Miyashiro H, Kimura T, Tomiyama M, Hattori M
Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan.
Nucleic Acids Symp Ser. 2000(44):55-6. doi: 10.1093/nass/44.1.55.
The RNase H activity of HIV-1 reverse transcriptase was examined using chemically synthesized deoxyribo.ribo-oligonucleotide hybrid duplex labeled with the fluorescence donor at the 5'-end and with the fluorescence acceptor at the 3'-end of DNA strand as a substrate. Fluorescence resonance energy transfer (FRET) between these fluorescent dyes was used to analyze the rate of the enzymatic reaction. Under excitation of the donor dye, that is 6-carboxyfluorescein (6-FAM), at 490 nm, the increase of the fluorescence resulting from the acceptor dye, that is 6-carboxytetramethylrhodamine (TAMRA), at 578 nm, was observed depending on the degradation of DNA.RNA hybrid duplex. This method can be introduced into the high throughput screening of the inhibitors against the RNase H activity for anti-HIV drug.
使用化学合成的、在DNA链的5'端标记有荧光供体且在3'端标记有荧光受体的脱氧核糖-核糖寡核苷酸杂交双链体作为底物，检测HIV-1逆转录酶的核糖核酸酶H活性。利用这些荧光染料之间的荧光共振能量转移（FRET）来分析酶促反应速率。在490nm激发供体染料（即6-羧基荧光素，6-FAM）时，观察到在578nm处受体染料（即6-羧基四甲基罗丹明，TAMRA）产生的荧光随着DNA-RNA杂交双链体的降解而增加。该方法可引入针对抗HIV药物的核糖核酸酶H活性抑制剂的高通量筛选中。
