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

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 opioids at cellular resolution. Here, we engineered an improved opioid-sensor, called M-SPOTIT2, which is 11x brighter than our previously engineered M-SPOTIT1.1. We engineered M-SPOTIT2 by adding the amino acids YNSH, located near the fluorophore of the enhanced green fluorescent protein, to the circular permuted green fluorescent protein in M-SPOTIT2. M-SPOTIT2 is 11x brighter than our previously engineered M-SPOTIT1.1 in HEK293T cell culture and 2.7x brighter in neuronal culture. M-SPOTIT2 will potentially be useful for the detection of opioids in cell culture for drug screening and the detection of opioids at cellular resolution in animal tissues. By using M-SPOTIT2, researchers can gain more understanding about the mechanisms of addiction, respiratory suppression, and pain-modulation involved in opioid signaling.