2024 AIChE Annual Meeting

(531c) Fluorescent Based Protease Detection Probe with Logic Gating Capabilities

Authors

Millicent O. Sullivan, University of Delaware
Wilfred Chen, University of Delaware
Proteases play a vital role in homeostasis. They are responsible for maintaining and restructuring tissue, processing and relaying cellular information, and regulating the activity of other proteases, among other roles. Accordingly, the dysregulation of protease activity is implicated in a vast and growing number of disease states, from chronic wounds, to cancer progression. Probing protease activity is highly desirable for applications such as screening therapeutic inhibitors and detecting viral infection, and more recently, probes of protease activity have garnered interest as prognostic markers for cancers and chronic wounds. One of the most widely used methods is Förster resonance energy transfer (FRET), which employs fluorophore and quencher moieties linked with a protease cleavable linker. While these probes are useful, they have several inherent shortcomings. Of particular concern, limitations in quenching efficiency result in substantial background fluorescence, and in addition, the stringent spacing requirements between the two moieties disallow modifications for logic gating.

Herein, we present a recombinant protease detection probe capable of larger dynamic range and logic gating. The probe uses a split fluorescent protein called fluorescence and absorbance shifting tag (FAST). Unlike other split fluorescent proteins, splitFAST’s fragment complementation is entirely reversible with rapid kinetics. We genetically fused the splitFAST fragments with various protease cleavable linkers that increased the apparent affinity to ‘turn-ON’ the probe. We then incubated each variant with its respective protease to cleave the linker and observed >20-fold reduction in fluorescence for all variants. This ‘turn-OFF’ behavior is only possible due to the reversibility of SplitFAST, and moreover, the response offers >4x higher dynamic range compared to commercially available FRET probes.

As compared with FRET probes, this design also has the added benefit that its linker can be modified to introduce logic gates. To our knowledge, this is the first protease detection probe capable of detecting multiple inputs (protease activity) and tranducing them into one output (fluorescence). The first logic introduced was a NOR-gate, where we demonstrated comparable turn-OFF behavior if any one of multiple proteases (TEVp, MMP3, Thrombin) were incubated with the probe. We then expanded the logic-toolbox by introducing a NAND-gate, which requires two orthogonal proteases to both be present in order to turn-OFF fluorescence and observed a <2-fold change in fluorescence if only one protease (TEVp or MMP3) were present and a >25-fold change if both were present. We are currently expanding the logic gating capabilities with several promising designs, and we envision this probe’s logic capabilities as a versatile and effective tool for screening protease activity.