Daube, Diane ; Vogel, Marc ; Suess, Beatrix ; Corzilius, Björn (2019)
Dynamic nuclear polarization on a hybridized hammerhead ribozyme: An explorative study of RNA folding and direct DNP with a paramagnetic metal ion cofactor.
In: Solid state nuclear magnetic resonance, 101
doi: 10.1016/j.ssnmr.2019.04.005
Artikel, Bibliographie

Kurzbeschreibung (Abstract)

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 C/N-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 C-C (PDSD) as well as N-C (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 Mg 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 Mg is substituted by paramagnetic Mn.

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