<b>Development of a Genetically Encoded NMT Indicator for Detection of Mercury Ions Based on the Green Fluorescent Protein mNeonGreen and Metallothionein II from Rat Liver</b>

Heavy metals, particularly mercury, rank as some of the most hazardous systemic toxicants known to cause multiple organ damage, even at lower levels of exposure. Its detection in the environment and in the live cells is an actual task. Here, we engineered a novel genetically encoded fluorescent NMT indicator for mercury ions by inserting the metallothionein II domain from rat liver into the bright green-yellow fluorescent protein mNeonGreen, followed by directed molecular evolution of the resulting sensor prototype in bacteria. In solution, the NMT indicator was 1.7-fold brighter than the standard eGFP fluorescent protein and responded to the addition of even 10^-18-10^-19 M mercury ions by quenching fluorescence with a 5-fold fluorescence response and extremely high affinity to mercury ions characterized by the K_d value of 0.50 ± 0.05 aM. We also characterized the selectivity of the NMT indicator to other metal cations. In cultured mammalian cells, the NMT indicator detected even an extracellular concentration of 0.1 fM mercury ions and achieved a 5.9-fold change in DF/F fluorescence intensity.