Development of a Universal RNA Dual-Terminal Labeling Method for Sensing RNA-Ligand Interactions

Dual labeling of an RNA can provide Förster resonance energy transfer(FRET)sensors for studying RNA folding,miRNA maturation,and RNA-protein interactions.Here,we report the development of a highly efficient strategy for direct dual-terminal labeling of any RNA of interest.We explored new Michael cycloaddition for facile labeling of 5′-terminal RNA with improved efficiency.Direct chemical tetrazinylation of RNA at the 3′-terminus was achieved with the highly efficient and catalysis-free tetrazine-cycloalkyne ligation.Both single-terminal labeling methods were combined for dual-terminal labeling of an RNA including short hairpin RNA,pre-miRNA,riboswitch,and noncoding RNA.Notably,these dual-labeled RNA-based FRET sensors were used to monitor RNA-ligand interactions in vitro and in live cells.It is anticipated that these universal RNA labeling strategies will be useful to study RNA structures and functions.