Engineering Ultrasonic Reporters for Imaging Cellular Enzyme Activity

Author: Yang, Jee Won

Year: 2026

Degree: Dissertation (Ph.D.)

Advisor: Shapiro, Mikhail G.

Committee Members: Tirrell, David A.; Phillips, Robert B.; Kennedy, Mary B.; Shapiro, Mikhail G.

Option: Chemical Engineering

Abstract

Enzyme activity governs virtually every aspect of cell behavior, from signal transduction and gene regulation to tissue remodeling and disease progression. Yet visualizing when and where specific enzymes act inside living organisms remains a fundamental challenge: optical reporters lack tissue penetration, nuclear imaging requires exogenous radioactive tracers, and MRI-based reporters suffer from low sensitivity. Ultrasound offers a compelling alternative, combining deep tissue penetration, high spatiotemporal resolution, and broad clinical accessibility — but has historically lacked the genetically encoded molecular reporters needed to connect acoustic contrast to specific cellular activities.

Gas vesicles (GVs) — air-filled protein nanostructures derived from buoyant microbes — have recently bridged this gap, enabling genetically encoded ultrasound contrast and providing initial demonstrations of acoustic biosensing for protease activity and calcium dynamics. However, extending GV-based biosensors to detect diverse enzyme activities in mammalian systems, with the specificity and sensitivity required for biological discovery, has remained an open challenge.

This thesis addresses this challenge by engineering three classes of GV-based acoustic biosensors targeting distinct enzyme families. UReKA (Ultrasonic Reporter of Kinase Activity) is the first genetically encoded ultrasonic reporter of protein kinase A activity, in which phosphorylation of an engineered GvpC substrate motif triggers dissociation from the GV shell and produces a quantitative, reversible increase in nonlinear acoustic contrast in purified GVs and mammalian cells. eUROP (evolved Ultrasound Reporter of Orthogonal Protease) extends this strategy to designed proteolytic activity by combining rational substrate engineering with directed evolution of plum pox virus protease, yielding a low-background, mammalian-compatible acoustic biosensor responsive to constitutive, inducible, and stimulus-driven gene expression. PAINTUR (Protease-Activated Imaging of Nonlinear Tumor Ultrasound Response) extends the platform from intracellular signaling to the extracellular tumor microenvironment, where an MMP9-cleavable GvpC enables acoustic imaging of endogenous protease activity in an orthotopic glioblastoma model while preserving the mechanical robustness needed for deep-tissue ultrasound. Together, these three sensor systems establish GvpC engineering as a generalizable platform for converting enzymatic activity into quantitative, noninvasive acoustic signals, opening new avenues for molecular imaging of cellular function in opaque tissues.