Chemical biology based determination of DNA local structures in vivo

介绍了在对DNA局部结构进行研究的基础上，对DNA功能和DNA结构分析方法的研究进展。8-溴基和5-卤脲嘧啶等功能修饰碱基的加入促进了DNA局部构象的结构分析。用含8-溴的DNA分析人类端粒g -四重体的结构。已知人类端粒区单链3 '-悬垂形成K^+离子协调的稳定g -四重体。研究表明，与planer化合物结合稳定g -四重体对端粒酶活性有较强的抑制作用。已发现人类端粒序列d[AGGG (TTAGGG)_3]形成不同类型的g -四重体结构。核磁共振显示，在Na^+溶液中，这22个核苷酸（nt）序列表现出反平行结构，而在K^+存在下的晶体学研究显示出明显不同的平行结构。由于细胞内K^+的浓度大于Na^+，因此在K^+存在的情况下，这个22nt序列的结构引起了强烈的兴趣。然而，在K^+溶液中，22nt端粒序列的结构类型仍然是一个开放的问题。我们用8-溴鸟嘌呤取代了序列中的Gs，并通过圆二色性（CD）光谱检测了合成的结构和热稳定性。我们发现在K^+溶液中，22nt以混合平行/反平行和chair型g -四重体的混合形式存在。利用5-卤脲嘧啶dna分析dna -蛋白相互作用研究了5-卤脲嘧啶dna -蛋白复合物的光反应性。发现从Sso7d的色氨酸残基到5-溴嘧啶的有效电子转移产生尿嘧啶自由基DNA。由于DNA中的胸腺嘧啶残基可以被5-卤脲嘧啶取代，进一步证实了该方法可用于DNA的体内构象探测。

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.