Environment-Sensitive Base-Modified Fluorescent Ribonucleoside Analogue Probes: Synthesis, Incorporation and Applications

Abstract

Several biophysical tools have been developed to uncover the fundamentals of nucleic acid folding and recognition processes. In particular, fluorescent nucleoside analogues that report changes in their conformation and surrounding environment in the form of changes in the fluorescence properties such as quantum yield, emission maximum, lifetime and anisotropy have found wide applications in developing tools to investigate the structure, dynamics and function of nucleic acids. However, the majority of nucleoside analogues either have excitation and emission maximum in UV region and or exhibit progressive fluorescence quenching upon incorporation into single stranded and double stranded oligonucleotides, which preclude their implementation in certain fluorescence methods (e.g., single-molecule spectroscopy and cell microscopy). The overall aim of this thesis is to develop nucleoside probes that (i) are structurally minimally invasive, (ii) have emission maximum in the visible region, (iii) retain appreciable fluorescence efficiency when incorporated into oligonucleotides and (iv) importantly, report changes in microenvironment via changes in the photophysical properties. This thesis illustrates the design strategy, synthetic methodology, photophysical characterizations, enzymatic incorporation into RNA oligonucleotides and applications of two new base-modified fluorescent nucleoside analogues derived by conjugating benzothiophene and selenophene moieties at the 5 position of uracil.