SUMMARY: Circular RNAs are an important class of RNAs that have fundamental cellular functions and have the potential to be novel types of RNA-based therapeutics. Circular RNAs are difficult to synthesize and purify due to low yields after gel extraction. Resolving these issues could be a major benefit to academic and biotechnology research on circular RNA. The goal of this proposal is to make RNA synthesis as simple as a DNA plasmid prep kit. To do this, we will develop two new high-impact, market-ready kits. Our first kit is the “Chimerna In-cell circular RNA purification kit,” which uses our “Tornado” technology for expressing RNAs as a circle. When used in E. coli, the Tornado technology results in the production of RNA at high levels not previously seen with conventional linear RNAs. The stability of circular RNA allows it to accumulate to high levels in E. coli, and also confers high stability in vitro and when transfected into mammalian cells, thus mitigating the persistent degradation problem of conventional linear RNAs. We also developed a new RNA-purification aptamer which we incorporate into our circles to achieve high purity with a simple one-step elution protocol. Thus, our kit will overcome the problems of in vitro transcription: low yield, complex enzymatic synthesis reaction, RNA degradation, and the problem of laborious RNA purification. We will also prepare the Chimerna RNA labeling kit that repurposes a tRNA-modifying enzyme for RNA labeling. This companion product, comprising the enzyme and suitable substrates, will markedly enhance the ability to impart biotin, fluorescent dyes, cell-targeting moieties, and other useful tags to RNA. In order to develop these new commercial products, our specific aims are: (1) To optimize a kit for high-yield circular RNA synthesis and purification without in vitro transcription. We will optimize the E. coli cell line, culturing conditions, dextran binding and elution conditions, and RNA storage conditions. We will also establish protocols, manuals, reproducibility and stability conditions for kitting; (2) To develop an approach for simple and efficient RNA labeling. We will optimize reaction labeling conditions and target RNA sequences, develop protocols, manuals, and establish stability conditions that will allow us to create a user-ready RNA labeling kit; (3) To develop a system for highly efficient elution of tag-free RNA circles from dextran beads. We will perform pilot experiments to test a completely new RNA purification kit involving on- bead RNA circularization and elution triggered by a small molecule. This system is highly simplified and fast, with even fewer contaminants and produces RNA without any affinity tags. Taken together, this project will result in the first kit for synthesizing circular RNA, an especially challenging but highly important type of RNA. This technology is extended with our innovative RNA labeling kit, enabling researchers to label, tag, or track RNAs for div...