Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR

Abstract

Solution NMR spectroscopy is a well-established tool with unique advantages for structural studies of RNA molecules. However, for large RNA sequences, the NMR resonances often overlap severely. A reliable way to perform resonance assignment and allow further analysis despite spectral crowding is the use of site-specific isotope labeling in sample preparation. While solid-phase oligonucleotide synthesis has several advantages, RNA length and availability of isotope-labeled building blocks are persistent issues. Purely enzymatic methods represent an alternative and have been presented in the literature. In this study, we report on a method in which we exploit the preference of T7 RNA polymerase for nucleotide monophosphates over triphosphates for the 5’ position, which allows 5’-labeling of RNA. Successive ligation to an unlabeled RNA strand generates a site-specifically labeled RNA. We show the successful production of such an RNA sample for NMR studies, report on experimental details and expected yields, and present the surprising finding of a previously hidden set of peaks which reveals conformational exchange in the RNA structure. This study highlights the feasibility of site-specific isotope-labeling of RNA with enzymatic methods.

Extending the toolbox for RNA biology with SegModTeX: a polymerase-driven method for site-specific and segmental labeling of RNA

“…First, one can synthetize individual RNA fragments and then ligate with T4 DNA or RNA ligase — Puglisi and coworkers used this approach to probe a 100 kDa HCV IRES RNA by solution NMR 7 , 8 . Others, by extension, have added single isotopically labeled and modified bases at specific ligation sites using chemical or T7 RNA polymerase synthesis 9 , 10 . However, ligation itself has many shortcomings that severely limit its use including low yields in individual ligation and preparatory steps, sequence constraints at and around the ligation site, and minimum segment lengths 7 .…”