Preparation of Novel Modified Oligonucleotides and Their Application to Man-made Enzymes with Sequence-Specific RNA-Cleaving Activity

新型修饰寡核苷酸的制备及其在具有序列特异性 RNA 切割活性的人造酶中的应用

• 批准号: 09450337
• 负责人: SAWAI Hiroaki
• 金额: $ 8.64万
• 依托单位: Gunma University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-09450337/
• 关键词: DNA; Modified Nucleic Acids; 5-Substituted 2'-Deoxyuridines; 2'-Modiifed Nucleosides; 2-Substituted Deoxyuanosine Derivatives; RNA Cleaving Activity; Tris-Amine; Labeled DNA; トリスアシン; 2'-位置換スクレオシド; 2重鎖形成能; 2'デオキシウリジン誘導体; α-ヌクレオシド; アンチセンス核酸; 標

We have developed facile synthesis of new 5-substituted thymidine derivatives, which are key compounds for the synthesis of modified DNAs. The modified oligodeoxyribonucleotides were prepared by a DNA synthesizer using the 5-substituted thymidine derivatives and the commercial available nucleoside phosphoramidites. The functional molecules were introduced into the modified oligonucleotides by pre- or post-synthetic modification methods. Post-synthetic modification is useful for the synthesis of various modified DNA in a final step of the DNA synthesis. Multi-functionalization of oligodeoxyribonucleotides could be accomplished by the new post-synthetic modification method. Various functional molecules such as a fluorescent agent, a chelating agent, a catalyst or an enzyme were incorporated into the modified oligodeoxyribonucleotides at the 5-position of the thymidine derivatives. The substituent groups on the modified oligodeoxyribonucleotides have positive or negative effect on the dup … More lex stability depending on the nature of the substituent group. Modified oligodeoxyribonucleotides bearing fluorescence were used for the studies of structure of DNA and of interaction of DNA and DNA binding protein. Complexes of a radioactive metal ion, Y-90 or In-111, with the modified oligodeoxyribonucleotides bearing a chelating agent whose sequence is complementary to N-Myc gene were used for fundamental studies of diagnosis and radiotherapy of some cancer related to the N-Myc. Other new modified oligonucleotides bearing α-nucleosides, 2-substituted deoxyguanosine, sugar-part 2'-modifed deoxyuridine or arabinosyl uridine were also prepared and their double-helix formation with the complementary DNAs and RNAs were studied. Tris (2-aminoethylamine), which was proved to be an acid-base catalyst for the cleavage of RNA in our previous study, was introduced into the modified oligodeoxyribonucleotides at 5-position of thymidine, 5-position of α-thymidine, 2-position of deoxyguanosine or 2'-position of uridine, and their RNA cleaving activities were assessed. The modified oligodeoxyribonucleotides bearing tris (2-aminoethylamine) at 2-modified deoxyguanosine showed strong sequence-specific RNA cleaving activity, if the bulge-out was created at the target site of RNA. Less

我们开发了新的5-取代胸苷衍生物的简易合成，这是合成修饰DNA的关键化合物。使用5-取代的胸苷衍生物和市售核苷亚磷酰胺，通过DNA合成仪制备修饰的寡脱氧核糖核苷酸。通过合成前或合成后修饰方法将功能分子引入修饰的寡核苷酸中。合成后修饰对于在DNA合成的最后一步中合成各种修饰的DNA是有用的。这种新的合成后修饰方法可以实现寡脱氧核苷酸的多功能化。将各种功能性分子如荧光剂、螯合剂、催化剂或酶掺入到修饰的寡脱氧核糖核苷酸的胸苷衍生物的5-位。修饰后的寡核苷酸上的取代基对dup有正或负的影响 ...更多信息 Lex稳定性取决于取代基的性质。修饰的带有荧光的寡脱氧核苷酸被用于研究DNA的结构以及DNA与DNA结合蛋白的相互作用。本文报道了放射性金属离子Y-90或In-111与带有螯合剂的修饰的寡脱氧核苷酸（其序列与N-Myc基因互补）的配合物，用于与N-Myc相关的某些癌症的诊断和放射治疗的基础研究。本文还制备了其它新的修饰寡核苷酸，如α-核苷、2-取代脱氧鸟苷、2 '-糖基修饰脱氧尿苷和阿拉伯糖基修饰尿苷，并研究了它们与互补DNA和RNA的双螺旋形成。在修饰后的寡脱氧核苷酸中，分别在胸苷5位、α-胸苷5位、脱氧鸟苷2位和尿苷2 '位引入三（2-氨基乙胺），测定其对RNA的切割活性。在2-修饰脱氧鸟苷上带有三（2-氨基乙胺）的修饰寡核苷酸显示出强的序列特异性RNA切割活性，如果在RNA的靶位点产生凸起。少

项目成果

M.Mine, H.Ozaki, H.Sawai: "Effect of an Linker Arm at C5 Position of Pyrimidine Nucleoside on Thermal Stability of Duplexes"Nucleic Acids.Symposium Series. 39. 53-54 (1998)

M.Mine、H.Ozaki、H.Sawai：“嘧啶核苷 C5 位点连接臂对双链体热稳定性的影响”核酸。研讨会系列。

H.Sawai, H.Wakai, A.Nakamura-Ozaki: "Synthesis and Reactions of Nucleoside 5'-Diphosphate Imidazolide. A Nonenzymatic Capping Agent for 5'-Monophosphorylated oligoribonucleotides in Aqueous Solution"J.Org.Chem.. 64. 5836-5840 (1999)

H.Sawai、H.Wakai、A.Nakamura-Ozaki：“核苷 5-二磷酸咪唑化物的合成和反应。水溶液中 5-单磷酸化寡核糖核苷酸的非酶加帽剂”J.Org.Chem.. 64. 5836

H.Ozaki,S.Moriyama,K.Yokotsuka,H.Sawai: "Synthesis of Oligodeopxyribonucleotides Bearing a Functional Group at 2'-Position by Post-Synthetic Modification"Nucleic Acids,Symposium Series. 42. 47-48 (1999)

H.Ozaki、S.Moriyama、K.Yokotsuka、H.Sawai：“通过合成后修饰合成 2 位带有官能团的寡脱氧核糖核苷酸”核酸，研讨会系列。

H.Ozaki,Y.Ogawa,M.Mine,H.Sawai: "Synthesis of Oligodeoxyribonucleotide Bearing 2'-S-Alkyl Residue and Its Effect on the Duplex Stability"Nucleosides and Nucleotides. (in press).

H.Ozaki，Y.Okawa，M.Mine，H.Sawai：“带有2-S-烷基残基的寡脱氧核糖核苷酸的合成及其对双链体稳定性的影响”核苷和核苷酸。

N.Watanabe,H.Sawaki,K.Endo,K.Shizozuka,H.Ozaki,S.Tanaka,H.Murai,Y.Sasaki: "Labeliong of Phosphotothioate Antisense Oligonucleotides with Yttrium-90"Nuclaer Med.Biol.. 26. 239-243 (1999)

N.Watanabe、H.Sawaki、K.Endo、K.Shizozuka、H.Ozaki、S.Tanaka、H.Murai、Y.Sasaki：“用钇 90 标记硫代磷酸酯反义寡核苷酸”Nuclaer Med.Biol.. 26