Development of indole-based hydration-sensitive fluorescent nucleoside analogues: experimental and computational...

Natural nucleobases lack the appropriate photophysical properties to serve as intrinsic fluorescence reporters. Therefore, fluorescence studies of DNA/RNA conformations and/or conformational changes often rely on the use of fluorescent nucleoside analogues (FNAs). While many FNAs have been developed thus far, they usually suffer from various limitations. Thus, further development of new FNAs with different fluorescence properties and hence tailored applications is required. Herein, we synthesize and characterize two indole-based hydration-sensitive nucleoside analogues, indole-4-carboxaldehyde-2'-deoxyribonucleoside (I4A-NS) and 4-acetylindole-2'-deoxyribonucleoside (4AcI-NS). These probes expand the toolkit of fluorescent nucleotides by offering several advantages: (1) they can equivalently pair with native nucleobases, without perturbing the native double-helical structure of DNA; (2) they emit strongly only in protic solvents with a large Stokes shift (e.g., the fluorescence quantum yield, fluorescence lifetime and maximum emission wavelength of 4AcI-NS are ca. 0.6, 6.8 ns and 487 nm, respectively, in water); (3) when incorporated into single- and double-stranded DNA, their fluorescence properties (intensity and wavelength) undergo significant changes, due to a transition into a dehydrated environment and fluorescence quenching by nearby natural nucleobases. Furthermore, using density functional theory calculations, we determine that their high fluorescence quantum yields in water arise from hydrogen-bonding interactions between the carbonyl group of these FNAs and solvent molecules, which alter the molecular orbital type of the lowest singlet excited state, effectively suppressing the non-radiative intersystem crossing process.