Development and applications of preparation methods of stable-isotopically labeled nucleic acids for structural analysis of high-molecular-weight nucleic acid and nucleic acid-protein complexes.

用于高分子量核酸和核酸-蛋白质复合物结构分析的稳定同位素标记核酸制备方法的开发和应用。

• 批准号: 08680725
• 负责人: ONO Akira
• 金额: $ 1.54万
• 依托单位: TOKYO METROPOLITAN UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08680725/
• 关键词: nuclear magnetic resonances; DNA; stable isotope; structural analysis; chemical synthesis; homeodomain; 化学合成; DNAオリゴマー; RNAオリゴマー; 多次元NMR; ヘア-ピン構造; 微生物発酵; ヌクレオシド

(1) Establishment of synthetic route of ^<13>C-labeled nucleosides from labeled glucose ; The stable-isotopically labeled uridine and thymidine are synthesized from labeled glucose in good overall yields. In contrast, the overall yields of the chemical syntheses of labeled purine nucleosides are not satisfactory. In order to overcome this drawback, we examined an enzymatic method for synthesizing purine nucleosides from pyrimidine nucleosides (uridine and thymidine) and purine bases (adenine and diaminopurine) by enzymatic trans-glycosylation reactions. Thus, adenosine, guanosine, 2'-deoxyadenosune, 2'-deoxyguanosine were obtained in over 90% yields from uridine and thymidine.(2) Application of labeled DNA oligomers to the structural analysis of DNA-protein complexes ; One of the goal of this study is the completion of the structural analysis of high-molecular-weight DNA-protein complexes. As molecular weight become higher, NMR signals become more broad and overlapped more severely. The signal overlaps can be reduced by using a set of residue-selectively labeled DNA oligomers, then the structural information obtained from isotopomers are accumulated to get more precise three dimensional structures. In order to estimate value of above methodology, the complex of 13C/15N-labeled 14mer DNA duplex and antennapedia homeodomain was used for NMR studies. One of the results, the sugar-phosphate backbone structure, BI or BII,of the DNA duplex can be assigned.

(1)以标记葡萄糖为原料合成13 C标记核苷的路线的建立；由标记的葡萄糖合成稳定同位素标记的尿苷和胸苷，总收率良好。相比之下，标记嘌呤核苷的化学合成的总产率并不令人满意。为了克服这个缺点，我们研究了一种通过酶转糖基化反应从嘧啶核苷（尿苷和胸苷）和嘌呤碱基（腺嘌呤和二氨基嘌呤）合成嘌呤核苷的酶法。由此，从尿苷和胸苷中得到腺苷、鸟苷、2'-脱氧腺苷、2'-脱氧鸟苷，收率超过90%。 (2)标记DNA寡聚物在DNA-蛋白质复合物结构分析中的应用；本研究的目标之一是完成高分子量DNA-蛋白质复合物的结构分析。随着分子量变高，NMR 信号变得更宽且重叠更严重。通过使用一组残基选择性标记的DNA寡聚物可以减少信号重叠，然后积累从同位素异构体获得的结构信息以获得更精确的三维结构。为了评估上述方法的价值，使用 13C/15N 标记的 14mer DNA 双链体和触角同源域的复合物进行 NMR 研究。结果之一是可以指定 DNA 双链体的糖-磷酸主链结构 BI 或 BII。

项目成果

Akira Ono: "Synthesis of (5'S)-[5'-^2H_1 ; 1',2',3',4',5'-^<13>C_5]-Thymidine via Stereoselective Deuteration of a 5-Oxoribose Derivative" Tetrahedron Letters. 38・3. 395-398 (1997)

Akira Ono：“通过 5-氧化核糖衍生物的立体选择性氘化合成 (5S)-[5-^2H_1 ; 1,2,3,4,5-^<13>C_5]-胸苷”四面体信件 38・3。

Ono,A: "Synthesis of (5′S)-〔5′^2Hi;1′,2′,3′,4′,5′,-^<13>C_5〕-thymidine Via stereoselective deuteration of a 5-oxoribose derivatire" Tetrahedron Letters. 38. 395-398 (1997)

Ono,A：“通过 5- 的立体选择性氘化合成 (5′S)-[5′^2Hi;1′,2′,3′,4′,5′,-^<13>C_5]-胸苷氧化核糖衍生物”四面体快报。38. 395-398 (1997)

Toko Shiina: "Structural analysis of DNA by isotope-aided NMR : application of ^<13>C/^2H-doubly labeled DNA oligomers" Nucleic Acids Symposium Series. 35. 17-18 (1996)

Toko Shiina：“通过同位素辅助NMR进行DNA结构分析：^ 13 C/^ 2 H-双标记DNA寡聚物的应用”核酸研讨会系列。

Szyperski, T.: "Measurement of ^3Jcip scalar couplings in a 17kDa protein complex with ^<13>C,^<15>N-labeled DNA distingishes the BI and BII phosphate conformation of the DNA" Journal of American Chemical Society. 119. 9901-9902 (1997)

Szyperski, T.：“用^ 13 C、^ 15 N标记的DNA测量17kDa蛋白质复合物中的^ 3Jcip标量耦合可区分DNA的BI和BII磷酸构象”美国化学会杂志。

Fernandes,C.: "NMR with ^<13>C/^<15>N-doubly labeled DNA:the anten napedia Homeodomain complex with a 14mer DNA duplex" Journal of Biomolecular NMR. (in press). (1998)

Fernandes，C.：“具有^ 13 C/^ 15 N-双标记DNA的NMR：具有14聚体DNA双链体的触角napedia同源结构域复合物”《生物分子NMR杂志》。