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Synthesis and applications of quinazoline-based fluorescent nucleoside analogues

Abstract

A family of quinazoline-based fluorescent nucleoside analogues is synthesized for photophysical studies and applications in probing nucleic acid structure, dynamics, and recognition. These size-expanded U analogues exhibit fluorescent emission wavelengths that span 155 nm, from 335 to 490 nm. Each nucleoside has unique characteristic response to changes in its microenvironment. These distinct features lead to a variety of applications in biological assays, many of which have been explored. The fluorescent nucleoside analogues are useful in Förster Resonance Energy Transfer (FRET) experiments. The 5- methoxyquinazoline-2,4(1H,3H)-dione based nucleoside acts as the donor fluorophore to commercially available 7- diethylaminecoumarin. The FRET pair is used in a robust analysis and discovery platform for antibiotics targeting the bacterial ribosomal RNA A-site. The emissive U surrogate is incorporated into a model RNA construct of the A-site and the aminoglycosides are labeled with the 7- diethylaminecoumarin fluorescence acceptor. Titrating the coumarin labeled aminoglycosides into the emissive A-site construct shows a decrease in donor emission and concurrent increase of the acceptor emission. Titration curves faithfully generate EC50 values. Titration of unlabeled ligands into the pre-formed FRET complex yields valuable data regarding competitive displacement of aminoglycosides. Furthermore, an orthogonal FRET assembly reports antibiotic affinities to two different RNA targets. A binder was labeled with a fluorophore that acts both as an acceptor for the emissive nucleoside on the bacterial A-site and a donor fluorophore for the terminally-labeled human A-site. Unlabeled drugs were used to dissociate the labeled antibiotic. The nucleoside based on 5-aminoquinazoline-2,4(1H,3H)-dione makes a good FRET acceptor to tryptophan, one of the most infrequently occurring amino acids in proteins. The FRET pair facilitates the study of RNA-protein interactions, which is demonstrated in studying the binding of the Rev peptide to the RRE. Additionally, upon incorporation into a RNA oligonucleotide, 5-aminoquinazoline-2,4(1H,3H)-dione detects the presence of a RNA bulge by fluorescence enhancement and hypsochromic wavelength shift. In another single fluorophore experiment, the fluorescent U-analogue 7-aminoquinazoline-2,4(1H,3H)-dione detects mismatch G upon incorporation into a DNA oligonucleotide by displaying G-specific fluorescence enhancement

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