Wednesday, September 16, 2015

Dynamic nuclear polarization of nucleic acid with endogenously bound manganese


Wenk, P., et al., Dynamic nuclear polarization of nucleic acid with endogenously bound manganese. J Biomol NMR, 2015: p. 1-13.


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 epsilon = 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 epsilon approximately 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.