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Description
Fluorescent nucleoside analogues are chemically modified variants of natural nucleosides, which have been very valuable in the study of nucleic acid structure and function. Fluorescent nucleobases are useful because many of them structurally resemble natural bases, and also display useful properties, such as large Stoke shifts and sensitivity to changes in the local chemical environment. Unlike traditional fluorescence probes, the majority of which do not resemble natural biological structures and mostly report on longer-range changes, nucleoside analogues when incorporated into oligonucleotides can report changes at the nucleotide level. Shifting the absorption and emission spectra of a fluorescent nucleoside to the red was the inspiration for making a resorufin nucleoside analogue. A red-shifted analogue would enable new FRET applications, while also reducing background fluorescence in biological media. Through our research on synthetic approaches, we were able to functionalize resorufin with a bromide ortho to the hydroxyl. The functionalization of resorufin will make it possible to glycosylate a ribose sugar via Heck reaction and make it possible to conduct further photophysical studies. A second project in this thesis is to further the photophysical and lifetime fluorescent studies of the known 8-DEA-tC. This tricyclic cytidine analogue is known for its fluorescent turn-on effect upon DNA-DNA duplex formation with up to a 20-fold increase in fluorescence quantum yield. We furthered the research by studying 8-DEA-tC in DNA-RNA heteroduplexes and conducted photophysical and lifetime fluorescent studies. Our results showed that the 10-mer GXC DNA-RNA exhibited the largest Φem of 0.22 while other sequences had Φem values within 0.10 – 0.17. The least fluorescent sequence CXT had comparable Φem values to ssDNA and had the smallest fluorescence turn-on. Overall DNA-RNA experienced greater fluorescence turn-on effects than DNA-DNA.
