Daube, Diane, M. Vogel, Beatrix Suess, and Bjoern Corzilius. “Dynamic Nuclear Polarization on a Hybridized Hammerhead Ribozyme: An Explorative Study of RNA Folding and Direct DNP with a Paramagnetic Metal Ion Cofactor,” n.d., 43.
While uniform isotope labeling of ribonucleic acids (RNA) can simply and efficiently be achieved by in-vitro transcription, the specific introduction of nucleotides in larger constructs is non-trivial and often ineffective. Here, we demonstrate how a medium-sized (67-mer), biocatalytically relevant RNA (hammerhead ribozyme, HHRz) can be formed by spontaneous hybridization of two differently isotope-labeled strands, each individually synthesized by in-vitro transcription. This allows on the one hand for a significant reduction in the number of isotope-labeled nucleotides and thus spectral overlap particularly under magic-angle spinning (MAS) dynamic nuclear polarization (DNP) NMR conditions, on the other hand for orthogonal 13C/15N-labeling of complementary strands and thus for specific investigation of structurally or functionally relevant inter-strand and/or inter-stem contacts. By this method, we are able to confirm a non-canonical interaction due to single-site resolution and unique spectral assignments by two-dimensional 13C–13C (PDSD) as well as 15N–13C (TEDOR) correlation spectroscopy under “conventional” DNP enhancement. This contact is indicative of the ribozyme’s functional conformation, and is present in frozen solution irrespective of the presence or absence of a Mg2+ co-factor. Finally, we use different isotope-labeling schemes in order to investigate the distance dependence of paramagnetic interactions and direct metal ion DNP if the diamagnetic Mg2+ is substituted by a paramagnetic Mn2