EAGER: Developing a Highly Selective, Orthogonal, Enzymatic RNA Labeling Technology via Directed Evolution of an RNA Transglycosylase

Tools that facilitate the study of individual RNAs are critical for understanding their mechanisms of action and biological significance. RNAs are difficult to study as they vary in size, function, location, abundance, and lifetime. This project intends to engineer new enzymes that insert novel, small molecule reporters into an RNA in a manner that is appropriate for studying any RNA of interest.

These enzymes will insert reporters that are small enough to not interfere with RNA function and do so in a manner that is specific to that individual RNA. These never-before-seen enzymes and small molecules will be programmed to work alongside their native counterparts without unwanted crosstalk, allowing researchers to efficiently investigate how location and abundance of RNAs influences their role in genetic mechanisms.

The project will have broad impacts including improving societal literacy surrounding genetic mechanisms through presentation in accessible media platforms and increasing STEM exposure of underrepresented groups through local high school outreach.

This research project intends to use directed evolution to discover new RNA modifying enzymes capable of exchanging a nucleobase in an RNA for a functional small molecule reporter. The directed evolution workflow will involve iterative rounds of site saturation mutagenesis and a unique high throughput screen to probe mutant transglycosylases for their ability to accept novel substrates.

This technology avoids the limitations of state-of-the-art technologies, including bulky reporters, transient association, and off target installation. The enzyme will be evolved to act orthogonally to endogenous RNA modification enzymes and their substrates, facilitating high fidelity tagging and low background. The versatility of this RNA modification strategy will provide investigators with an unprecedented tool to study their transcript of choice.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.