Automated 3D Design and Evaluation of RNA Nanostructures with RNAMake

Despite growing interest in applying RNA's unique structural characteristics to solve diverse biotechnology and nanotechnology problems, there are few computational tools for targeted tertiary design. As a result, RNA 3D design is traditionally slow, resource-consuming, and dependent on expert modeling. In this chapter, we discuss our recently developed software package: RNAMake, a set of applications capable of designing RNA tertiary structures to solve various relevant nanotechnology problems and provide basic thermodynamic calculations for the generated designs. We provide in-depth examples and instructions for designing example RNA nanostructures such as minimal RNA sequences containing a single tertiary contact, generating RNAs that stabilize small-molecule ligands, and building tethers that link ribosomal subunits together. We also highlight the addition of a new Monte Carlo design algorithm and the ability to estimate the thermodynamic contribution of helical elements in RNA 3D structures.