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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenine*
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Isotope Labeling
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Isotopes
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Magnetic Resonance Spectroscopy / methods
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Nuclear Magnetic Resonance, Biomolecular / methods
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Nucleic Acid Conformation
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RNA* / genetics
Grants and funding
JS: Marie Sklodowska-Curie IF (EU H2020, MSCA-IF Project No. 747446). European Research Executive Agency
https://ec.europa.eu/research/mariecurieactions/ KP: Swedish Research Council (grant number 2014-04303, and NT-2018-15),
https://www.vr.se/english.html the Swedish Foundation for Strategic Research (project number ICA14-0023 and FFL15-0178),
https://strategiska.se/en/ Harald och Greta Jeansson Stiftelse (JS20140009),
https://jeanssonsstiftelser.se/om-jeanssons-stiftelser/ Eva och Oscar Ahréns Stiftelse,
https://www.ahrenstiftelse.se Åke Wiberg Stiftelse (467080968 and M14-0109),
https://ake-wiberg.se Cancerfonden (CAN 2015/388, CAN 2018/715),
https://www.cancerfonden.se the Karolinska Institute (KID 2016-00062),
https://staff.ki.se/kid-funding the Ragnar Söderberg Stiftelse (M91/14),
https://ragnar.soderbergs.org Knut och Alice Wallenberg foundation [project grant KAW 2016.0087]
https://kaw.wallenberg.org The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.