新一代基因编码5-羟色胺荧光探针优化及应用
Optimization of a genetically encoded fluorescent sensor for the detection of 5-HT
摘要
Abstract
Objective·To optimize iSeroSnFR,a genetically encoded 5-hydroxytryptamine(5-HT)fluorescent sensor based on bacterial periplasmic binding proteins(PBPs),to enhance its performance for both in vivo and in vitro 5-HT detection.Methods·iSeroSnFR1.2 was engineered by replacing the circularly permuted superfolder green fluorescence protein(cpsfGFP)sequence in iSeroSnFR1.0 with that from the acetylcholine sensor iAChSnFR using Gibson assembly.The fluorescence response and kinetic properties of iSeroSnFR1.0 and iSeroSnFR1.2 were compared by overexpressing the sensors in HEK293 cells and puffing with exogenous 5-HT.Additionally,to mimic physiological conditions,cultured mouse cortical neurons infected with Sindbis virus carrying each sensor were electrically stimulated to induce endogenous 5-HT release and further evaluate sensor performance.Results·iSeroSnFR1.2 showed significantly improved performance over iSeroSnFR1.0.In HEK293 cells,it exhibited a 1.5-fold increase in fluorescence response(ΔF/F0)to exogenous 5-HT,along with faster kinetics(rise time:36.3 ms vs 44.9 ms;decay time:1 003.6 ms vs 1 730.4 ms).In cortical neurons,it demonstrated a 2.7-fold increase in response to endogenously released 5-HT,with rise and decay times reduced by 44.0%and 26.7%,respectively.Notably,iSeroSnFR1.2 showed increased basal fluorescence,enabling better imaging in high-background environments.Conclusion·The optimized iSeroSnFR1.2 sensor offers a markedly improved fluorescent response and temporal resolution for 5-HT detection,providing an advanced tool for studying 5-HT dynamics in neuroscience and psychiatric research.关键词
基因编码探针/5-羟色胺/循环重排超折叠绿色荧光蛋白/细菌周质结合蛋白Key words
genetically encoded sensor/5-hydroxytryptamine(5-HT)/circularly permuted superfolder GFP/periplasmic binding protein分类
医药卫生引用本文复制引用
徐慕凡,张坤,王静怡,高欣柯,程傲冰,张鹏..新一代基因编码5-羟色胺荧光探针优化及应用[J].上海交通大学学报（医学版）,2025,45(5):529-539,11.基金项目
国家自然科学基金(82102184) （82102184）
上海市高水平地方高校建设项目(PT21011) （PT21011）
2023年上海"科技创新行动计划"自然科学基金项目(23ZR1436800) （23ZR1436800）
国家中医药管理局高水平中医药学科建设项目(ZYYZDXK-2023070) （ZYYZDXK-2023070）
阿尔茨海默病协会研究基金(AARF-19-619387) （AARF-19-619387）
上海市高水平地方高校建设项目2024年"中西医结合研究平台"建设项目(ZXY24006). Funding Information]National Natural Science Foundation of China(82102184) （ZXY24006）
Shanghai High-level Local University Construction Project(PT21011) （PT21011）
Shanghai 2023"Science and Technology Innovation Action Plan"Natural Science Foundation Project(23ZR1436800) （23ZR1436800）
NATCM's Project of High-level Construction of Key TCM Disciplines(ZYYZDXK-2023070) （ZYYZDXK-2023070）
Alzheimer's Association Research Fellowship(AARF-19-619387) （AARF-19-619387）
Shanghai High-Level Local Universities Construction Project 2024 Integrated Traditional Chinese and Western Medicine Research Platform' Construction Project(ZXY24006). （ZXY24006）
