Synthesis of (6-(13)C)pyrimidine nucleotides as spin-labels for RNA dynamics |
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Authors: | Wunderlich Christoph H Spitzer Romana Santner Tobias Fauster Katja Tollinger Martin Kreutz Christoph |
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Institution: | Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria. |
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Abstract: | We present a (13)C-based isotope labeling protocol for RNA. Using (6-(13)C)pyrimidine phosphoramidite building blocks, site-specific labels can be incorporated into a target RNA via chemical oligonucleotide solid-phase synthesis. This labeling scheme is particularly useful for studying milli- to microsecond dynamics via NMR spectroscopy, as an isolated spin system is a crucial prerequisite to apply Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion type experiments. We demonstrate the applicability for the characterization and detection of functional dynamics on various time scales by incorporating the (6-(13)C)uridine and -cytidine labels into biologically relevant RNAs. The refolding kinetics of a bistable terminator antiterminator segment involved in the gene regulation process controlled by the preQ(1) riboswitch class I was investigated. Using (13)C CPMG relaxation dispersion NMR spectroscopy, the milli- to microsecond dynamics of the HIV-1 transactivation response element RNA and the Varkud satellite stem loop V motif was addressed. |
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