Isothiazologuanosine (^tzG) as Environmentally Sensitive Fluorescent Reporter in RNA

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 ^tzG standing out due to its brightness. Here, we report the first synthesis of a ^tzG phosphoramidite and its incorporation into RNA via solid-phase synthesis. Comprehensive biochemical and photophysical analyses demonstrate that ^tzG 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 ^tzG as a valuable emissive nucleoside analog for the interrogation of RNA modifications at defined positions.