Synthesis of (6-13C)Pyrimidine Nucleotides as Spin-Labels for RNA Dynamics
2012
Wunderlich, Christoph H. | Spitzer, Romana | Santner, Tobias | Fauster, Katja | Tollinger, Martin | Kreutz, Christoph
We present a ¹³C-based isotope labeling protocol for RNA. Using (6-¹³C)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 (6-¹³C)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 preQ₁ riboswitch class I was investigated. Using ¹³C 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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