + D20R S4.

Claims (6)

  Complementary strand A having target DNA (target DNA) having a sequence complementary to the 5 'end region of the target portion of the target DNA and a sequence complementary to the 3' end region of the target portion of the target DNA In the method of cleaving the target DNA by hybridizing with B to obtain a nucleic acid complex and then contacting the nucleic acid complex with cerium (IV) ion or cerium (IV) complex, the 5 ′ end of the complementary strand A and / or Alternatively, a method for cleaving DNA, wherein a phosphate group or a derivative containing the same is introduced into the 3 ′ end of the complementary strand B.   A gap exists between the 5 ′ end region of the target DNA to which the complementary strand A hybridizes and the 3 ′ end region of the target DNA to which the complementary strand B hybridizes, and a desired cleavage point exists in the gap. 2. The method for cleaving DNA according to claim 1.   The method for cleaving DNA according to claim 1-2, wherein the cerium (IV) complex is a complex of cerium (IV) and polyamine-N-polycarboxylic acid.   4. The method for cleaving DNA according to claim 1, wherein both or one of the complementary strand A and the complementary strand B is DNA.   4. The method for cleaving DNA according to claim 1, wherein both or one of the complementary strand A and the complementary strand B is a peptide nucleic acid (PNA).   Complementary strand A having target DNA (target DNA) having a sequence complementary to the 5 'end region of the target portion of the target DNA and a sequence complementary to the 3' end region of the target portion of the target DNA In the method of cleaving the target DNA by hybridizing with B to obtain a nucleic acid complex and then contacting the nucleic acid complex with cerium (IV) ion or cerium (IV) complex, the 5 ′ end of the complementary strand A and / or Alternatively, a kit wherein a phosphate group or a derivative containing the same is introduced at the 3 ′ end of the complementary strand B.

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