Rapid NMR screening of RNA secondary structure and binding
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Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3′ end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields μmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA.
KeywordsNMR spectroscopy RNA secondary structure In vitro transcription Riboswitch RNA High throughput method
We greatly acknowledge support for RNA purification by Elke Stirnal and Christian Richter for continuous support with the NMR-spectrometers. This work was supported by Deutsche Forschungsgemeinschaft (DFG) in the SFB902: Molecular principles of RNA-based regulation and in Graduate College: CLIC. C.H. is supported by a fellowship of the Fonds der Chemischen Industrie. Work at the Center for Biomolecular Magnetic Resonance (BMRZ) is supported by the state of Hesse. H.S. and M.H. are members of the DFG-funded Cluster of Excellence: Macromolecular Complexes (EXC115).