Fluorometric measurement of reverse transcriptase activity with 4',6-diamidino-2-phenylindole.
We describe a rapid fluorometric assay for reverse transcriptase (RT) activity. After RT is incubated in the presence of poly(A).oligo(dT) and dTTP for up to 1 h, the reaction is stopped with EDTA and aliquots are added to cuvettes containing 4',6-diamidino-2-phenylindole (DAPI). DAPI fluorescence, which is increased upon binding the RNA.DNA heteroduplex, is measured after 30 min and is linearly dependent on the enzymatic reaction time and the amount of active RT added to the enzyme assay. The increased fluorescence correlates well with the incorporation of [alpha-32P]dTTP into DNA (r2 = 0.986). However, similar assays with the Klenow fragment using poly(dA).oligo(dT) did not result in increased fluorescence under conditions wherein incorporation of [alpha-32P]dTTP into DNA was documented. Thus, the poly(A).poly(dT) [RNA.DNA] heteroduplex must differ from the poly(dA).poly(dT) [DNA.DNA] duplex in a manner that allows for a perturbation of DAPI fluorescence. The relative specific activities of RT in crude preparations measured with the fluorometric assay were comparable to conventional isotopic enzyme assays as were determinations for the type of inhibition and the kinetic constants of purified RT with inhibitors such as zidovudine 5'-triphosphate, nevirapine, and oltipraz.(ABSTRACT TRUNCATED AT 250 WORDS)
Chavan
S J
SJ
Molecular Pharmacology and Therapeutics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 1002, USA.
Prochaska
H J
HJ
D000998:Antiviral Agents / Q000494:pharmacology*; D054306:Dideoxynucleotides; D005456:Fluorescent Dyes; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology; D007202:Indicators and Reagents; D007211:Indoles; D007700:Kinetics; D019829:Nevirapine; D010761:Phosphorus Radioisotopes; D011719:Pyrazines / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012194:RNA-Directed DNA Polymerase / Q000032:analysis* / Q000378:metabolism; D011865:Radioisotope Dilution Technique; D018894:Reverse Transcriptase Inhibitors; D013050:Spectrometry, Fluorescence / Q000379:methods; D013871:Thiones; D013876:Thiophenes; D013942:Thymine Nucleotides / Q000494:pharmacology; D015215:Zidovudine / Q000031:analogs & derivatives / Q000494:pharmacology
D000998:Antiviral Agents; D054306:Dideoxynucleotides; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007211:Indoles; D010761:Phosphorus Radioisotopes; D011719:Pyrazines; D011725:Pyridines; D018894:Reverse Transcriptase Inhibitors; D013871:Thiones; D013876:Thiophenes; D013942:Thymine Nucleotides; C007293:DAPI; D015215:Zidovudine; C026209:oltipraz; C052873:zidovudine triphosphate; D019829:Nevirapine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase