BME PhD Dissertation Defense: McKayla Vlasity

Title: "Programmable Control of mRNA Stability and Translation Through RNA Structure and Context"

Advisory Committee: Alexander A. Green, PhD – BME (Research Advisor) Wilson Wong, PhD – BME (Chair) Liang Hao, PhD – BME John Ngo, PhD – BME Julien Berro, PhD – Yale MB&B

Abstract: Messenger RNA (mRNA) is a versatile platform for controlling gene expression, yet the ability to precisely regulate its stability, translation, and responsiveness to intracellular signals remains a central challenge in synthetic biology and therapeutic design. This thesis presents a framework for engineering RNA systems that achieve optimized and conditional gene expression through coordinated structural and sequence-level design. First, conditional control is achieved through the development of ARES (Aptamer Ribozyme Expression Switch) systems, which couple RNA aptamers to hammerhead ribozymes to create protein-responsive regulators of mRNA stability. These switches operate through mutually exclusive folding, enabling conditional self-cleavage controlled by RNA-binding protein occupancy. Second, conditional translational control is demonstrated through engineered iron-responsive reporters based on the IRE-IRP regulatory system, which confer iron concentration-dependent regulation of translation. Third, a transcript-level design strategy employing the SANDSTORM–DIGs framework reveals that translation efficiency is governed by coherency across the 5’ UTR, coding sequence, and 3’ UTR. Co-optimized transcript architectures consistently outperformed independently optimized elements, and intermediate coding sequence structure was found to maximize translational output across cell types. Finally, engineered pseudoknot-based RNA structures are shown to enhance transcript stability and translation while reducing dsRNA accumulation during in vitro transcription. These elements provide a structural alternative to nucleotide modification, preserving functional RNA motifs while improving expression across both linear and circular RNA formats. Collectively, this work establishes that RNA regulatory behavior emerges from coordinated interactions among structural elements and transcript context. By integrating protein-responsive switching, endogenous regulatory motifs, transcript-level optimization, and structural stabilization, this thesis expands the design space for programmable RNA systems and provides a foundation for future applications in synthetic biology, diagnostics, and RNA therapeutics.