Engineering a Synthetic Fluorogenic RNA Switch for Oxidized Guanine.

Reactive oxygen species generate a family of oxidized guanine species that serve as important biomarkers of oxidative damage. Here, we report the development of a fluorogenic RNA switch that selectively recognizes oxidized guanine species. We first systematically characterized an oxidized guanine RNA aptamer, defining its minimum functional binding domain and molecular recognition profile. Structural minimization produced a compact aptamer with higher affinity than the full-length sequence that retained broad recognition of structurally diverse oxidized guanine species while discriminating against their unmodified counterparts. We subsequently incorporated the optimized aptamer into fluorogenic RNA switch architectures, revealing that fluorescence activation required iterative optimization of aptamer topology, scaffold architecture, and communication-linker design. The resulting fluorogenic RNA switch exhibited target-dependent fluorescence activation while retaining the aptamer's molecular recognition profile. This work establishes a fluorogenic RNA switch for oxidized guanine species and highlights the importance of systematic aptamer characterization and optimization in fluorogenic RNA switch development.