The rational design of fluorescent nucleoside analogues is greatly hampered by the lack of ag eneral method to predict their photophysics, ap roblem that is especially acute when base pairing and stacking changef luorescence. To better understand these effects, as eries of tricyclic cytidine (tC and tC O )a nalogues ranging from electron-rich to electron-deficient was designed and synthesized.T hey were then incorporated into oligonucleotides,a nd photophysical responses to base pairing and stackingw ere studied.W hen insertedi nto double-stranded DNA oligonucleotides, electron-rich analogues exhibit af luorescencet urn-on effect, in contrast with the electron-deficient compounds, whichs how diminished fluorescence. The magnitude of these fluores-cence changes is correlated with the oxidation potential of nearest neighbor nucleobases. Moreover,m atched base pairing enhances fluorescenceturn-on for the electron-rich compounds, and it causesaf luorescence decrease for the electron-deficientc ompounds. For the tC O compounds, the emergence of vibrational fine structure in the fluorescence spectra in response to base pairing and stacking was observed, offering ap otentialn ew tool for studying nucleic acid structurea nd dynamics. These results, supported by DFT calculations,h elp to rationalize fluorescencec hangesi n the base stacka nd will be useful for selectingt he best fluorescent nucleoside analogues for ad esired application.

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.

DEAtC is a tricyclic 2’-deoxycytidine analogue that can be incorporated into oligonucleotides by solid-phase synthesis and that exhibits a large fluorescence enhancement when correctly base-paired with guanine base in a DNA–DNA duplex. The synthesis of DEAtC begins with 5-amino-2-methylbenzothiazole and provides the DEAtC nucleobase analogue over four synthetic steps. This nucleobase analogue is then silylated using BSA and conjugated to Hoffer’s chlorosugar to provide the protected DEAtC nucleoside in good yield. Following protective group removal and chromatographic isolation of the β-anomer, dimethoxytritylation and phosphoramidite synthesis offered the monomer for solid-phase DNA synthesis. Solid-phase DNA synthesis conditions using extended coupling of the DEAtC amidite and a short deprotection time are used to maximize efficiency. By following the protocol described in this unit, the DEAtC fluorescent probe can be synthesized and incorporated into any desired synthetic DNA oligonucleotide.