基于阳离子共轭聚合物比色检测HIV逆转录酶的RNase H活性

Colorimetric detection of RNase H activity of reverse transcriptase-HIV with cationic polythiophene derivative

利用阳离子聚噻吩衍生物与单链DNA和杂合体DNA/RNA通过静电相结合时所产生的紫外吸收变化,建立了一种检测HIV逆转录酶(RT-HIV)的RNase H活性的方法。阳离子聚噻吩衍生物的紫外吸收最大波长位于短波385nm,与单链DNA结合会使聚噻吩衍生物的紫外吸收最大波长红移至525nm;而与杂合体DNA/RNA结合时对其紫外吸收最大波长几乎没有影响,当利用RT-HIV的核糖核酸酶RNase H活性水解掉杂合体中的RNA时,杂合体溶液又会使聚噻吩衍生物的紫外吸收最大波长发生红移。结果表明,紫外吸收最大波长变化明显,甚至直观用肉眼就可以观察到杂合体水解前后溶液颜色的变化。同时还测定了不同时间下RNase H酶水解杂合体中RNA的吸光度变化曲线,计算出了RNase H酶水解的动力学常数和最大初速度。

A rapid and convenient method was established for the detection of RNase H activity of reverse transcriptase-HIV（RT-HIV） based on the absorption changes of the cationic conjugated polymer PMNT. The maximum UV absorbance of PMNT itself was at 385nm; the maximum UV absorbance of PMNT solution exhibited a marked red shift to 525nm after combing with ssDNA; but the maximum UV absorbance of the triplex DNA/RNA/PMNT was almost the same as that of PMNT itself （385nm）. When the DNA/RNA hybrids were hydrolyzed by RNase H of RT-HIV and then added to the PMNT solution, the maximum absorbance of the solution exhibited a marked red shift（525nm） once again. As a result, the maximum absorbance displayed an obvious change. The kinetic constants and the maximum initial velocity of RNase H were also obtained according to this method.