Site-Specific Labeling of RNA

Abstract

In this protocol, RNAs containing a specific internally labeled phosphate are generated. The entire transcript of interest is synthesized using in vitro transcription. It is then digested with RNase H in the presence of a complementary chimeric oligonucleotide of the sequence 5′-NNNddddNNNNNNN-3′, where N is a 2′-O-methyl (2′ OMe) ribonucleotide and d is a de­oxy­nu­cle­o­tide. RNase H specifically cleaves the RNA in the oligonucleotide–RNA hybrid at the phosphodiester bond opposite the 5′-most deoxynucleotide, creating 3′-hydroxyl and 5′-phosphate termini. After dephosphorylation of the 5′ end of the 3′ fragment with phosphatase, this terminus is labeled to high specific activity with [γ-32P]ATP. The fragments of the original RNA are then resealed with T4 DNA ligase in the presence of a splint (or bridge) oligonucleotide. These labeled RNAs can be used in subsequent procedures to probe RNA–protein or RNA–RNA interactions.