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

Abstract

Dual labeling of an RNA can provide FRET sensors for studying RNA folding, miRNA maturation, and RNAprotein 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 3'-terminus was achieved with the highly efficient and catalysis-free tetrazine-cycloalkyne ligation. The both single-terminal labeling methods were combined for dual-terminal labeling of an RNA including shRNA, pre-miRNA, riboswitch and ncRNA. Notably, these dual-labeled RNA-based FRET sensors could be used to monitor RNA-ligand interactions in vitro and in live cells. It is anticipated that these universal RNA labeling strategies would be useful to study RNA structures and functions.

A unique reaction of diphenylcyclopropenone and 1,2-aminothiol with the release of thiol for multiple bioconjugation

“…5 a Researchers have also achieved multiple labelling and imaging of glycans, 6 plant cell walls, 7 and nucleic acids. 8 In prior work, we reported a new Michael cycloaddition and its application for dual labelling of an RNA in one pot. 8 c Here, we report mechanistic insights into the reaction of DPCP ( 1 ) with 1,2-aminothiol and the dual labelling of N -terminal Cys ( N -Cys).…”