A genetically encoded sensor with improved fluorescence intensity for opioid detection at cellular resolution

Abstract

The mu-opioid receptor (MOR) regulates the neuronal pathways involved in pain, reward, and respiration. To increase our understanding of MOR’s roles in these pathways, there is a need to detect...

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

“…17 This sensor motif has previously been used to engineer an opioid sensor, called SPOTIT. 17,18 In this paper, we demonstrate that this sensor motif is highly modular; cpGFP-Nb39 fusion results in low fluorescence, and removal of Nb39 from cpGFP leads to high fluorescence activation. Here, we show that the removal of Nb39 can occur in two ways: by the binding of Nb39 to a higher-affinity binding protein or by peptide cleavage (Figure 1).…”

“…These values are summarized in Figure D, along with information for the limit of detection (LOD), dynamic range, and sensitivity values for these sensors. The SPOTon opioid sensor has a larger linear dynamic range but lower opioid sensitivity and a larger EC 50 value than SPOTIT . The decreased sensitivity is possibly because the geometry of the FKBP-FRB interaction pair is not optimal for Nb39 to reach the activated MOR.…”

“…The SPOTon opioid sensor has a larger linear dynamic range but lower opioid sensitivity and a larger EC 50 value than SPOTIT. 18 The decreased sensitivity is possibly because the geometry of the FKBP-FRB interaction pair is not optimal for Nb39 to reach the activated MOR. Decreased sensitivity delays the saturation point of the sensor, where concentrations from 0.1 μM to 1 μM can give distinguishable signal differences from the saturation points at 10 μM and 50 μM.…”

“…Therefore, the mechanism of sensor activation is a result of both an increase in cpGFP fluorophore formation and cpGFP protein level. Compared to our previously designed SPOTIT sensor for detecting opioids, 18 SPOTon opioid sensors have many advantages. First, the chemical-dependent temporal gating enables the opioid signal integration within a specific time window, which could be extremely advantageous for detecting opioid peptide release in animal models only during specific behaviors.…”

“…We previously engineered SPOTIT, an irreversible integration sensor, for detecting opioids. , We reasoned it would be useful to engineer a version of SPOTIT with temporal gating to enable opioid signal detection within a specific time window. To engineer SPOTIT with temporal gating, we took advantage of the rapamycin-induced heterodimerization of FRB and FKBP and the observation that cpGFP-Nb39 exhibits virtually no background fluorescence .…”