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Synthesis and Photophysical Studies of Tricyclic Cytidine Analogues in Oligonucleotides
Fluorescence spectroscopy is a sensitive, versatile, and straightforward analytical technique used extensively in modern research. The inherent emissions of some biomolecules, such as tryptophan, allow fluorescence spectroscopy to probe the inner workings of biological systems involving these molecules. However, the lack of intrinsic fluorescence from native nucleosides has made observing nucleic acid interactions problematic. This challenge has motivated extensive research in developing and studying emissive nucleoside analogues. 1,3-diaza-2-oxophenothiazine and 1,3-diaza-2-oxophenoxazine (tC and tCo, respectively) are part of a class of fluorescent nucleoside analogues known as tricyclic cytidines. The fluorescence of these two molecules are not significantly quenched in duplex nucleic acids, a property not usually demonstrated by other fluorescent nucleosides. Inspired by past work on tricyclic cytidine analogues, this study aimed to establish structural-photophysical relationships using derivatives of tC and tCo incorporated into short oligonucleotide sequences. These derivatives consisted of substituent modification at the C8 position of the tricyclic cytidine nucleobase. Results showed that modifications at the C8 position of the tC nucleoside with electron donating groups decreased the fluorescence quantum yield as the substituent strength effects increased. Nucleobase stacking as a result of incorporation into single- and double-stranded oligonucleotides displayed negligible influence on the fluorescence quantum yields of oligonucleotides containing 8-OCH3 tC. This was contrasted by the tCo scaffold. The quantum yields for 8-Cl tCo containing oligonucleotides exhibited greater sensitivity to base pairing associated with oligonucleotide hybridization as well as to the identities of the neighboring nucleobases stacked against fluorophore. The emissive properties of the reported tricyclic cytidine analogues in oligonucleotides were then applied to examine their usefulness in reporting on mismatched base pairing caused by tautomerism as well as abasic sites consisting of a tetrahydrofuran analogue. The data obtained yielded promising results in the detection of single nucleotide polymorphisms.
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