Probing Nucleic Acid Structure and Recognition in Cell-Free and Cellular Environments Using Environment-Sensitive Nucleoside Probes

Abstract

In addition to the classical double helical structure, nucleic acid adopts various secondary and tertiary structures like hairpin, pseudoknot, bulge, triplex, tetraplex (G-quadruplex and i-motif) etc. to perform their cellular functions. Biophysical techniques like fluorescence, NMR, EPR, X-ray have been used widely to study nucleic acids in vitro. However, understanding the structure-function relationship of nucleic acid motifs in native cellular environment has remained a major challenge due to the paucity of chemical probes that can be used both in cell-free and cellular systems. Therefore, development of biophysical tools, which will enable the probing of nucleic acid structure, dynamics and recognition properties in both cell-free and cellular environments, will be highly beneficial in nucleic acid-targeted drug discovery. My thesis describes the development of biophysical platforms to study nucleic acid structure and recognition in cell-free and cellular environments by using conformation-sensitive nucleoside probes. The nucleoside probes are derived by attaching heterobicycles like benzofuran, 5-fluoro-benzofuran and 5-methoxybenzofuran at the 5 position of 2’-deoxyurindine and uridine. The conformation sensitivity of fluorescent benzofuran-modified nucleosides was utilized in probing the GQ structure and ligand binding properties of the human telomeric DNA and RNA repeats in aqueous buffer and cell-like confined environment. 5-Fluorobenzofuran-modified nucleoside analogs serve as dual-app probes composed of a microenvironment-sensitive fluorophore and a 19F NMR label. In particular, distinct signatures displayed by the 19F-labeled nucleoside for different GQs enabled a systematic study in Xenopus laevis oocytes to provide new structural insights into the GQ topologies adopted by human telomeric overhang in cells, which so far has remained unclear. The scope of this dual-app probe was further expanded in studying the conformational changes occurring in the internal ribosomal entry site of hepatitis C-virus RNA genome. Notably, the fluorescence properties of 5-methoxybenzofuran-modified uridine analog were found to be highly sensitive to subtle changes in the polarity of the surrounding environment. This probe photophysically reported the pyrimidine-pyrimidine mismatch and metal-mediated base pairing in an RNA-DNA duplex. The design, synthesis and use of environment-sensitive nucleoside probes in studying nucleic acid structure and recognition in vitro and in native cellular environment are discussed in the thesis.