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 0.09-5 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.

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue DEAtC

“…37 By expanding on the parent tC molecular scaffold with a series of chemical modifications, we have developed novel derivatives with environmentally sensitive fluorescent properties. 25,34,38 One of these compounds, the tC analogue DEA tC, is nearly non-emissive as a free nucleoside in aqueous solution (Φ em = 0.006) but experiences up to a 20fold fluorescence turn-on effect (up to Φ em = 0.12) when basestacked in double-stranded DNA (dsDNA) and base paired with guanine (Figure 1), with some dependence on the identity of neighboring bases. 39 This fluorescence turn-on response to base pairing and stacking is rare; a few other base analogues that exhibit significant fluorescent increases in response to duplex formation have been reported; their turnon results for local rigidification.…”

“…FBAs have been used to report on local structure, conformation, and dynamics of nucleic acids, sequence (mis)­matches, enzyme-mediated nucleobase modifications, and changes in local polarity or pH. − Some nucleobase analogues such as 2AP experience nearly complete emission quenching when base-stacked in double-stranded nucleic acids. − However, others may remain emissive when base-stacked, − including the largely environmentally insensitive tricyclic cytidine (tC) nucleobase . By expanding on the parent tC molecular scaffold with a series of chemical modifications, we have developed novel derivatives with environmentally sensitive fluorescent properties. ,, One of these compounds, the tC analogue DEA tC, is nearly non-emissive as a free nucleoside in aqueous solution (Φ em = 0.006) but experiences up to a 20-fold fluorescence turn-on effect (up to Φ em = 0.12) when base-stacked in double-stranded DNA (dsDNA) and base paired with guanine (Figure ), with some dependence on the identity of neighboring bases . This fluorescence turn-on response to base pairing and stacking is rare; a few other base analogues that exhibit significant fluorescent increases in response to duplex formation have been reported; their turn-on results for local rigidification. , …”