Fully Reversible Regulation of 8‐17 DNAzyme Activity Using Nucleoside‐Based Diarylethenes

Controlling enzyme activity with external triggers is a central goal in chemical biology. Light is an especially attractive stimulus because it is noninvasive, offers high spatiotemporal precision, and can reversibly modulate molecular function. In this study, we use nucleoside‐based diarylethenes (DAEs) to achieve light‐dependent control of the RNA‐cleaving deoxyribozyme (DNAzyme) 8–17. Three high‐performing DAE‐modified 2′‐deoxyuridines were incorporated at structurally informed positions within the DNAzyme. A single optimal combination, dU‐Ph t Bu at position 2.1, afforded efficient, reversible photocontrol: in the photostationary state after ultraviolet irradiation (PSS UV ), the catalytic rate is reduced ∼11‐fold relative to the all‐open form (OF), while comparison of isolated OF and closed form (CF) reveals an ∼200‐fold difference in activity. The switch is thermally robust and can be cycled repeatedly with minimal loss of function. In contrast, seemingly minor changes in DAE substitution, such as addition of a single methyl group, abolish useful regulation, underscoring the critical interplay between photoswitch structure and biomolecular context.