Resolving excited states of a ribozyme by NMR

Using chemical RNA synthesis isolated 13C- 1 H spin systems will be introduced into the extended hammerhead ribozyme and mutants thereof. For that purpose, 13C modified purine and pyrimidine building blocks have been synthesized. The labels represent isolated 13C- 1 H spin pairs, for which relaxation dispersion NMR methods can be carried out in a straightforward manner. Using sophisticated NMR methods the dynamics in the micro- to millisecond time frame of the catalytically active RNA will be probed. We plan to compare the conformational dynamics of the more recently characterized extended hammerhead ribozyme (eHHRz), which comprises a loop- receptor interaction, and mutants thereof with changes on a functional group and nucleotide level. Comparing the results of the various RNAzymes will give a first comprehensive NMR based analysis of the structural dynamics of a ribozyme and its tertiary contacts that are required for activating catalysis. The tertiary interaction between a loop region and its receptor found in the extended hammerhead ribozyme, although distant from the catalytic core residues, very likely primes the RNA for catalysis by increasing the population of a conformation close to the transition state. This explicit hypothesis will be tested in the proposed research.

The originally proposed research goals visualizing transiently populated conformational states of the hammerhead ribozyme could not be achieved due to the low quality of the nuclear magnetic resonance (NMR) spectra of all tested hammerhead ribozyme constructs. Thus, the research plan was adapted and the goals were transferred to a recently discovered ribozyme class the pistol ribozyme. After several rounds of optimization, a pistol ribozyme complex was obtained, that gave high quality NMR spectra. This complex was investigated by various biophysical methods, including x-ray crystallography and solution NMR spectroscopy. Thereby, a detailed mechanism of the self-cleavage reaction of the pistol ribozyme could be obtained. We currently refine the mechanism of the cleavage reaction by carrying out additional NMR experiments. This refined, detailed atomic movie of the catalytic process will give the possibility to use the pistol ribozyme in biochemical/- technological applications. Additionally, as the pistol ribozyme is presumably involved in gene regulatory processes of pathogenic bacteria, small molecules can be designed to interact/block transitions states of the self-cleavage reaction, thus giving access to novel RNA-targeting drugs.