Development of novel, user-centric technologies for detection and single-molecule analysis of RNA

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Our understanding of RNA and its complex roles in biology is constantly evolving. We know that most of the genome is transcribed into RNA, but only a small fraction of these transcripts code for proteins. The functions of the remaining non-coding RNAs are still largely unknown, but increasing evidence points to important regulatory roles. Thus, there is increasing interest in exploiting RNA for the improvement of human health, using RNA as potential disease biomarkers, therapeutics, or therapeutic targets. My lab is focused on developing novel, user-centric technologies for detection and analysis of RNA to accelerate the pace of discovery in RNA research and ultimate realize the goal of improving human health. First, we are developing a “smart reagent” for simple, cost effective, and instrument-free RNA detection. This reagent consists of nanoscale devices that we call DNA nanoswitches. The DNA nanoswitches self- assemble by DNA hybridization into molecular switches that change conformations upon binding to molecules of interest. These conformations can be easily identified using one of the most common lab tools – gel electrophoresis. The result is that users will be able to detect RNA sequences from biological samples using a one-step reaction. Over the next five years we will optimize these tools for microRNA, mRNA, long non-coding RNA, and viral RNA. Second, we are developing methods for high-throughput single-molecule analysis of RNA. This will be accomplished by continued development of the Centrifuge Force Microscope (CFM) and associated assays for RNA analysis. The CFM integrates a centrifuge and a microscope to enable thousands of simultaneous single-molecule experiments as tethered beads are monitored in real time. Importantly, recent developments in our lab have enabled these experiments to be performed in a standard benchtop centrifuge, making such experiments user friendly even at the undergraduate level. Over the next five years, we will develop CFM features and single molecule assays for three distinct projects: 1) mechanisms of transcription termination, 2) therapeutic ribozyme optimization, and 3) structure/function probing of a 3’ UTR in mRNA. These tools will provide new ways for researchers to detect and analyze various RNAs without expensive equipment or special training. For each of the technology development projects, we have engaged regional RNA researchers in collaborations to provide materials, biological context, and feedback to ensure that we are achieving our goals and the having the broadest possible impact.