Designing a Single Protein‐Chain Reporter for Opioid Detection at Cellular Resolution

Abstract

Mu-opioid receptor (MOR) signaling regulates multiple neuronal pathways, including those involved in pain, reward, and respiration. To advance the understanding of MORsr oles in pain modulation, there is an eed for highthroughput screening methods of opioids in vitro and highresolution mapping of opioids in the brain. To fill this need, we designed and characterized ag enetically encoded fluorescent reporter,c alled Single-chain Protein-based Opioid Transmission Indicator Tool for MOR (M-SPOTIT). M-SPOTIT represents an ew and unique mechanism for fluorescent reporter design and can detect MOR activation, leaving ap ersistent green fluorescence mark for image analysis.M -SPOTIT showed an opioid-dependent signal to noise ratio (S/ N) up to 12.5 and was able to detect as fast as a3 0-second opioid exposure in HEK293T cell culture.A dditionally,i t showed an opioid-dependent S/N up to 4.6 in neuronal culture and detected fentanyl with an EC 50 of 15 nM. M-SPOTIT will potentially be useful for high-throughput detection of opioids in cell cultures and cellular-resolution detection of opioids in vivo.M-SPOTITsnovel mechanism can be used as aplatform to design other G-protein-coupled receptor-based sensors.

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

“…To engineer SPOTIT with temporal gating, we took advantage of the rapamycin-induced heterodimerization of FRB and FKBP 19 and the observation that cpGFP-Nb39 exhibits virtually no background fluores- cence. 17 We tethered FKBP to the N-terminus of cpGFP-Nb39 and tethered FRB to the C-terminus of the mu, kappa, and delta opioid receptors (MOR, KOR, and DOR, respectively; Figure 2A). Without rapamycin-induced heterodimerization of FRB and FKBP, Nb39 remains bound to cpGFP, inhibiting cpGFP fluorophore formation.…”

“…From previous studies, we've seen that it takes at least 3−4 h to see sufficient fluorophore formation to distinguish activation from the background signal. 17 To determine the minimum amount of time that is needed for the PPI to interact to see significant activation, we replaced FKPB and FRB with a light-activated PPI pair, iLiD 24 and sspB. We used the same sspB-cpGFP-Nb39 construct we designed for detecting sspB/ssrA PPI.…”

“…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 . We tethered FKBP to the N-terminus of cpGFP-Nb39 and tethered FRB to the C-terminus of the mu, kappa, and delta opioid receptors (MOR, KOR, and DOR, respectively; Figure A).…”

“…Hence, the cpGFP-Nb39 fusion protein has extremely low fluorescence, while cpGFP alone was found to be 507× brighter than the cpGFP-Nb39 fusion protein. 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.…”

“…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).…”