Spatiotemporal Control of RNA Functions via Bioorthogonal Chemistry for Activatable Biosensing

Spatiotemporal control of RNA function is critical but challenging for the advancement of RNA-based biosensing and therapeutic technologies. In this work, we developed a bioorthogonally activatable strategy based on trans-cyclooctene (TCO)-caged RNAs that can be specifically activated by tetrazine (TZ) through rapid inverse-electron-demand Diels–Alder (IEDDA) reactions. We demonstrated that the site-specific modification of the single RNA in the 8–17 DNAzyme enabled spatiotemporal activation of DNAzyme sensors for precise Zn2+ imaging in live cells, with minimizing background signal leakage in extracellular environments during the delivery process. We also showed that the effective modification of sgRNA enabled precise control of the CRISPR/dCas9 transcriptional system, allowing on-demand expression of fluorescent proteins for signal reporting. We anticipate that our strategy will provide a versatile and effective platform for spatiotemporal control of RNA-based sensing elements, highlighting its potential for precise bioimaging, disease diagnosis, and gene therapy.