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

Abstract

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 livecell 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 DEA tC that is included as a surrogate for natural cytidine in DNA probe strands and that reports directly on Watson−Crick base pairing. The DEA tCcontaining DNA oligonucleotide probes exhibit an average 8-fold increase in fluorescence intensity when hybridized to matched RNA with DEA tC base paired with G and little fluorescence turn-on when DEA tC 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 DEA tC's fluorescence turn-on response to RNA targets is typically 3-fold greater than for DNA targets.

The importance of molecular diagnostic techniques on evaluation of cancers

“…Then, a fluorescent signal that indicates the existence of the sequence in a person's DNA may be seen. The DNA probes will wash off, and no fluorescence will be seen if the subject's DNA does not contain the problematic DNA sequence [31,32].…”