Alkyne-Tagged Raman Probes for Local Environmental Sensing by Hydrogen-Deuterium Exchange

Alkyne-tagged Raman probes have shown high promise for noninvasive and sensitive visualization of small biomolecules to understand their functional roles in live cells. However, the potential for alkynes to sense cellular environments that goes beyond imaging remains to be further explored. Here, we report a general strategy for Raman imaging-based local environment sensing by hydrogen-deuterium exchange (HDX) of terminal alkynes (termed alkyne-HDX). We first demonstrate, in multiple Raman probes, that deuterations of the alkynyl hydrogens lead to remarkable shifts of alkyne Raman peaks for about 130 cm^-1, providing resolvable signals suited for imaging-based analysis with high specificity. Both our analytical derivation and experimental characterizations subsequently establish that HDX kinetics are linearly proportional to both alkyne pK_as and environmental pDs. After validating the quantitative nature of this strategy, we apply alkyne-HDX to sensing local chemical and cellular environments. We establish that alkyne-HDX exhibits high sensitivity to various DNA structures and demonstrates the capacity to detect DNA structural changes in situ from UV-induced damage. We further show that this strategy is also applicable to resolve subtle pD variations in live cells. Altogether, our work lays the foundation for utilizing alkyne-HDX strategy to quantitatively sense the local environments for a broad spectrum of applications in complex biological systems.