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) a nd 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 hetero duplex, is measured after 30 min and is linearly dependent on the enzy matic reaction time and the amount of active RT added to the enzyme as say, The increased fluorescence correlates well with the incorporation of [alpha-P-32]dTTP into DNA (r(2) = 0.986). However, similar assays with the Klenow fragment using poly(dA) oligo(dT) did not result in in creased fluorescence under conditions wherein incorporation of [alpha- P-32]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 r elative 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 kine tic constants of purified RT with inhibitors such as zidovudine 5'-tri phosphate, nevirapine, and oltipraz, This assay is far more rapid and less labor-intensive than standard isotopic assays for RT activity, an d is much less expensive to perform than nonisotopic assays that measu re the incorporation of nucleotide into DNA by immunological detection methods. This assay concept should allow for higher throughput screen ing of agents that inhibit RT, and may be useful for detecting RT acti vity in biological specimens, (C) 1995 Academic Press,Inc.