Serum-stable RNA origami nanodevices for sensing and targeting

Rational RNA design seeks to encode advanced functions in the RNA polymer for applications in medicine and biotechnology. However, current design methods such as RNA origami lack the ability to integrate chemical modifications to improve properties such as increased stability and reduced immunogenicity. Here we demonstrate 2'-fluoro pyrimidine RNA (FY-RNA) origamis that co-transcriptionally fold into well-defined serum-stable nanodevices. Cryogenic electron microscopy reveals how FY-RNA can alter folding pathways and tertiary structure compared to RNA, while molecular dynamics simulations pinpoint the effects from altered hydrogen bonding, sugar pucker, and helix-helix interactions. Despite these structural perturbations, converted RNA fluorogenic aptamers embedded within FY-RNA origami retain partial activity and enable logic-based molecular sensing in human serum. Further, a FY-RNA origami scaffold was designed to aid the structural characterization of an FY-RNA anti-Spike aptamer bound to the Spike protein, resolved to at 3.4 Å resolution, uncovering new fluorine-specific structural motifs and protein interactions. Together, our results establish design principles for nuclease-resistant RNA architectures and position FY-RNA as a versatile polymer for constructing medical nanodevices and environmental sensors. More broadly, this work provides a framework for systematically exploring the folding landscape of chemically modified RNAs, expanding the chemical and functional diversity accessible to nucleic acid nanotechnology and RNA medicine.