Design, Synthesis and Incorporation of Fluorescent Thiophene-Modified Nucleoside Analogs

The central dogma of molecular biology asserts the vastly important roles of nucleic acids in all domains of life. In order to explore and illuminate the intricate biomolecular interactions of nucleic acids with themselves and other biomolecules, isomorphic fluorescent nucleoside analogs have been developed. Fluorescence spectroscopy allows for the monitoring of molecular dynamics down to the subnanomolar concentration in real-time.;In order to continue to evolve as a field, novel isomorphic fluorescent nucleoside analogs must be synthesized and implemented in a variety of ways. Thiophene-modified fluorescent nucleoside analogs have proven to be of significant utility. However, the low quantum yield of several of these modified fluorescent nucleosides leaves room for further experimentation and improvement. To increase fluorescence signal sensitivity for several biophysical assays, multiple incorporations of a single thiophene-modified nucleoside were explored. These resulted in an enhanced on-signal for duplex melting and annealing as well as mismatch dC detection.;Additionally, in order to form pi-expanded chromophores, four new thiophene-modified fluorescent nucleoside analogs base on pyrrolo-cytidine (PyC) were synthesized and evaluated. Although these new nucleosides (fused and extended PyC analogs) are slightly larger than previous generations of Tor lab fluorescent nucleosides, they possess exceptional photophysical qualities such as unusually high brightness and sensitivity towards solvent polarity.;Finally, a novel fluorescent nucleoside analog FRET system was explored in hopes of monitoring RNA helicase activity in real-time. After synthesizing a novel fluorescent nucleic acid construct, a number of important factors were illustrated with regard to the need for a further red-shifted excitation wavelength and a higher quantum yield in the presence of endogenous proteins and their autofluorescence. Further studies into nucleic acid-protein interactions with a fluorescent nucleoside FRET pair hold great promise.