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.

(1)建立了以标记葡萄糖为原料合成^<13> c标记核苷的路线；稳定同位素标记的尿嘧啶和胸腺嘧啶是由标记的葡萄糖合成的，总收率很高。相比之下，化学合成标记嘌呤核苷的总体产率并不令人满意。为了克服这一缺点，我们研究了一种酶法，通过酶促反式糖基化反应，从嘧啶核苷（尿嘧啶和胸腺嘧啶）和嘌呤碱基（腺嘌呤和二氨基嘌呤）合成嘌呤核苷。因此，从尿苷和胸腺嘧啶得到的腺苷、鸟苷、2'-脱氧腺苷、2'-脱氧鸟苷的产率超过90%。(2)标记DNA低聚物在DNA-蛋白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 -蛋白复合物的结构分析。分子量越大，核磁共振信号越宽，重叠越严重。利用一组残基选择性标记的DNA低聚物可以减少信号重叠，然后将从同位素体中获得的结构信息进行积累，得到更精确的三维结构。为了评估上述方法的价值，使用13C/ 15n标记的14mer DNA双链和天线基同域的配合物进行NMR研究。结果之一，可以确定DNA双链的糖-磷酸主链结构BI或BII。

(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.