Fluorescence-based detection of specific DNA/RNA sequences in biological samples using classic fluorescence in situ hybridization (FISH) has been pivotal in optically detecting specific sequences. Common biological targets may include specific mRNA, genomic DNA, or cloning vectors. The probe strands are initially bound to a complement with a quencher, but upon binding to the target complement sequence, the quenching strand becomes dissociated and fluorescence can be observed. This method can yield false positives by hybridizing to similar sequences that differ little in their duplex thermodynamic stability. These mismatch hybrids may differ by 1-2 bases, yet exhibit similar thermostability to the desired hybrid, and highlight the major limitation of FISH in that fluorescence is the direct result of preferential binding and not necessarily sequence recognition. Probes with our lab’s tricyclic cytidine (tC) derivatives can potentially discriminate sequences more accurately than current hybridization methods. In our lab’s previous work, the tC derivative 8-diethylamino-tC (8-DEA-tC) was shown to have a fluorescence turn-on property in double-stranded DNA. This analogue has a low intrinsic emission of fluorescence (Φ<sub>em</sub>=0.010), however the emission can increase up to 0.12 when 8-DEA-tC pairs with guanosine in double-stranded DNA (dsDNA). The fluorescence turn-on does not occur when 8-DEA-tC is mismatched across a non-guanosine base, which may lead to more accurate hybridization probes. In this current work, we measure the photophysical properties of 8-DEA-tC DNA-RNA heteroduplexes using RNA. The magnitude of increase depends on the bases flanking 8-DEA-tC, however of the sequences tested, the ratio of emission is at least 0.10, which is more than double the average quantum yield measured for dsDNA. Though the sequences are identical as originally measured with DNA, the drastic change in fluorescence turn-on may be the product of overall duplex structure. 8-DEA-tC heteroduplexes are shown to adopt A-form DNA, like dsRNA, with a characteristic transition around 210 nm in the CD spectra. 8-DEA-tC probes may become especially useful for recognizing specific RNA in biological samples given the large increase in fluorescence upon forming DNA-RNA heteroduplexes.

Increasing the fluorescence turn-on of the nucleoside analogue 8-diethlamino-tC in duplex nucleic acids using DNA-RNA heteroduplexes

2019 Fall National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.