Control of RNA function by conformational design
Final Report Abstract
In recent years, a number of studies in the field of RNA engineering have shown that functional properties of RNAs can be varied in a defined way by careful sequence design. However, most attempts to rationally encode structure and function into nucleic acid sequences have focused on self-assembly into predefined target structures, rather than on engineering transient system dynamics. It has become evident that more complex design targets are not amenable to manual sequence design, but require the use of computer based optimization methods. On the other hand, since even small errors in the energy model can significantly shift the balance between alternative conformations, computational RNA structure prediction is often not accurate enough to yield immediately functional RNA molecules. The computational models are, however, very efficiently able to reduce the search space to a small number of candidates that can be probed experimentally. We have made use of computer-aided design for the refinement of hairpin ribozyme like RNA structures with pre-defined properties. Previously, we had developed descendants of the hairpin ribozyme that support the circularization and oligomerization of linear RNA precursors. However, at that time, we were not able to direct activity towards either one of the two activities. In collaboration with the laboratory of Ivo Hofacker at Vienna University we successfully predicted RNA sequences with improved properties, in particular regarding the preference of a certain hairpin ribozyme variant to undergo intramolecular ligation (circularization) or intermolecular ligation (oligomerization). Experimental characterization of the designed RNA sequences on one hand confirmed the theoretical prediction. On the other hand, it still showed some deviations, what however, is still a valuable result concerning the improvement/refinement of the computational models, and in turn, refining model setup and parameters. The results of this work have been published in a common paper between the two collaborating laboratories. Furthermore we have developed hairpin ribozyme descendants, which support RNA self-ligation and recombination. Thus, in the aggregate, our work demonstrates that a number of activities can originate from a simple RNA structure. In RNA world scenarios, these activities may be regarded as being relevant to the extension of genetic space and diversity, and thus to the generation of RNAs with new functionalities.
Publications
- RNA self-ligation: from oligonucleotides to full length ribozymes. Biochimie, 2012, 94, 1457 – 1463
S. Gwiazda, K. Salomon, B. Appel, S. Müller
(See online at https://doi.org/10.1016/j.biochi.2012.03.015) - RNA self-processing: formation of cyclic species and concatemers from a small engineered RNA. FEBS Lett., 2013, 587, 2435-2440
S. Petkovic, S. Müller
(See online at https://doi.org/10.1016/j.febslet.2013.06.013) - RNA-Engineering - Rationales Design verschiedener Varianten des Hairpinribozyms. Bioforum, 2014, 37, 28-30
S. Müller
- Engineering of ribozymes with useful activities in the ancient RNA world. Ann. N. Y. Acad. Sci. 2015, 1341, 54-60
S. Müller
(See online at https://doi.org/10.1111/nyas.12695) - Sequencecontrolled RNA self-processing: computational design, biochemical analysis and visualization by AFM. RNA, 2015, 21, 1249-1260
S. Petkovic, S. Badelt, S. Block, C. Flamm, M. Delcea, I. Hofacker, S. Müller
(See online at https://doi.org/10.1261/rna.047670.114) - Hairpin ribozyme mediated RNA recombination. Chem. Commun. 2016, 52, 4365-4368
R. Hieronymus, S. P. Godehard, D. Balke, S. Müller
(See online at https://doi.org/10.1039/C6CC00383D)
