PyFuRNAce: an integrated design engine for RNA origami.

Recent developments in medicine and biotechnology have revealed the transformative power of RNA design. To realize the full potential of RNA nanotechnology and RNA origami, user-friendly design tools are needed. Here, we present pyFuRNAce, an open-source, Python-based software package with a graphical user interface that enables the design of complex RNA nanostructures, with particular focus on co-transcriptional RNA origami. PyFuRNAce integrates the entire RNA origami workflow-from motif definition and blueprint design to sequence generation and primer selection-into a single, user-friendly platform. Built around a motif-based assembly paradigm, the software enables users to create and modify custom RNA nanostructures through an intuitive web interface with streamlined design steps and real-time 3D visualization. We use pyFuRNAce to design three distinct RNA nanostructures, including self-assembling RNA filaments, RNA droplets, and the largest co-transcriptional RNA origami to date, consisting of 2501 nucleotides. The structures and their high-yield assembly are validated experimentally with atomic force microscopy and confocal fluorescence imaging. By consolidating multiple design stages into a unified environment, pyFuRNAce broadens the scope and reduces the barrier of entry for RNA nanotechnology, accelerating the development of functional RNA origami structures for applications in medicine, biotechnology, and synthetic biology.