Development of Solid State NMR Methods and Technology

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Progress in FY2025 was in the following areas: (1) TIME-RESOLVED SOLID STATE NMR OF DNA HYBRIDIZATION: In previous years, we developed time-resolved solid state NMR methods for identifying and structurally characterizing transient intermediates in fast, time-dependent processes in which biological macromolecules change their conformations or states of assembly. We applied these methods to the folding and tetramerization of melittin, the formation of calmodulin/Ca2+/peptide complexes, folding of villin HP35, and self-assembly of amyloid-beta peptides. For each of these processes, the time-resolved solid state NMR data revealed unanticipated aspects of their mechanisms. In FY2025, we have extended the time-resolved solid state NMR methods to nucleic acids, starting with hybridization of 12-mer DNA oligonucleotides. So far, we have found that single-stranded states of an oligonucleotide are readily distinguished from double-stranded, fully base-paired states through their 1D and 2D solid state NMR spectra in freeze-trapped solutions. Preliminary data for intermediate states, created by heating a solution of complementary oligonucleotides to 90 C for ~200 ms, then rapidly cooling to 30 C for a variable time period before rapid freezing, indicate that partially hybridized states can be trapped on time scales less than 10 ms. We expect to complete these studies and submit a manuscript for publication early in FY2026. (2) OPTIMIZATION OF 31P-13C DISTANCE MEASUREMENTS BY SOLID STATE NMR: The rotational-echo double-resonance (REDOR) technique is one of the most widely used solid state NMR techniques for measuring interatomic distances in biomolecular assemblies. Typically, REDOR is used to measure nitrogen-carbon (15N-13C) distances. In our recent studies of RNA-protein assemblies, we were interested in identifying contacts between the phosphorus sites in the phosphodiester backbone of RNA and carbon sites in the protein through 31P-13C REDOR. We found that the 31P-13C REDOR data were unreliable unless very strong radio-frequency pulses at the 31P NMR frequency were used. This finding led us to characterize the dependence of 31P-13C distances by REDOR on radio-frequency pulse amplitudes and develop a theoretical explanation for the results. The experiments and theory show that the large chemical shift anisotropies of 31P nuclei in solids produce imperfections in spin rotations produced by the radio-frequency pulses, but only for a subset of molecular orientations in the sample under study. A paper describing this work was published in the Journal of Magnetic Resonance.