Rolling circle RNA synthesis catalysed by RNA

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Abstract

RNA-catalysed RNA replication is widely considered a key step in the emergence of life's first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. We observe sustained RCS by a triplet polymerase ribozyme beyond full-length circle synthesis with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularisation. Thus, all steps of a viroid-like RNA replication pathway can be catalysed by RNA alone. Finally, we explore potential RCS mechanisms by molecular dynamics simulations, which indicate a progressive build-up of conformational strain upon RCS' with destabilisation of nascent strand 5'- and 3'-ends. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.

Data availability

All data generated or analyzed in this manuscript is supplied within the manuscript or supporting file; Source Data files containing original unedited gels images as well as numeric data have been provided for Figures 1,2,4 and 5, as well as figure supplements when relevant.Modelling data and sequencing data are provided as described in the data availability section in the manuscript.

The following data sets were generated

1. Kristoffersen EL
(2021) Deep Sequencing data for document titled: Rolling Circle RNA Synthesis Catalysed by RNA
Dryad Digital Repository, doi:10.5061/dryad.tht76hf10.

https://doi.org/10.5061/dryad.tht76hf10

Article and author information

Author details

Emil Laust Kristoffersen

MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-8965-8201
Matthew Burman

Department of Physics, University of York, York, United Kingdom

Competing interests
The authors declare that no competing interests exist.
Agnes Noy

Department of Physics, University of York, York, United Kingdom

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-0673-8949
Philipp Holliger

MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

For correspondence
ph1@mrc-lmb.cam.ac.uk

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-3440-9854

Funding

Carlsbergfondet (CF17-0809)

Emil Laust Kristoffersen

Medical Research Council (MC_U105178804)

Philipp Holliger

Engineering and Physical Sciences Research Council (EP/N027639/1)

Agnes Noy

Engineering and Physical Sciences Research Council (EP/R513386/1)

Matthew Burman

Engineering and Physical Sciences Research Council (EP/T022205/1)

Agnes Noy

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.