The goal of the proposed program is to design, synthesize and implement new fluorescent nucleoside analogs as probes for nucleic acids structure, dynamics and recognition. The main criteria directing the proposed work are to maintain the highest possible structural similarity to the natural nucleobases, to shift the emission to longer wavelengths, and to retain an adequate emission quantum efficiency.
The specific aims of this project are: (a) To design """"""""ideal"""""""" fluorescent nucleoside analogs that will possess the following characteristics: (i) A high structural similarity to the native nucleobases to faithfully mimic their size and shape, as well as hybridization and recognition properties, (ii) A red shifted absorption spectrum to minimize overlap with the absorption of the natural bases, (iii) Respectable emission quantum efficiency and a long emission wavelength (preferably in the visible range); (b) To devise concise synthetic approaches to the desired nucleobase analogs; (c) To examine the photophysical characteristics of the new nucleosides; (d) To convert the most promising ones into phosphoramidites for automated DNA and RNA synthesis; (e) To incorporate the fluorescent nucleobases into oligonucleotides; (f) To examine the photophysical characteristics of the modified oligonucleotides; and (g) To develop fluorescence-based assays that utilize the newly developed novel nucleosides. The increased appreciation for nucleic acids modification (e.g., methylation) in gene expression and its impact on diseases, as well as the rapid emergence of resistant pathogens, necessitate the development of new tools for the effective study of fundamental processes and the discovery of lead compounds as novel drugs. The successful analogs will be implemented into novel fluorescence-based assays. These assays will facilitate: (i) the study of nucleic acids modifying enzymes (e.g. DNA methyl transferases) that play crucial roles in development, genetic diseases and cancers, (ii) the discovery of novel anti-HIV agents assisted by a fluorescent TAR construct, an essential viral regulatory element, and (iii) the discovery of novel antibiotics targeted at the bacterial ribosome assisted by a fluorescent A-site analog. These investigations will advance the fundamental understanding of key biological processes and will have long-term impact on improving human health.
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