Project Summary Our vision is to develop, apply, and advance new technologies in the broad thematic program of the manipulation of nucleic acids. The research largely focuses on RNA-based applications, which is both driven and enhanced by our unique environment at The RNA Institute, enabling easy collaboration and cross pollination of ideas and techniques across fields. The importance of RNA and RNA technologies has never been clearer, with a pandemic RNA virus (SARS-CoV-2) being managed by RNA biosensing tools and mRNA-based vaccines. It is now well accepted that RNA plays a complex role in biology that goes far beyond the “central dogma”. Elucidating many RNA functions and biomedical applications requires investment in technology development. In this proposal, carried out over the next five years, we aim to advance technology in three main areas: 1) DNA nanoswitches as programmable nanoscale reagents for reporting molecular interactions, 2) the Centrifuge Force Microscope (CFM) for high throughput single molecule probing and analysis, and 3) DNA nanotechnology for applications in drug delivery and sensing. Each of these areas builds on our strengths and past performance. In the first area, we use DNA nanoswitches as “smart” reagents that change conformation upon binding a target molecule. Building on past success, we will expand this innovative method to enable low cost and simple RNA detection, quantification, and purification for various RNAs. These include detection of RNA modifications, detection of regulatory lncRNAs, and purification of RNA and RNA complexes. In the second area, we will develop and execute assays for the Centrifuge Force Microscope (CFM) in RNA and DNA interaction analysis and RNA structure-function probing. In particular, we will investigate fundamental thermodynamics of weak interactions in DNA and RNA, develop a new method for single-molecule structural probing of RNA, and study the structure-function relationship of a pre-clinical therapeutic ribozyme. In the third area, relatively new for our lab, we expand work to analyze and develop DNA nanotechnology for applications including drug delivery. Specifically, we are interested in understanding and “programming” biostability into DNA nanostructures, and in coordinating chemical attachment and triggered release of molecular payloads from DNA nanostructures. This proposal aims to carry out ambitious research and development plans performed within the context of several biomedical projects. The impact of the developments will extend beyond the individual projects, as our core technologies and methods are increasingly adopted by other groups. In the first MIRA term we have proven our ability to develop impactful technology and advance innovative science on several fronts. In this second term, we aim show sustainability of our research endeavors, solidifying our niches, maintaining high productivity, and ultimately producing knowledge and tools to benefit the scientific com...