Isothiazologuanosine ( tz G) as Environmentally Sensitive Fluorescent Reporter in RNA

ABSTRACT Site‐specific labeling of RNA with emissive, structurally non‐perturbing nucleosides is critically needed for probing RNA structure, dynamics, and function. Isothiazolopyrimidine‐based nucleoside surrogates are leading examples of fluorescent RNA building blocks, with the C‐nucleosidic guanosine analogue tz G standing out due to its brightness. Here, we report the first synthesis of a tz G phosphoramidite and its incorporation into RNA via solid‐phase synthesis. Comprehensive biochemical and photophysical analyses demonstrate that tz G acts as an isomorphic and isofunctional substitute for guanosine, with fluorescence properties that are sensitive to local environmental changes. These features enable fluorescence‐based monitoring of RNA cleavage by deoxyribozymes and of site‐specific RNA methylation by a methyltransferase ribozyme. Our findings establish tz G as a valuable emissive nucleoside analog for the interrogation of RNA modifications at defined positions.