Co-transcriptional folding of a bio-orthogonal fluorescent scaffolded RNA origami

Torelli, E.; Kozyra, J.; Shirt-Ediss, B.; Piantanida, L.; Voïtchovsky, K.; Krasnogor, N.

doi:10.1021/acssynbio.0c00009

Co-transcriptional folding of a bio-orthogonal fluorescent scaffolded RNA origami

Torelli, E.; Kozyra, J.; Shirt-Ediss, B.; Piantanida, L.; Voïtchovsky, K.; Krasnogor, N.

Authors

E. Torelli

J. Kozyra

B. Shirt-Ediss

L. Piantanida

Professor Kislon Voitchovsky kislon.voitchovsky@durham.ac.uk
Professor

N. Krasnogor

Abstract

The scaffolded origami technique is an attractive tool for engineering nucleic acid nanostructures. This paper demonstrates scaffolded RNA origami folding in vitro in which, for the first time, all components are transcribed simultaneously in a single-pot reaction. Double-stranded DNA sequences are transcribed by T7 RNA polymerase into scaffold and staple strands able to correctly fold in high yield into the nanoribbon. Synthesis is successfully confirmed by atomic force microscopy and the unpurified transcription reaction mixture is analyzed by an in gel-imaging assay where the transcribed RNA nanoribbons are able to capture the specific dye through the reconstituted split Broccoli aptamer showing a clear green fluorescent band. Finally, we simulate the RNA origami in silico using the nucleotide-level coarse-grained model oxRNA to investigate the thermodynamic stability of the assembled nanostructure in isothermal conditions over a period of time. Our work suggests that the scaffolded origami technique is a viable, and potentially more powerful, assembly alternative to the single-stranded origami technique for future in vivo applications.

Citation

Torelli, E., Kozyra, J., Shirt-Ediss, B., Piantanida, L., Voïtchovsky, K., & Krasnogor, N. (2020). Co-transcriptional folding of a bio-orthogonal fluorescent scaffolded RNA origami. ACS Synthetic Biology, 9(7), 1682-1692. https://doi.org/10.1021/acssynbio.0c00009

Journal Article Type	Article
Acceptance Date	May 29, 2020
Online Publication Date	May 29, 2020
Publication Date	Jul 17, 2020
Deposit Date	May 31, 2020
Publicly Available Date	May 29, 2021
Journal	ACS Synthetic Biology
Publisher	American Chemical Society
Peer Reviewed	Peer Reviewed
Volume	9
Issue	7
Pages	1682-1692
DOI	https://doi.org/10.1021/acssynbio.0c00009

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Copyright Statement
This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS synthethic biology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acssynbio.0c00009