Fluorescent Tricyclic Cytidine Analogues as Substrates for Retroviral Reverse Transcriptases

Abstract

We report on the ability of the reverse transcriptases (RTs) from avian myeloblastosis virus (AMV), Moloney murine leukemia virus (M-MLV), and human immunodeficiency virus 1 (HIV-1) to generate labeled DNA using the fluorescent tricyclic cytidine analogues d(tC)TP and d(^DEA tC)TP as substrates. Michaelis-Menten kinetics for the insertion of these analogues show V_max /K_M from 0.0-5 times that of natural dCTP across from G, depending on the polymerase and whether the template is RNA or DNA. The analogues are prone to misinsertion across from adenosine with both RNA and DNA templates. Elongation after analogue insertion is efficient with RNA templates, but the analogues cause stalling after insertion with DNA templates. A model reverse transcription assay using HIV-1-RT, including RNA-dependent DNA synthesis, degradation of the RNA template by the RT's RNase H activity, and synthesis of a second DNA strand to form fluorescently labeled dsDNA, shows that d(tC)TP and d(^DEA tC)TP are compatible with a complete reverse transcription cycle in vitro.

Keywords: fluorescence; nucleotide analogue; polymerase; retrovirus; reverse transcriptase.