Journal
ACS SYNTHETIC BIOLOGY
Volume 9, Issue 7, Pages 1682-1692Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.0c00009
Keywords
cotranscriptional folding; scaffolded RNA origami; bio-orthogonal; split Broccoli aptamer; oxRNA simulation
Categories
Funding
- Engineering and Physical Sciences Research Council [EP/N031962/1]
- Royal Academy of Engineering Chair in Emerging Technology award
- Biotechnology and Biological Sciences Research Council [BB/M024830/1]
- BBSRC [BB/M024830/1] Funding Source: UKRI
- EPSRC [EP/N031962/1] Funding Source: UKRI
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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 a high synthesis 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 dear 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.
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