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

Mu‐opioid receptor (MOR) signaling regulates multiple neuronal pathways, including those involved in pain, reward, and respiration. To advance the understanding of MOR's roles in pain modulation, there is a need for high‐throughput screening methods of opioids in vitro and high‐resolution mapping of opioids in the brain. To fill this need, we designed and characterized a genetically encoded fluorescent reporter, called Single‐chain Protein‐based Opioid Transmission Indicator Tool for MOR (M‐SPOTIT). M‐SPOTIT represents a new and unique mechanism for fluorescent reporter design and can detect MOR activation, leaving a persistent 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 a 30‐second opioid exposure in HEK293T cell culture. Additionally, it showed an opioid‐dependent S/N up to 4.6 in neuronal culture and detected fentanyl with an EC50 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‐SPOTIT's novel mechanism can be used as a platform to design other G‐protein‐coupled receptor‐based sensors. A genetically‐encoded fluorescent sensor was designed for the mu‐opioid receptor using a novel sensor mechanism. In the absence of opioids, Nb39 interacts with the circular permuted green fluorescent protein (cpGFP) and inhibits the fluorophore maturation of cpGFP. When the opioid binds to the opioid receptor (OR), Nb39 binds to the intracellular side of the activated OR. This removes Nb39 from cpGFP, allowing the cpGFP fluorophore to mature, resulting in an opioid‐induced fluorescence increase.