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.

RNA折纸是纳米支架模块化设计的框架，可以从单链RNA折叠并用于以纳米级精度组织分子成分。必须以共转录方式折叠的可遗传表达的 RNA 折纸的设计需要同时考虑热力学、折叠途径、序列约束和假结优化的建模和设计工具。在这里，我们描述了 RNA 折纸自动化设计软件 (ROAD)，它从结构模块库中构建折纸模型，识别潜在的折叠障碍并设计优化的序列。使用 ROAD，我们扩展了 RNA 支架的规模和功能多样性，创造了多达 2,360 个核苷酸的 32 种设计，其中 5 种为两种蛋白质提供支架，7 种在精确距离处为两种小分子提供支架。优化和未优化结构的显微照片和色谱比较验证了我们的链路由和序列设计原则显着提高了产量。通过提供高效的 RNA 折纸设计，ROAD 可以简化用于纳米医学和合成生物学的定制 RNA 支架的构建。