RNA origami design tools enable cotranscriptional folding of kilobase-sized nanoscaffolds

RNA origami can be used for the modular design of RNA nanoscaffolds but can be challenging to design. Newly developed computer-aided design software has now been shown to improve the folding yield of kilobase-sized RNA origami. These structures fold from a single strand during transcription by an RNA polymerase, and are able to position small molecules and protein components with nanoscale precision. RNA origami is a framework for the modular design of nanoscaffolds that can be folded from a single strand of RNA and used to organize molecular components with nanoscale precision. The design of genetically expressible RNA origami, which must fold cotranscriptionally, requires modelling and design tools that simultaneously consider thermodynamics, the folding pathway, sequence constraints and pseudoknot optimization. Here, we describe RNA Origami Automated Design software (ROAD), which builds origami models from a library of structural modules, identifies potential folding barriers and designs optimized sequences. Using ROAD, we extend the scale and functional diversity of RNA scaffolds, creating 32 designs of up to 2,360 nucleotides, five that scaffold two proteins, and seven that scaffold two small molecules at precise distances. Micrographic and chromatographic comparisons of optimized and non-optimized structures validate that our principles for strand routing and sequence design substantially improve yield. By providing efficient design of RNA origami, ROAD may simplify the construction of custom RNA scaffolds for nanomedicine and synthetic biology.

Automated summary

RNA origami is a framework for the modular design of nanoscaffolds that can be folded from a single strand of RNA and used to organize molecular components with nanoscale precision. The newly developed computer-aided design software has been shown to improve the folding yield of kilobase-sized RNA origami, simplifying the construction of custom RNA scaffolds for nanomedicine and synthetic biology. The design of genetically expressible RNA origami requires modeling and design tools that simultaneously consider thermodynamics, the folding pathway, sequence constraints and pseudoknot optimization.