BME PhD Dissertation Defense: Jiayi Li

Title: "RNA Reprogramming for RNA Sensing and Imaging in Live Cells"

Advisory Committee: Alexander A. Green, PhD – BME (Research Advisor) Mary Dunlop, PhD – BU BME (Chair) Ahmad (Mo) Khalil, PhD – BU BME Liangliang Hao, PhD - BU BME Daniel Cifuentes, PhD – BU Biochemistry & Cell Biology

Abstract: RNA plays central roles in diverse cellular processes, including gene expression and regulation at multiple levels. However, commonly used live-cell RNA imaging tools often require genetic modification of endogenous targets or exogenous probe delivery that complicates the detection process. Technologies that specifically and robustly sense RNA in its native state are thus highly desirable. This dissertation develops two genetically encoded RNA-based biosensors capable of RNA sensing in live prokaryotic and eukaryotic cells, respectively. First, I present tSENSE (tRNA-Scaffolded Engineered Nucleic acid Sensor), a class of programmable RNA switches that leverage conditional formation of tRNA scaffolds to improve fluorescence output in live E. coli. I demonstrate tSENSE design generalizability to accommodate diverse fluorogenic aptamer structures, programmability for dual-color sensing, and robustness in detecting functional RNA targets. Second, I introduce STARS (Switchable Tandem Aptamer for RNA Sensing), a class of de novo designed RNA switches that modulate tandem MS2 aptamer structure to enable sequence-specific RNA sensing in live HEK293T cells. I demonstrate both ON and OFF switch configurations with robust fold changes and apply STARS ON switches to detect exogenously expressed mRNA. Together, these two platforms provide distinct and broadly applicable design principles for engineering genetically encoded RNA biosensors, expanding the toolkit for monitoring RNA dynamics in living cells.