Engineering MRI-Based Programmable Genetic Sensors Using the MAPPER Platform

Genetically encodable reporters that produce signals detectable in deep tissues offer a powerful tool for noninvasive monitoring of molecular events in vivo. Although magnetic resonance imaging (MRI) is a standard technique for noninvasive clinical imaging, its wider application in detecting molecular activities has been constrained by the lack of programmable sensors. This limitation is in stark contrast to the widespread use of fluorescent reporter-derived sensors in cultured cells and in transparent specimens. To overcome this limitation, we recently developed the modular aquaporin-based protease-activatable probe for enhanced reporting (MAPPER) platform. This sensor engineering framework integrates a metal-free MRI reporter derived from human aquaporin-1 (hAqp1) with synthetic protease-based circuits. This integration facilitates the modular and scalable creation of a wide range of sensors by regulating protease activity through precise molecular events, such as protein-protein interactions, pharmacological inhibition, and second messenger signaling. In this paper, we present a detailed protocol for constructing and deploying sensors using the MAPPER paradigm. The protocol encompasses genetic design, lentiviral production, stable cell line generation, biochemical and microscopic validation of sensor function, diffusion-weighted MRI, and MR image analysis to quantify sensor signals in terms of the apparent diffusion coefficient. We describe two distinct MAPPER architectures: DD-MAPPER, which leverages protease-controlled protein degradation, and ER-MAPPER, which utilizes protease-controlled, subcellular trafficking. The MAPPER framework allows adaptation to various molecular targets without the need to redesign the core MRI reporter mechanism, making MAPPER a versatile platform for noninvasive biosensing in living cells and tissues. Key features • MAPPER enables programmable, protease-controlled switching of aquaporin-1-based diffusion weighted-MRI signals in genetically modified mammalian cells. • The protocol covers two complementary biosensor architectures (DD-MAPPER and ER-MAPPER) that exploit different post-translational regulatory mechanisms to modulate MRI signals. • Stable MAPPER cell lines are generated via lentiviral transduction, allowing long-term, selection-free biosensor expression across multiple mammalian cell types. • The sensor is fully modular; proteases and protease-based logic circuits can be substituted without altering the hAqp1 reporter, enabling rapid adaptation to new molecular targets.