Dynamic nuclear polarization on a hybridized hammerhead ribozyme: An explorative study of RNA folding and direct DNP with a paramagnetic metal ion cofactor.

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.

Heteronuclear Cross-Relaxation under Solid-State Dynamic Nuclear Polarization.

We report on the spontaneous polarization transfer from dynamically hyperpolarized H to C during magic-angle spinning dynamic nuclear polarization (DNP) at temperatures around 100 K. The transfer is mediated by H-C cross-relaxation within methyl groups due to reorientation dynamics, and results in an inverted C NMR signal of enhanced amplitude. Further spreading of transferred polarization can then occur via C-C spin-diffusion.