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

Sequence-specific fluorescent probes for RNA are widelyused inmicroscopy applications such as fluorescence in situ hybridizationand a growing number of newer approaches to live-cell RNA imaging.The sequence specificity of most of these approaches relies on differentialhybridization of the probe to the correct target. Competing sequenceswith only one or two base mismatches are prone to causing off-targetrecognition. Here, we report the sequence-specific fluorescent detectionof model RNA targets using a tricyclic cytidine analogue (DEA)tC that is included as a surrogate for natural cytidine in DNA probestrands and that reports directly on Watson-Crick base pairing.The (DEA)tC-containing DNA oligonucleotide probes exhibitan average 8-fold increase in fluorescence intensity when hybridizedto matched RNA with (DEA)tC base paired with G and littlefluorescence turn-on when (DEA)tC is base paired with A.Duplex structure determination by NMR, time-resolved fluorescencestudies, and Stern-Volmer quenching experiments suggest thatthe combination of greater pi stacking and narrower grooves inthe A-form DNA-RNA heteroduplex provides additional shieldingand favorable electronic interactions between bases, explaining why (DEA)tC's fluorescence turn-on response to RNA targetsis typically 3-fold greater than for DNA targets.