Novel FRET-based assay to detect reverse transcriptase activity using modified dUTP analogues.
We have developed a novel continuous assay to measure reverse transcriptase (RT) polymerase activity. The assay uses fluorescence energy transfer measurements to detect the incorporation of complementary pairs of fluorescently labeled deoxyuridine into cDNA product. The fluorescently labeled dUTP substrates were prepared using commercially available reagents with a simple coupling reaction. The fluorescent dye pairs have significant spectral overlap which allows FRET interaction between dyes incorporated into the cDNA. Using a polyA/oligo dT primer/template, the assay can readily detect DNA polymerase activity from any viral reverse transcriptase enzyme. The reaction proceeds linearly over time, and the rate is proportional to the enzyme concentration. We used the assay to compare the thermostability of a number of wild-type and mutant viral RT enzymes. Our results indicate that the wild-type AMV (avian myeloblastosis virus) enzyme is slightly more stable at 43 degrees C than the HIV-1 (human immunodeficiency virus) or MMLV (Moloney murine leukemia virus) enzymes. The thermostability of the RT enzyme was dramatically increased by the presence of primer/template with the enzyme. We also used the assay to study the effects of inhibitors on HIV-1 RT polymerase activity. This assay may be highly useful for the identification and characterization of potent RT inhibitors which could be candidates for development as therapeutic antiviral agents.
Krebs
Joseph F
JF
Ambion/Applied Biosystems, Research and Development Department, 2130 Woodward Austin, Texas 78744, USA. jkrebs@ambion.com
Kore
Anilkumar R
AR
D000818:Animals; D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D003856:Deoxyuracil Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D054306:Dideoxynucleotides / Q000378:metabolism; D004795:Enzyme Stability; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D015497:HIV-1 / Q000201:enzymology; D006358:Hot Temperature; D006801:Humans; D051379:Mice; D008979:Moloney murine leukemia virus / Q000201:enzymology; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D013942:Thymine Nucleotides / Q000378:metabolism; D015215:Zidovudine / Q000031:analogs & derivatives / Q000378:metabolism
D003856:Deoxyuracil Nucleotides; D054306:Dideoxynucleotides; D005456:Fluorescent Dyes; D018894:Reverse Transcriptase Inhibitors; D013942:Thymine Nucleotides; C027078:deoxyuridine triphosphate; D015215:Zidovudine; C052873:zidovudine triphosphate; D012194:RNA-Directed DNA Polymerase