Abstract

Biophysical probes for the study of nucleic acids are essential tools in the endeavor to understand the regulation and expression of the genetic code. While many capable fluorescent nucleoside analogues exist for these applications, it has been especially challenging to design analogues that maintain Watson–Crick hydrogen bonding, offer fluorescence brightness similar to conventional probes, and that absorb and emit at long wavelengths. Moreover, it is not yet possible to predict how the fluorescence of nucleobase analogues will respond to base pairing and stacking. Towards our goal of developing nucleoside analogues with new and enhanced fluorescent properties and plugging knowledge gaps in the relationship between base analogue structure and photophysics, we have designed a series of cytidine analogues with a range of structural modifications and studied their fluorescent responses to base pairing and stacking and the efficiency of their incorporation by DNA and RNA polymerases. This series of cytidine analogues shows clear relationships between analogue electronic properties, the electronics of neighboring bases, and the fluorescent responses to base pairing and stacking, including with mismatches. One of the analogues, DEA -tC, offers a powerful, sequence-specific fluorescence turn-on response to base pairing and stacking that is further enhanced in DNA /RNA heteroduplexes. The combination of photophysical studies, NMR structure determination of representative duplexes, and computational work helps to explain the observed photophysical properties and relate them back to cytidine analogue structure and neighboring base effects.