Oxidation of guanine in DNA by Ru(phen)(2)(dppz)(3+) using the flash-quench technique
Oxidation of guanine in DNA by Ru(phen)(2)(dppz)(3+) using the flash-quench technique
复制标题
DOI：
10.1021/ja963606p
复制
发表时间：
1997-03-26
影响因子：
15
通讯作者：
Barton, JK
中科院分区：
文献类型：
--
作者：
Stemp, EDA;Arkin, MR;Barton, JK
研究人员开发了一种快速灭闪法来探测DNA的氧化损伤。光激发的Ru（II）插入剂在DNA中被弱束缚的电子转移猝灭剂猝灭，生成强大的基态氧化剂Ru（III）。一旦生成，与poly（dG-dC）结合的Delta-Ru(phen)(2)(dppz)（3+）迅速氧化DNA双链中的鸟嘌呤。瞬态吸收光谱显示在DNA双链中中性鸟嘌呤自由基的快速形成。用合成寡核苷酸双链的凝胶电泳监测闪灭实验造成的永久性损伤。通过哌啶处理，氧化损伤选择性地发生在5‘- ggg -3’位点的5'-G位点以及5‘- ggg -3’三胞胎的5'-G和中心G位点；在没有哌啶处理的情况下，酶切反应以Ru(NH3)(6)（3+）为猝灭剂生成8-氧-2'-脱氧鸟苷。此外，电子转移猝灭器的选择可调节基损伤的屈服。损伤量子产率的变化顺序为Ru(NH3)(6)(3+) <甲基紫素(2+)< Co(NH3)(5)Cl2+，并与还原猝灭剂的不稳定性有关。闪灭法结合了光谱学和产物分析，为探索双螺旋DNA的电子转移化学提供了一种新颖而可调的方法。
A flash-quench method has been developed to probe oxidative damage to DNA. A photoexcited Ru(II) intercalator is quenched in DNA by a weakly bound, electron-transfer quencher to generate Ru(III), a powerful ground-state oxidant. Once generated, Delta-Ru(phen)(2)(dppz)(3+) bound to poly(dG-dC) rapidly oxidizes guanine within the DNA duplex. Transient absorption spectroscopy indicates rapid formation of the neutral guanine radical within the DNA duplex. Permanent damage resulting from the flash-quench experiment is monitored by gel electrophoresis of synthetic oligonucleotide duplexes. Oxidative damage, visualized by treatment with piperidine, occurs selectively at the 5'-G of 5'-GG-3' sites and at the 5'- and central G of 5'-GGG-3' triplets; enzymatic digestion in the absence of piperidine treatment shows formation of 8-oxo-2'-deoxyguanosine with Ru(NH3)(6)(3+) as quencher. The yield of base damage is, furthermore, modulated by the choice of electron-transfer quencher. Quantum yields for damage vary in the order Ru(NH3)(6)(3+) < methyl viologen(2+) < Co(NH3)(5)Cl2+ and correlate with the instability of the reduced quencher. The flash-quench method, combining spectroscopy and product analysis, offers a novel and tunable approach to explore electron transfer chemistry on double helical DNA.