Alkyne-Tagged Raman Probes for Local Environmental Sensing byHydrogen-Deuterium Exchange

Alkyne-tagged Raman probes have shown highpromise for noninvasive and sensitive visualization of smallbiomolecules to understand their functional roles in live cells.However, the potential for alkynes to sense cellular environmentsthat goes beyond imaging remains to be further explored. Here, wereport a general strategy for Raman imaging-based local environ-ment sensing by hydrogen-deuterium exchange (HDX) ofterminal alkynes (termed alkyne-HDX). Wefirst demonstrate, inmultiple Raman probes, that deuterations of the alkynyl hydrogenslead to remarkable shifts of alkyne Raman peaks for about 130cm-1, providing resolvable signals suited for imaging-based analysiswith high specificity. Both our analytical derivation andexperimental characterizations subsequently establish that HDXkinetics are linearly proportional to both alkyne pKas 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 tovarious DNA structures and demonstrates the capacity to detect DNA structural changesin situfrom UV-induced damage. Wefurther show that this strategy is also applicable to resolve subtle pD variations in live cells. Altogether, our work lays the foundationfor utilizing alkyne-HDX strategy to quantitatively sense the local environments for a broad spectrum of applications in complexbiological systems.