Preparation of large RNA oligonucleotides with complementary isotope-labeled segments for NMR structural studies

Abstract

RNA structure determination by solution NMR spectroscopy is often restricted to small RNAs (13C- and the other ¹⁵N-labeled, are prepared by in vitro transcription from a single plasmid DNA. The desired RNA fragments are excised from the primary transcript by two cis-acting hammerhead ribozymes, yielding the required engineered ends for subsequent, complementary ligation. The resulting RNA oligonucleotides display NMR spectra with greatly reduced resonance overlap and thus enable NMR studies of smaller labeled RNA segments within the native context of a large RNA. The procedure is expected to take 3–4 weeks to implement.