Optimization of a genetically encoded fluorescent sensor for the detection of 5-HT
10.3969/j.issn.1674-8115.2025.05.001
- VernacularTitle:新一代基因编码5-羟色胺荧光探针优化及应用
- Author:
Mufan XU
1
;
Kun ZHANG
;
Jingyi WANG
;
Xinke GAO
;
Aobing CHENG
;
Peng ZHANG
Author Information
1. 上海交通大学基础医学院药理学与化学生物学系,上海 200025
- Publication Type:Journal Article
- Keywords:
genetically encoded sensor;
5-hydroxytryptamine(5-HT);
circularly permuted superfolder GFP;
periplasmic binding protein
- From:
Journal of Shanghai Jiaotong University(Medical Science)
2025;45(5):529-539
- CountryChina
- Language:Chinese
-
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.
