Probing DNA G-Quadruplex and I-Motif Structures Via a Fluorescent Nucleoside Analogue:...

Since the building blocks of DNA are nonfluorescent, various external fluorescence reporters have been employed to investigate the structure, dynamics, and function of DNA G-quadruplexes (GQs) and i-motifs (iMs), which play an important role in gene regulation and expression. However, most of those fluorescence reporters lack the ability to provide site-specific structural information of interest. Therefore, it is necessary to develop fluorescent nucleoside analogues that can be covalently inserted into oligonucleotides, which not only serve this purpose, but minimize any potential perturbation towards the native structure of the DNA systems in question. Herein, we characterize the spectroscopic utility of a high quantum yield fluorescent nucleoside analogue, 4-cyanoindole-2'-deoxyribonucleoside (4CNI-NS). We show that (1) incorporation of 4CNI-NS into various oligonucleotides does not alter their ability to fold into their respective native structures, nor does it affect the overall stability of those structures and (2) the fluorescence property of 4CNI-NS is sensitive to its local environment, and the fluorescence intensity and decay kinetics of the 4CNI-NS-containing oligonucleotides exhibit a clear dependence on their secondary structure formation. Collectively, our results demonstrate that 4CNI-NS can be used as a sensitive, isomorphic probe in the study of noncanonical DNA structures.