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

Sequence-specific fluorescent probes for RNA are widely used in microscopy applications such as fluorescence in situ hybridization and a growing number of newer approaches to live-cell RNA imaging. The sequence specificity of most of these approaches relies on differential hybridization of the probe to the correct target. Competing sequences with only one or two base mismatches are prone to causing off-target recognition. Here, we report the sequence-specific fluorescent detection of model RNA targets using a tricyclic cytidine analogue DEAtC that is included as a surrogate for natural cytidine in DNA probe strands and that reports directly on Watson-Crick base pairing. The DEAtC-containing DNA oligonucleotide probes exhibit an average 8-fold increase in fluorescence intensity when hybridized to matched RNA with DEAtC base paired with G and little fluorescence turn-on when DEAtC is base paired with A. Duplex structure determination by NMR, time-resolved fluorescence studies, and Stern-Volmer quenching experiments suggest that the combination of greater π stacking and narrower grooves in the A-form DNA-RNA heteroduplex provides additional shielding and favorable electronic interactions between bases, explaining why DEAtC's fluorescence turn-on response to RNA targets is typically 3-fold greater than for DNA targets.

Fluorescent Tricyclic Cytidine Analogues as Substrates for Retroviral Reverse Transcriptases.

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 Vmax /KM 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.

Electronic Modifications of Fluorescent Cytidine Analogues Control Photophysics and Fluorescent Responses to Base Stacking and Pairing.

The rational design of fluorescent nucleoside analogues is greatly hampered by the lack of a general method to predict their photophysics, a problem that is especially acute when base pairing and stacking change fluorescence. To better understand these effects, a series of tricyclic cytidine (tC and tCO ) analogues ranging from electron-rich to electron-deficient was designed and synthesized. They were then incorporated into oligonucleotides, and photophysical responses to base pairing and stacking were studied. When inserted into double-stranded DNA oligonucleotides, electron-rich analogues exhibit a fluorescence turn-on effect, in contrast with the electron-deficient compounds, which show diminished fluorescence. The magnitude of these fluorescence changes is correlated with the oxidation potential of nearest neighbor nucleobases. Moreover, matched base pairing enhances fluorescence turn-on for the electron-rich compounds, and it causes a fluorescence decrease for the electron-deficient compounds. For the tCO compounds, the emergence of vibrational fine structure in the fluorescence spectra in response to base pairing and stacking was observed, offering a potential new tool for studying nucleic acid structure and dynamics. These results, supported by DFT calculations, help to rationalize fluorescence changes in the base stack and will be useful for selecting the best fluorescent nucleoside analogues for a desired application.

Synthesis of Fluorescence Turn-On DNA Hybridization Probe Using the DEA tC 2'-Deoxycytidine Analog.

DEA tC is a tricyclic 2'-deoxycytidine analog that can be incorporated into oligonucleotides by solid-phase synthesis and that exhibits a large fluorescence enhancement when correctly base-paired with a guanine base in a DNA-DNA duplex. The synthesis of DEA tC begins with 5-amino-2-methylbenzothiazole and provides the DEA tC nucleobase analog over five synthetic steps. This nucleobase analog is then silylated using N,O-bis(trimethylsilyl)acetamide and conjugated to Hoffer's chlorosugar to provide the protected DEA tC nucleoside in good yield. Following protective-group removal and chromatographic isolation of the ß-anomer, dimethoxytritylation and phosphoramidite synthesis offer the monomer for solid-phase DNA synthesis. Solid-phase DNA synthesis conditions using extended coupling of the DEA tC amidite and a short deprotection time are employed to maximize efficiency. By following the protocols described in this unit, the DEA tC fluorescent probe can be synthesized and can be incorporated into any desired synthetic DNA oligonucleotide. © 2018 by John Wiley & Sons, Inc.