Base-Functionalized Nucleoside Analog Probes: Design, Synthesis and Applications in Nucleic Acid Labeling and Diagnosis

Abstract

Environment- and conformation-sensitive fluorescent nucleoside analogs are very useful in studying the structure and recognition properties of nucleic acids. Although several structurally diverse nucleoside analogs exhibiting probe-like properties have been developed over the years, majority of these analogs have absorption maximum in the UV region and importantly, display drastic fluorescence quenching upon incorporation into nucleic acids. Due to these shortcomings many of these analogs cannot be easily implemented in nucleic acid analysis and in cell based studies. Therefore, development of robust nucleoside analog probes and new labeling techniques suitable for in vitro as well as in cell analysis will greatly advance the fundamental understanding of nucleic acid structure and function. This thesis illustrates the design, synthesis and photophysical characterization of environment-sensitive fluorescent nucleoside analogs obtained by attaching heterocyclic rings onto the 5-position of a pyrimidine base. Some of these analogs are moderately emissive and are highly sensitive to solvent polarity and viscosity changes. Utilizing the probe-like properties of these analogs we have devised simple fluorescence-based assays to detect abasic sites in nucleic acids and discriminate different nucleic acid topologies (e.g., DNA/RNA G-quadruplex (GQ) and i-motif structures). We have also developed a simple and practical posttranscriptional chemical labeling method for RNA by using bioorthogonal reactions. In this strategy novel azide-modified uridine triphosphates have been incorporated into RNA by transcription reaction. The azide-modified RNA transcripts are conveniently labeled with a variety of biophysical probes in a modular fashion by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) and azide-phosphine Staudinger ligation reactions. Taken together, our results highlight the potential of these novel functionalized nucleoside analogs as efficient tools to study the structure and function of nucleic acids.