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Nucleic Acids Res. 2018 Nov 20. doi: 10.1093/nar/gky1118. [Epub ahead of print]

Design of highly active double-pseudoknotted ribozymes: a combined computational and experimental study.

Author information

1
Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA.
2
Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.
3
Department of Biochemistry & Biophysics and Center for RNA Biology, University of Rochester Medical Center, Rochester, New York, NY 14642, USA.
4
Department of Biostatistics & Computational Biology, University of Rochester Medical Center, Rochester, New York, NY 14642, USA.
5
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.

Abstract

Design of RNA sequences that adopt functional folds establishes principles of RNA folding and applications in biotechnology. Inverse folding for RNAs, which allows computational design of sequences that adopt specific structures, can be utilized for unveiling RNA functions and developing genetic tools in synthetic biology. Although many algorithms for inverse RNA folding have been developed, the pseudoknot, which plays a key role in folding of ribozymes and riboswitches, is not addressed in most algorithms. For the few algorithms that attempt to predict pseudoknot-containing ribozymes, self-cleavage activity has not been tested. Herein, we design double-pseudoknot HDV ribozymes using an inverse RNA folding algorithm and test their kinetic mechanisms experimentally. More than 90% of the positively designed ribozymes possess self-cleaving activity, whereas more than 70% of negative control ribozymes, which are predicted to fold to the necessary structure but with low fidelity, do not possess it. Kinetic and mutation analyses reveal that these RNAs cleave site-specifically and with the same mechanism as the WT ribozyme. Most ribozymes react just 50- to 80-fold slower than the WT ribozyme, and this rate can be improved to near WT by modification of a junction. Thus, fast-cleaving functional ribozymes with multiple pseudoknots can be designed computationally.

PMID:
30462314
DOI:
10.1093/nar/gky1118

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