Analysis of the RNase H activity by fluorescence resonance energy transfer.
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.
Miyashiro
H
H
Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan.
Kimura
T
T
Tomiyama
M
M
Hattori
M
M
D001483:Base Sequence; D005452:Fluoresceins; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D066298:In Vitro Techniques; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism; D012235:Rhodamines; D016914:Ribonuclease H / Q000378:metabolism*; D013379:Substrate Specificity
C106141:6-carboxytetramethylrhodamine; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D009843:Oligoribonucleotides; D012235:Rhodamines; C024098:6-carboxyfluorescein; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H