Chemical synthesis of modified twister ribozymes

Very recently, a novel phosphodiester self-cleaving ribozyme class termed `twister has been discovered (Roth, A. et al. Nat. Chem. Biol. 2014, 10, 56). Our preliminary result on a 2.9 Å X-ray structure of the env22 twister ribozyme that we have obtained in collaboration with the Patel group, has generated contrasting views of the catalytic pocket architecture (Ren, A., Kosutic, M., Rajashankar, K. R., Frener, M., Santner, T., Westhof, E., Micura*, R., Patel*, D. J. Nat. Commun. 2014, doi: 10.1038/ncomms6534). The near in-line alignment of the attacking 2-OH and the to-be-cleaved PO5 bond observed in our study contrasts the off-line orthogonal alignments in the 2.3 Å structure of the Oryza sativa twister ribozyme (Liu, Y. et al Nat. Chem. Biol. 2014, 10, 739) and the 4.1 Å structure of the env twister ribozyme (Eiler, D. et al. PNAS 2014, 111, 13028). A further disquieting feature of these structures reflects the different positioning of key residues lining the cleavage site and the absence/presence of a divalent cation at the cleavage site. On the other hand all three structures emphasize a key role for the same invariant guanosine in catalysis. The here proposed experimentation is required to resolve these contrasting views of alignments within the cleavage site between the three crystal structures of the twister ribozyme and the possible mechanistic scenarios by using a battery of chemical and biophysical methods. The methods we will use range from atom-specific mutagenesis and nucleoside mutagenesis, to bulk and single-molecule fluorescence spectroscopy, to NMR spectroscopy, based on tailored, chemically modified RNAs. The overall goal of the proposed research is to obtain a thorough understanding of the chemical mechanism of twister ribozymes. This objective will be reached by a multidisciplinary systemic approach harnessing the power of organic synthesis (i.e. synthesis of modified nucleosides; synthesis of site-specifically labeled RNA by solid-phase synthesis), bioorganic chemistry and chemical biology (i.e. targeted, atom-specific mutagenesis and nucleoside mutagenesis), and biophysics (i.e. 2-aminopurine fluorescence, single-molecule Fluorescence-Resonance-Energy-Transfer (smFRET) and NMR studies to elucidate the functional dynamics of the ribozyme in vitro, based on labeled RNAs with newly designed, photostable fluorophores and specific isotope patterns, respectively). Thus, we will achieve an unprecedented level of understanding of the chemical concepts that the new twister ribozymes apply for phosphodiester self-cleavage, thus paving the way for their efficient applicability as biotechnological tools.

The overall scientific goal of the project was to obtain a thorough understanding of the chemical mechanism of twister ribozymes. This objective has been reached by a multidisciplinary systemic approach harnessing the power of organic synthesis (i.e. synthesis of modified nucleosides; synthesis of site-specifically labeled RNA by solid-phase synthesis), bioorganic chemistry and chemical biology (i.e. targeted, atom-specific mutagenesis and nucleoside mutagenesis), biophysics (i.e. 2-aminopurine fluorescence, single-molecule Fluorescence-Resonance-Energy-Transfer (smFRET), and NMR studies to elucidate the functional dynamics of the ribozyme in vitro, based on labeled RNAs with newly designed, photostable fluorophores and specific isotope patterns, respectively). The following goals were reached successfully: Goal 1 - Identifying the nucleosides and structural elements critical for twister ribozyme cleavage. Goal 2 - Developing efficient routes for deaza-nucleoside phosphoramidites to enable atomic mutagenesis experiments on twister and other ribozymes. Goal 3 - Developing a chemical probing approaches to analyze small ribozyme folding. Goal 4 - Revealing twister ribozyme folding by a single-molecule FRET spectroscopy approach. Goal 5 - Thorough comparison of twister to other recently discovered small ribozymes.