Isomeric Indole-Chalcone Surrogates as Complementary Reporters of Nucleic Acid Binding Interactions

Abstract

We report isomeric indole-6-hydroxyindanone (Ind6HI) nucleobase surrogates as complementary environment-sensitive fluorescent reporters for probing nucleic acid (NA) binding interactions directly within the native helix. Both probes act as base-stacked surrogates to occupy similar microenvironments within the helix. However, the 3Ind6HI isomer exhibits pronounced fluorescent molecular rotor (FMR) character that senses microviscosity changes associated with helix rigidification during hybridization. In contrast, the 4Ind6HI isomer functions as a solvatofluorochromic (SFC) probe that reports changes in local polarity. Thus, the FMR-active 3Ind6HI surrogate far outperforms the SFC-active 4Ind6HI surrogate for sensing hybridization, while the 4Ind6HI surrogate markedly outperforms the 3Ind6HI for sensing binding to non-NA targets, which generate hydration and polarity changes through the release of ordered water molecules and counterions from the helix. We demonstrate this ability through its emission response to model systems known to induce dehydration upon NA binding and include nonspecific protein binding by bovine serum albumin (BSA) and hen egg lysozyme (HEL) as well as minor-groove recognition by 4′,6-diamidino-2-phenylindole (DAPI) and distamycin (Dst). These findings highlight the utility of SFC surrogates for monitoring hydration changes in NA binding interactions and establish 4Ind6HI as a benchmark polarity-sensing nucleobase surrogate.