1. Biochemistry and Chemical Biology
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Ribozyme-catalysed RNA synthesis using triplet building blocks

  1. James Attwater
  2. Aditya Raguram
  3. Alexey S Morgunov
  4. Edoardo Gianni
  5. Philipp Holliger  Is a corresponding author
  1. MRC Laboratory of Molecular Biology, United Kingdom
Research Article
  • Cited 48
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Cite this article as: eLife 2018;7:e35255 doi: 10.7554/eLife.35255

Abstract

RNA-catalyzed RNA replication is widely believed to have supported a primordial biology. However, RNA catalysis is dependent upon RNA folding, and this yields structures that can block replication of such RNAs. To address this apparent paradox we have re-examined the building blocks used for RNA replication. We report RNA-catalysed RNA synthesis on structured templates when using trinucleotide triphosphates (triplets) as substrates, catalysed by a general and accurate triplet polymerase ribozyme that emerged from in vitro evolution as a mutualistic RNA heterodimer. The triplets cooperatively invaded and unraveled even highly stable RNA secondary structures, and support non-canonical primer-free and bidirectional modes of RNA synthesis and replication. Triplet substrates thus resolve a central incongruity of RNA replication, and here allow the ribozyme to synthesise its own catalytic subunit '+' and '-' strands in segments and assemble them into a new active ribozyme.

Data availability

All data generated during this study and collated sequencing results are included in the manuscript and its supporting files. Source data files have been provided for Figures 1-4, 8 and 9.

Article and author information

Author details

  1. James Attwater

    Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Aditya Raguram

    Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Alexey S Morgunov

    Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Edoardo Gianni

    Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Philipp Holliger

    Protein and Nucleic Acid Chemistry, 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.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3440-9854

Funding

Medical Research Council (MC_U105178804)

  • Philipp Holliger

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

Reviewing Editor

  1. Anna Marie Pyle, Yale University, United States

Publication history

  1. Received: January 19, 2018
  2. Accepted: May 9, 2018
  3. Accepted Manuscript published: May 15, 2018 (version 1)
  4. Version of Record published: June 15, 2018 (version 2)

Copyright

© 2018, Attwater et al.

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.

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  1. Further reading

Further reading

  1. Did life on Earth start with RNA?

    1. Biochemistry and Chemical Biology
    Xavier Portillo et al.
    Research Article Updated

    An RNA polymerase ribozyme that has been the subject of extensive directed evolution efforts has attained the ability to synthesize complex functional RNAs, including a full-length copy of its own evolutionary ancestor. During the course of evolution, the catalytic core of the ribozyme has undergone a major structural rearrangement, resulting in a novel tertiary structural element that lies in close proximity to the active site. Through a combination of site-directed mutagenesis, structural probing, and deep sequencing analysis, the trajectory of evolution was seen to involve the progressive stabilization of the new structure, which provides the basis for improved catalytic activity of the ribozyme. Multiple paths to the new structure were explored by the evolving population, converging upon a common solution. Tertiary structural remodeling of RNA is known to occur in nature, as evidenced by the phylogenetic analysis of extant organisms, but this type of structural innovation had not previously been observed in an experimental setting. Despite prior speculation that the catalytic core of the ribozyme had become trapped in a narrow local fitness optimum, the evolving population has broken through to a new fitness locale, raising the possibility that further improvement of polymerase activity may be achievable.