2003 Fiscal Year Annual Research Report

人工制限酵素によるDNAの位置特異的切断と機能性核酸の合成

Research Project

Project/Area Number	13132204
Research Institution	The University of Tokyo
Principal Investigator	小宮山真東京大学, 先端科学技術研究センター, 教授 (50133096)
Co-Investigator(Kenkyū-buntansha)	須磨岡淳東京大学, 大学院・工学系研究科, 助手 (10280934) 浅沼浩之東京大学, 先端科学技術研究センター, 助教授 (20282577)
Keywords	DNA / RNA / PNA / リン酸エステル / 加水分解 / セリウム / アクリジン / 制限酵素
Research Abstract	昨年度までの結果に基づき、より高活性な人工核酸切断酵素を開発することに力を注ぎ以下の結果を得た。 1.リン酸モノエステル修飾DNAを相補鎖として用いたDNAのギャップ部位選択的切断リン酸修飾DNAを相補鎖として基質一本鎖にギャップ構造を取らせ、ここにCe(IV)/EDTA錯体を添加すると、高い活性でギャップ部位選択的な切断が実現すること明らかにした。この切断効率は、非修飾DNAを用いたて形成したギャップに比べて約10倍であった。 2.PNAを相補鎖として用いたDNAのギャップ部位選択的切断 PNAを相補鎖として用いて形成したギャップは、Ce(IV)/EDTA錯体を添加するとDNAを用いたて形成した場合に比べて高い効率で切断されることが明らかになった。 3.pseudo-complementary PNAのstrand invasionを利用した二本鎖DNAの位置選択的切断積極的に一本鎖部位を形成させるように設計したpseudo-complementary PNAを二本鎖DNAにdoublc-duplex invasionを起こさせ、Ce(IV)/EDTA錯体を添加すると、一本鎖部分で選択的に加水分解することに成功した。二本鎖のそれぞれの鎖を切断するで、結果的に二本鎖DNAを同時切断にも成功した。 4.配位子修飾PNAを用いたDNAのギャップ部位選択的切断配位子修飾PNAを用いてDNAにギャップ構造を形成させると、非修飾のPNAとに比較して、ギャップ部位での切断が非常に効率よくおきることを明らかにした。 5.配位子修飾pseudo-complementary PNAを用いた二本鎖DNAの高活性な位置選択的切断 3項と4項の結果をさらに拡張し、配位子修飾pseudo-complementary PNAを二本鎖DNAにdouble-duplex invasionさせ、高い活性で二本鎖DNAを切断することに成功した。 6.DNA切断人工酵素による切断断片の酵素による再結合 Ce(IV)/EDTAによって切断したギャップ部位での切断断片は、酵素によって望みのDNAと再結合できることを明らかにした。 7.高次構造を持つRNAをターゲットとした人工RNA切断酵素による切断複雑な立体構造をとる酵母tRNA^<Phe>の配列を持つRNAに対しても我々の開発した人工RNA切断酵素は適用可能であることが明らかになった。

Research Products
(21 results)

All Other

All Publications (21 results)

[Publications] A.Kuzuya et al.: "Site-selective RNA scission at two sites for precise genotyping of SNPs by mass spectroscopy."Chem.Commun.. 770-771 (2003)
[Publications] Y.Shi et al.: "Stereochemically pure acridine-modified DNA for site-selective activation and scission of RNA."Chem.Lett.. 32. 464-465 (2003)
[Publications] Y.Yamamoto et al.: "Oligoamine-acridine conjugates for promotion of gap-selective DNA hydrolysis by Ce(IV)/EDTA complex."Nucleic Acids Res.. 31. 4497-4502 (2003)
[Publications] Y.Kitamura et al.: "Hydrolysis of DNA by cerium(IV)/EDTA complex."Tetrahedron. 59. 10403-10408 (2003)
[Publications] H.Arishima et al.: "Site-selective DNA hydrolysis by the combination of Ce(IV) and oligonucleotide bearing EDTA Groups"Nucleic Acids Res.Supple. 3. 137-138 (2003)
[Publications] A.Kuzuya et al.: "Tandem site-selective RNA scission utilizing acridine-DNA conjugates."Nucleic Acids Res.Supple. 3. 167-168 (2003)
[Publications] S.Ye et al.: "Simultaneous detection of multiple single nucleotide polymorphism by single-strand-specific nuclease and PNA probe."Nucleic Acids Res.Supple. 3. 185-186 (2003)
[Publications] Y.Yamamoto et al.: "Peptide nucleic acid for rapid gap-selective hydrolysis of DNA by Ce(IV)/EDTA complex."Chem.Lett.. 32. 76-77 (2003)
[Publications] A.Kuzuya.et al.: "Selective activation of two sites of RNA by acridine-bearing oligonucleotide for clipping of designated RNA fragment."J.Am.Chem.Soc. 126. 1430-1436 (2004)
[Publications] W.Chen et al.: "Monophosphate as eminent ligand to bind Ce(IV)/EDTA complex for site-selective DNA hydrolysis."Chem.Lett. 33(in press). (2004)
[Publications] S.Ye et al.: "DNA protection by PNA from enzymatic digestion for mass-spectroscopic genotyping"Chem.Lett.. 32. 10-11 (2003)
[Publications] M.Komiyama et al.: "PNA for one-base differentiating protection of DNA from nuclease and its use for SNPs detection"J.Am.Chem.Soc. 125. 3758-3762 (2003)
[Publications] B.Ren et al.: "Acridine-bearing PNA for efficient protection of designated site of DNA from nuclease S1."Chem.Lett.. 32. 714-715 (2003)
[Publications] B.Ren et al.: "Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system."Nucleic Acids Res.. 32(in press). (2004)
[Publications] H.Asanuma et al.: "DNA-Dye conjugate for controllable H^* aggregation"J.Am.Chem.Soc.. 125. 2217-2223 (2003)
[Publications] H.Kashida et al.: "DNA-Naphthyl Red conjugate as a visualizing probe of DNA hybridization."Chem.Commun.. 1536-1537 (2003)
[Publications] H.Kashida et al.: "Development of a probe DNA which accompanies color change on hybridization."Nucleic Acids Res.Supple. 3. 143-144 (2003)
[Publications] H.Asanuma et al.: "Photo-regulation of DNA function by azobenzene-tethered oligonucleotides."Nucleic Acids Res.Supple. 3. 117-118 (2003)
[Publications] M.Liu et al.: "Effective photo-regulation of transcription reaction by SP6 RNA polymerase with modified DNA tethering multiple azobenzenes"Nucleic Acids Res.Supple. 3. 265-266 (2003)
[Publications] X.Liang et al.: "NMR study on the photoresponsive DNA tethering an azobenzene. Assignment of the absolute configuration of two diastereomers and structure determination of their duplexes in the trans-form"J.Am.Chem.Soc. 125. 16408-16415 (2003)
[Publications] M.Liu et al.: "Synergistic effect of the two azobenzenes in the promoter on the photo-regulation of transcription reaction with SP6 RNA polymerase."Chem.Lett. 32. 1174-1175 (2003)

2003 Fiscal Year Annual Research Report

人工制限酵素によるDNAの位置特異的切断と機能性核酸の合成

Principal Investigator

小宮山 真 東京大学, 先端科学技術研究センター, 教授 (50133096)

Research Products

[Publications] A.Kuzuya et al.: "Site-selective RNA scission at two sites for precise genotyping of SNPs by mass spectroscopy."Chem.Commun.. 770-771 (2003)

[Publications] Y.Shi et al.: "Stereochemically pure acridine-modified DNA for site-selective activation and scission of RNA."Chem.Lett.. 32. 464-465 (2003)

[Publications] Y.Yamamoto et al.: "Oligoamine-acridine conjugates for promotion of gap-selective DNA hydrolysis by Ce(IV)/EDTA complex."Nucleic Acids Res.. 31. 4497-4502 (2003)

[Publications] Y.Kitamura et al.: "Hydrolysis of DNA by cerium(IV)/EDTA complex."Tetrahedron. 59. 10403-10408 (2003)

[Publications] H.Arishima et al.: "Site-selective DNA hydrolysis by the combination of Ce(IV) and oligonucleotide bearing EDTA Groups"Nucleic Acids Res.Supple. 3. 137-138 (2003)

[Publications] A.Kuzuya et al.: "Tandem site-selective RNA scission utilizing acridine-DNA conjugates."Nucleic Acids Res.Supple. 3. 167-168 (2003)

[Publications] S.Ye et al.: "Simultaneous detection of multiple single nucleotide polymorphism by single-strand-specific nuclease and PNA probe."Nucleic Acids Res.Supple. 3. 185-186 (2003)

[Publications] Y.Yamamoto et al.: "Peptide nucleic acid for rapid gap-selective hydrolysis of DNA by Ce(IV)/EDTA complex."Chem.Lett.. 32. 76-77 (2003)

[Publications] A.Kuzuya.et al.: "Selective activation of two sites of RNA by acridine-bearing oligonucleotide for clipping of designated RNA fragment."J.Am.Chem.Soc. 126. 1430-1436 (2004)

[Publications] W.Chen et al.: "Monophosphate as eminent ligand to bind Ce(IV)/EDTA complex for site-selective DNA hydrolysis."Chem.Lett. 33(in press). (2004)

[Publications] S.Ye et al.: "DNA protection by PNA from enzymatic digestion for mass-spectroscopic genotyping"Chem.Lett.. 32. 10-11 (2003)

[Publications] M.Komiyama et al.: "PNA for one-base differentiating protection of DNA from nuclease and its use for SNPs detection"J.Am.Chem.Soc. 125. 3758-3762 (2003)

[Publications] B.Ren et al.: "Acridine-bearing PNA for efficient protection of designated site of DNA from nuclease S1."Chem.Lett.. 32. 714-715 (2003)

[Publications] B.Ren et al.: "Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system."Nucleic Acids Res.. 32(in press). (2004)

[Publications] H.Asanuma et al.: "DNA-Dye conjugate for controllable H^* aggregation"J.Am.Chem.Soc.. 125. 2217-2223 (2003)

[Publications] H.Kashida et al.: "DNA-Naphthyl Red conjugate as a visualizing probe of DNA hybridization."Chem.Commun.. 1536-1537 (2003)

[Publications] H.Kashida et al.: "Development of a probe DNA which accompanies color change on hybridization."Nucleic Acids Res.Supple. 3. 143-144 (2003)

[Publications] H.Asanuma et al.: "Photo-regulation of DNA function by azobenzene-tethered oligonucleotides."Nucleic Acids Res.Supple. 3. 117-118 (2003)

[Publications] M.Liu et al.: "Effective photo-regulation of transcription reaction by SP6 RNA polymerase with modified DNA tethering multiple azobenzenes"Nucleic Acids Res.Supple. 3. 265-266 (2003)

[Publications] X.Liang et al.: "NMR study on the photoresponsive DNA tethering an azobenzene. Assignment of the absolute configuration of two diastereomers and structure determination of their duplexes in the trans-form"J.Am.Chem.Soc. 125. 16408-16415 (2003)

[Publications] M.Liu et al.: "Synergistic effect of the two azobenzenes in the promoter on the photo-regulation of transcription reaction with SP6 RNA polymerase."Chem.Lett. 32. 1174-1175 (2003)

小宮山真東京大学, 先端科学技術研究センター, 教授 (50133096)