Fluorescent nucleosides, nucleotides and oligonucleotides

PROJECT SUMMARY Nucleic acids and their building blocks play central roles in all cellular events and, as such, have immense impact on the emergence of diseases and, in turn, on human health. Studying such events is complicated by the non-emissive nature of the natural nucleobases, which frequently deprives researchers from the use of

modern fluorescence-based techniques. Faithful minimally perturbing emissive nucleoside surrogates can thus facilitate the monitoring of nucleoside, nucleotides and nucleic acids-based transformations at nucleoside/tide- “resolution”, and advance basic research, diagnostic tools and drug discovery efforts.

The goal of the proposed program is to design and synthesize new isomorphic emissive nucleoside and nucleotide analogs and implement them as probes for monitoring nucleoside- and nucleotide-based transformations as well as nucleic acids function, structure, dynamics and recognition. Specifically, major

contemporary challenges will be tackled in an attempt to bridge major gaps, among them: (a) Powerful biophysical techniques, such as Fluorescence-Detected Circular Dichroism (FDCD), introduced nearly five decades ago, remains practically unexplored; (b) Multiphoton, imaging and single molecule spectroscopy-

based experiments, using native or minimally perturbed oligonucleotides or nucleotide cofactors, are severely underutilized; (c) Similarly, single molecule enzymology of nucleoside/tide processing enzymes has not advanced; (d) Probes for real time exploration of fundamental processes such as peptidyl transferase, phase

separated membrane-less organelle formation and mRNA decay are lacking; (e) Nucleoside/tide-based metabolic processes and nucleotide-based signaling events cannot be directly monitored; and (f) High throughput screening for nucleosides and nucleosides processing enzymes cannot be performed in real-time

and in a high throughput manner without the use of faithful emissive surrogate substrates. Capitalizing on several useful families of emissive nucleoside surrogates developed in our laboratory, we will further refine our “designer” emissive and isomorphic nucleosides/tides and apply them to advance

solutions to the challenges articulated above. We will pursue the advancement of new physical and biochemical methods, as well as effective real-time screening and diagnostic tools. These efforts will expand the community’s arsenal of emissive functional probes, driving future strides into discovery and imaging

applications. These innovations, in turn, will further fundamental understanding of key biological processes related to disease development and will have long-term impact on improving human health. This applications seeks funding to replace an old, automated chromatography unit, which is critically important for all aspects of the project as they critically rely on the synthesis and purification of nucleosides.