Dynamic nuclear polarization of nucleic acid with endogenously bound manganese
We report the direct dynamic nuclear polarization (DNP) of 13C nuclei of a uniformly [13C,15N]-labeled, paramagnetic full-length hammerhead ribozyme (HHRz) complex with Mn2+ where the enhanced polarization is fully provided by the endogenously bound metal ion and no exogenous polarizing agent is added. A 13C enhancement factor of ε = 8 was observed by intra-complex DNP at 9.4 T. In contrast, “conventional” indirect and direct DNP experiments were performed using AMUPol as polarizing agent where we obtained a 1H enhancement factor of ε ≈ 250. Comparison with the diamagnetic (Mg2+) HHRz complex shows that the presence of Mn2+ only marginally influences the (DNP-enhanced) NMR properties of the RNA. Furthermore two-dimensional correlation spectra (15N–13C and 13C–13C) reveal structural inhomogeneity in the frozen, amorphous state indicating the coexistence of several conformational states. These demonstrations of intra-complex DNP using an endogenous metal ion as well as DNP-enhanced MAS NMR of RNA in general yield important information for the development of new methods in structural biology.
KeywordsDynamic nuclear polarization (DNP) Solid-state NMR EPR RNA Transition metal Polarizing agent
This work was supported by the Deutsche Forschungsgemeinschaft (DFG) via Emmy Noether Grant CO802/2-1 issued to B.C. as well as DFG collaborative research center (Sonderforschungsbereich) 902, and by the Center for Biomolecular Magnetic Resonance (BMRZ). We thank J. Becker-Baldus and T. Gutmann for access to DNP spectrometers and technical assistance. Help from D. Akhmetzyanov during 180 GHz EPR experiments is gratefully acknowledged. We thank T. Prisner for proposing HHRz as the target system of this study, as well as H. Schwalbe and J. Wöhnert for helpful discussions. The sweepable MAS DNP NMR spectrometer was Granted to G. Buntkowsky (Darmstadt) via DFG Grant BU911/20-1.
Deutsche Forschungsgemeinschaft (DFG) Grants CO802/2-1, SFB902, and BU911/20-1; Center for Biomolecular Magnetic Resonance (BMRZ).
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Conflict of interest
The authors declare that they have no conflict of interest.
- Abragam A, Proctor WG (1958) Une nouvelle méthode de polarisation dynamique des noyaux atomiques dans les solides. CR Hebd Acad Sci 246:2253–2256Google Scholar
- Bassi GS, Murchie AI, Lilley DM (1996) The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation. RNA 2:756–768Google Scholar
- Chi Y-I, Martick M, Lares M, Kim R, Scott WG, Kim S-H (2008) Capturing hammerhead ribozyme structures in action by modulating general base catalysis. PLoS Biol 6:2060–2068Google Scholar
- Hu KN, Debelouchina GT, Smith AA, Griffin RG (2011) Quantum mechanical theory of dynamic nuclear polarization in solid dielectrics. J Chem Phys 134:19Google Scholar
- Kessenikh AV, Lushchikov VI, Manenkov AA, Taran YV (1963) Proton polarization in irradiated polyethylenes. Sov Phys-Sol State 5:321–329Google Scholar