Site-specific labeling of RNA

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 de-oxy-nu-cle-o-tide. 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-(32)P]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.