Fluorometric Measurement of Reverse Transcriptase Activity with 4′,6-Diamidino-2-phenylindole

Abstract

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 [α- 32P]dTTP into DNA ( r 2 = 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 [α- 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. This assay is far more rapid and less labor-intensive than standard isotopic assays for RT activity, and is much less expensive to perform than nonisotopic assays that measure the incorporation of nucleotide into DNA by immunological detection methods. This assay concept should allow for higher throughput screening of agents that inhibit RT, and may be useful for detecting RT activity in biological specimens.