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Inverse RNA Folding Workflow to Design and Test Ribozymes that Include Pseudoknots

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Ribozymes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2167))

Abstract

Ribozymes are RNAs that catalyze reactions. They occur in nature, and can also be evolved in vitro to catalyze novel reactions. This chapter provides detailed protocols for using inverse folding software to design a ribozyme sequence that will fold to a known ribozyme secondary structure and for testing the catalytic activity of the sequence experimentally. This protocol is able to design sequences that include pseudoknots, which is important as all naturally occurring full-length ribozymes have pseudoknots. The starting point is the known pseudoknot-containing secondary structure of the ribozyme and knowledge of any nucleotides whose identity is required for function. The output of the protocol is a set of sequences that have been tested for function. Using this protocol, we were previously successful at designing highly active double-pseudoknotted HDV ribozymes.

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Acknowledgements

This study was supported by National Institutes of HealthGrants R35GM127064 to P.C.B. and R01GM076485 to D.H.M. R.Y. was supported by a JSPS Overseas Research Fellowship.

Author information

Author notes

  1. Mohammad Kayedkhordeh and Ryota Yamagami contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Biophysics and Center for RNA Biology, University of Rochester Medical Center, Rochester, NY, USA

    Mohammad Kayedkhordeh & David H. Mathews

  2. Department of Chemistry, Pennsylvania State University, University Park, PA, USA

    Ryota Yamagami

  3. Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA, USA

    Ryota Yamagami, Philip C. Bevilacqua & David H. Mathews

  4. Department of Chemistry, Pennsylvania State University, University Park, PA, USA

    Philip C. Bevilacqua

  5. Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, USA

    Philip C. Bevilacqua

  6. Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA

    David H. Mathews

  7. Department of Biostatistics and Computational Biology, University of Rochester Medical Center, Rochester, NY, USA

    David H. Mathews

Authors
  1. Mohammad Kayedkhordeh
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  2. Ryota Yamagami
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  3. Philip C. Bevilacqua
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  4. David H. Mathews
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Corresponding authors

Correspondence to Philip C. Bevilacqua or David H. Mathews .

Editor information

Editors and Affiliations

  1. Department of Microbiology and Immunology, Lady Davis Institute for Medical Research, Montréal, QC, Canada

    Robert J Scarborough

  2. Department of Microbiology and Immunology, Lady Davis Institute for Medical Research, Montréal, QC, Canada

    Anne Gatignol

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Kayedkhordeh, M., Yamagami, R., Bevilacqua, P.C., Mathews, D.H. (2021). Inverse RNA Folding Workflow to Design and Test Ribozymes that Include Pseudoknots. In: Scarborough, R.J., Gatignol, A. (eds) Ribozymes. Methods in Molecular Biology, vol 2167. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0716-9_8

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  • DOI: https://doi.org/10.1007/978-1-0716-0716-9_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0715-2

  • Online ISBN: 978-1-0716-0716-9

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