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.