Native Chemical Ligation of Hydrolysis-Resistant 3'-NH-Cysteine-Modified RNA.

Abstract

Hydrolysis-resistant RNA-peptide conjugates that contain a 3'-NH linkage between the adenosine ribose and the C-terminal carboxyl group of a peptide moiety instead of the natural ester mimic acylated tRNA termini. Their detailed preparation that combines solid-phase oligonucleotide synthesis and bioconjugation is described here. The key step is native chemical ligation (NCL) of 3'-NH-cysteine-modified RNA to highly soluble peptide thioesters. These hydrolysis-resistant 3'-NH-peptide-modified RNAs, containing the universally conserved 3'-CCA end of tRNA, are biologically active and can bind to the ribosome. They can be used as valuable probes for structural and functional studies of the ribosomal elongation cycle.