Chemical biology based determination of DNA local structures in vivo

在对DNA局部结构研究的基础上，综述了DNA功能和DNA结构分析方法的研究进展。8-溴G和5-卤尿嘧啶等功能性修饰碱基的引入，促进了DNA局部构象的结构分析。用含8-溴G的DNA分析人端粒G-四链体。已经表明，通过平面化合物的结合来稳定G-四链体显示出对端粒酶活性的强烈抑制。人类端粒序列d[AGGG（TTAGGG）_3]可形成不同类型的G-四链体结构。核磁共振显示，在Na^+溶液中，这22个核苷酸（nt）的序列呈现出反平行结构，而在K^+存在下的晶体学研究则显示出截然不同的平行结构。由于细胞内K^+的浓度大于Na^+的浓度，这段22 nt的序列在K^+存在下的结构引起了人们的极大兴趣。然而，K^+溶液中22 nt端粒序列的结构类型问题仍然悬而未决。我们用8-溴鸟嘌呤取代了序列中的Gs，并通过圆二色性（CD）光谱研究了所得的结构和热稳定性。我们发现22 nt在K^+溶液中以混合平行/反平行和椅子型G-四链体的混合物形式存在。用5-卤代尿嘧啶DNA分析DNA-蛋白质相互作用研究了5-卤代尿嘧啶DNA-蛋白质复合物的光反应性。发现Sso 7 d的色氨酸残基与5-溴尿嘧啶之间的有效电子转移产生了尿嘧啶自由基DNA。由于DNA中的胸腺嘧啶残基可被5-卤代尿嘧啶取代，进一步证实了该方法可用于体内DNA的构象探针。

Progress of the research on the DNA function and methodology of structural analysis of DNA, following investigation of local DNA structures was carried out. Incorporation of functional modified bases such as 8-bromoG and 5-halouracil facilitated structural analysis of DNA local conformations.Structural analysis of human telomere G-quadruplex using 8-bromoG-containing DNA.Single stranded 3 '-overhang of human telomere region is known to form K^+ ion coordinated stable G-quadruplex. It has been shown that stabilization of G-quadruplex by the binding of planer compound shows strong inhibition of telomerase activity. The human telomeric sequence d[AGGG (TTAGGG)_3] has been found to form different types of G-quadruplex structures. NMR revealed that in Na^+ solution this 22 nucleotide (nt) sequence exhibits an antiparallel structure, whereas crystallographic studies in the presence of K^+ showed a dramatically different parallel structure. The structure of this 22 nt sequence in the presence of K^+ has drawn intense interest as the intracellular K^+ concentration is greater than that of Na^+. However, the question of the type of structure for the 22 nt telomeric sequence in K^+ solution remains open. We substituted the Gs in the sequence with 8-bromoguanine and examined the resultant structures and thermal stabilities by circular dichroism (CD) spectroscopy. We discovered that the 22 nt in K^+ solution exists as a mixture of mixed-parallel/antiparallel and chair-type G-quadruplex.Analysis of DNA-protein interaction using 5-halouracil DNAInvestigation of the photoreactivity of 5-halouracil DNA-protein complex was carried out. It was found that efficient electron transfer from tryptophan residue of Sso7d to 5-bromouracil generate uracil radical DNA. Because thymine residue in DNA can be substituted with 5-halouracil, it was further confirmed that this method can be used for the conformational probe of DNA in vivo.