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In-ice evolution of RNA polymerase ribozyme activity

Abstract

Mechanisms of molecular self-replication have the potential to shed light on the origins of life. In particular, self-replication through RNA-catalysed templated RNA synthesis is thought to have supported a primordial ‘RNA world’. However, existing polymerase ribozymes lack the capacity to synthesize RNAs approaching their own size. Here, we report the in vitro evolution of such catalysts directly in the RNA-stabilizing medium of water ice, which yielded RNA polymerase ribozymes specifically adapted to sub-zero temperatures and able to synthesize RNA in ices at temperatures as low as −19 °C. The combination of cold-adaptive mutations with a previously described 5′ extension operating at ambient temperatures enabled the design of a first polymerase ribozyme capable of catalysing the accurate synthesis of an RNA sequence longer than itself (adding up to 206 nucleotides), an important stepping stone towards RNA self-replication.

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Figure 1: In-ice selection for polymerase ribozyme activity.
Figure 2: Cold adaptation of ribozyme activity.
Figure 3: Basis of cold adaptation.
Figure 4: Long-range RNA synthesis by ribozyme tC9Y.
Figure 5: Template selection.

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Acknowledgements

The authors thank J.N. Skepper (University of Cambridge) for help with SEM imaging, S. James and S. Brunner for help with MiSeq sequencing and analysis and M. Daly (MRC LMB) for help with FACS. This work was supported by a Homerton College, Cambridge Junior Research Fellowship (J.A.) and by the Medical Research Council (programme number U105178804).

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Contributions

J.A. and P.H. conceived and designed the experiments. J.A. and A.W. developed and validated the CBT selection system. J.A. performed the selection and subsequent experiments. All authors analysed data and co-wrote the paper.

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Correspondence to Philipp Holliger.

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The authors declare no competing financial interests.

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Attwater, J., Wochner, A. & Holliger, P. In-ice evolution of RNA polymerase ribozyme activity. Nature Chem 5, 1011–1018 (2013). https://doi.org/10.1038/nchem.1781

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