Directed Evolution of Key Residues in Fluorescent Protein Inverses the Polarity of Voltage Sensitivity in the Genetically Encoded Indicator ArcLight

作者：Platisa Jelena; Vasan Ganesh; Yang Amy; Pieribone Vincent A

来源：ACS Chemical Neuroscience, 2017, 8(3): 513-523.

DOI：10.1021/acschemneuro.6b00234

• Genetically encoded voltage indicators
• ArcLight
• green fluorescent protein
• biosensors
• directed evolution
• mutagenesis

Genetically encoded calcium indicators (GECIs) produce unprecedentedly large signals that have enabled routine optical recording of single neuron activity in vivo in rodent brain. Genetically encoded voltage indicators (GEVIs) offer a more direct measure of neuronal electrical status, however the signal-to-noise characteristics and signal polarity of the probes developed to date have precluded routine use in vivo. We applied directed evolution to target modulable areas of the fluorescent protein in GEVI ArcLight to create the first GFP-based GEVI (Marina) that exhibits a Delta F/Delta V with a positive slope relationship. We found that only three rounds of site-directed mutagenesis produced a family of "brightening" GEVIs with voltage sensitivities comparable to that seen in the parent probe ArcLight. This shift in signal polarity is an essential first step to producing voltage indicators with signal-to-noise characteristics comparable to GECIs to support widespread use in vivo.

• 出版日期2017-3