A Modular Fluorescent Sensor Motif Used to Detect Opioids, Protein–Protein Interactions, and Protease Activity

Abstract

Modular fluorescent sensor motifs are needed to design fluorescent sensors for detecting various cellular processes and functional molecules. Here, we took advantage of the versatility of a new sensor motif to design a series of sensors called SPOTon. SPOTon sensors integrate the signal from either opioids, protein−protein interactions, or protease activities to generate persistent green fluorescence. We demonstrate that SPOTon can be engineered with temporal gating to allow detection of these cellular events during a user-defined time window, providing temporal information about cellular processes and functional molecule release. These SPOTon sensors all show a high signal-to-noise ratio, up to 38 for chemical gated opioid detection, 147 for chemical gated protein−protein interaction detection, and 85 for protease activity detection.

Modular Peroxidase-Based Reporters for Detecting Protease Activity and Protein Interactions with Temporal Gating

“…Protease activity is involved in many biological processes, including apoptosis and viral infection. − Existing FP-based protease activity reporters require a long incubation time for fluorophore maturation. − , Existing luciferase-based protease activity reporters can measure luminescence with high sensitivity − , but are limited by their spatial resolution for imaging in tissues. Therefore, there is a need for reporters for detecting protease activity in live cells with an activation time of minutes and with cellular or sub-cellular spatial resolution.…”

“…Fluorescent protein (FP)-based reporters are frequently used to monitor biological processes. For example, split FPs have been applied to detect protein–protein interactions (PPIs). − A split FP that is sterically blocked from reconstitution was designed to detect protease activity. , Circularly permuted green FP (cpGFP) or FP fused with an inhibitory protein or peptide that can prevent the FP fluorophore maturation was also designed for detecting protease activity and PPIs. , FP-based reporters are advantageous and convenient to use because they provide a fluorescence readout directly. However, they do not amplify the signal, resulting in low fluorescence when the signal input is low.…”

“…4,5 Circularly permuted green FP (cpGFP) or FP fused with an inhibitory protein or peptide that can prevent the FP fluorophore maturation was also designed for detecting protease activity and PPIs. 6,7 FPbased reporters are advantageous and convenient to use because they provide a fluorescence readout directly. However, they do not amplify the signal, resulting in low fluorescence when the signal input is low.…”

Genetically encoded tools for in vivo G‐protein‐coupled receptor agonist detection at cellular resolution

“…Then, the opioid‐activated OR can recruit Nb39, allowing the cpGFP fluorophore to form. This sensor had a comparable opioid‐dependent SNR as the SPOTIT sensors and strong rapamycin‐dependence 125 . Due to rapamycin's low blood–brain barrier (BBB) penetrability, the engineering of a chemical‐gated SPOTon that is activated with a BBB penetrable molecule would be needed for animal studies.…”

“…Similarly, the background signal, which arises from the basal activity of the ORs, could also accumulate over time. To minimise the background signal accumulation, a chemical‐gated version of SPOTIT, called SPOTon, was designed 125 . In the SPOTon sensor design, SPOTIT is split into two components which are fused to a heterodimerising PPI pair, FK506 binding protein (FKBP) and FKBP‐rapamycin binding domain (FRB); the OR was fused to FRB and cpGFP‐Nb39 was fused to FKBP.…”