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- Feyrer, H, et al.
(författare)
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Enzymatic incorporation of an isotope-labeled adenine into RNA for the study of conformational dynamics by NMR
- 2022
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 17:7, s. e0264662-
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Tidskriftsartikel (refereegranskat)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.
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