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

Abstract

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. Design of genetically expressible RNA origami, which must cotranscriptionally fold, requires modeling and design tools that simultaneously consider thermodynamics, 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 2360 nucleotides, five that scaffold two proteins, and seven that scaffold two small molecules at precise distances. Micrographic and chromatographic comparison of optimized and nonoptimized structures validates that our principles for strand routing and sequence design substantially improve yield. By providing efficient design of RNA origami, ROAD may simplify construction of custom RNA scaffolds for nanomedicine and synthetic biology.

Algorithmic design of 3D wireframe RNA polyhedra

“…The versatility of the methodology was advanced by Liu et al (26), who introduced, among other things, a novel "branching" kissing-loop connector motif that can be used to create trivalent branches in the structures, leading to a richer design space than was previously available. A further improvement was presented by Geary et al (27) by enabling co-transcriptional folding of RNA origami of larger constructs compared to their earlier work (21). In the present work, we contribute a solution to a broad family of further design challenges in RNA nanotechnology by providing a fully general design scheme and automated software pipeline for designing arbitrary 3D RNA wireframe polyhedra.…”

Application of Nucleic Acid Frameworks in the Construction of Nanostructures and Cascade Biocatalysts: Recent Progress and Perspective

“…Remarkably, these RNA nanostructures can form by a co-transcriptional folding manner. Recently, they developed a tool called RNA Origami Automated Design (ROAD) for constructing RNA origami with expanded structural and functional diversity (Geary et al, 2021). Simmel and co-workers developed a similar tile-based RNA nanostructure extending in three dimensions, which could not only again assemble into a hexagonal plane via interaction between kissing loops, but also incorporate out-of-plane functionalization (Chopra et al, 2019).…”

“…However, more efforts still need to be made to make the procedures and the computational programs for the design and construction of complicated nanostructures more robust and user-friendly (Andronescu et al, 2003;Jabbari et al, 2015;Jun et al, 2019). For broader practical and even industrial applications of the nucleic acid nanostructures, the techniques for massive and cost-effective production of DNA and RNA building blocks also need to be developed and optimized (Chandler et al, 2020;Geary et al, 2021). Recently, biomass DNA directly extracted from living organisms was successfully converted into biodegradable materials, ranging from gels to plastics, at large scales with very low costs Han et al, 2021).…”

Engineered Riboswitch Nano-carriers as a Possible Disease-Modifying Treatment for Metabolic Disorders

“…The straightforward Watson-Crick base pairing has enabled exceptional control over the shape, size, and geometry of both DNA and RNA nanostructures. [1][2][3][4] This, has led to the rapid development of the nucleic-acids nanotechnology field with numerous applications. 3,[5][6][7][8][9][10] Yet, one of the greatest limitations of nucleic acid-based devices is their rapid nuclease-mediated degradation, when applied in vivo.…”

“…[1][2][3][4] This, has led to the rapid development of the nucleic-acids nanotechnology field with numerous applications. 3,[5][6][7][8][9][10] Yet, one of the greatest limitations of nucleic acid-based devices is their rapid nuclease-mediated degradation, when applied in vivo. 4,11 One way to overcome this hurdle is to chemically modify the nucleic acids which are costly.…”