A Förster Resonance Energy Transfer-based d-2-hydroxyglutarate Biosensor
A Förster Resonance Energy Transfer-based d-2-hydroxyglutarate Biosensor
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DOI：
10.1016/j.snb.2023.133681
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发表时间：
2023-03
期刊：
影响因子：
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通讯作者：
Dan Xiao;Xian-zhi Xu;Kaiyu Gao;Mengjiao Wang;Wen Zhang;Chuanjuan Lü;Xia Wang;Qian Wang;Ping Xu;Cuiqing Ma;Chao Gao
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文献类型：
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作者：
Dan Xiao;Xian-zhi Xu;Kaiyu Gao;Mengjiao Wang;Wen Zhang;Chuanjuan Lü;Xia Wang;Qian Wang;Ping Xu;Cuiqing Ma;Chao Gao
D-2-羟基戊二酸(d-2-HG)是一种在D-2-羟基戊二酸尿症或各种异柠檬酸脱氢酶突变相关癌症患者体内异常积聚的代谢物。D-2-HG的检测对这些疾病的诊断和治疗具有重要意义。为此，研制了一种基于Förster共振能量转移(FRET)的d-2-HG生物传感器。以脱氮Achromobacter nBRC 15125中的HED-2-HG特异性变构转录因子DhdR为识别元件，将其插入三叶草和mRuby2两个荧光蛋白之间。通过截断DhdR的末端氨基酸和添加人工连接物，提高了传感器的响应值。优化后的变异体DHGFR1.0具有良好的灵敏度和特异性，最大发射率变化(ΔRmax)为30.77%，半最大有效浓度(EC_(50))为2.78μM，动态检测范围为0.17-36.22μM。通过DhdR的定点突变，预测了DhdR的配体结合部位，并获得了几种低亲和力的Ford-2-HG生物传感器。
d-2-Hydroxyglutarate (d-2-HG) is an oncometabolite aberrantly accumulated in patients withd-2-hydroxyglutaric aciduria or various isocitrate dehydrogenases mutation-associated cancers. The detection ofd-2-HG is of great significance for the diagnostic and therapeutic aspects of these diseases. Herein, a Förster resonance energy transfer (FRET)-basedd-2-HGbiosensor was developed. Thed-2-HG-specific allosteric transcription factor DhdR fromAchromobacter denitrificansNBRC 15125 was used as its biorecognition element and inserted between two fluorescent proteins Clover and mRuby2. The response magnitude of the biosensor was improved by truncation of the terminal amino acids of DhdR and addition of artificial linkers. The optimized variant DHGFR1.0 exhibited excellent sensitivity and specificity with a maximum emission ratio change (ΔRmax) of 30.77%, a half-maximal effective concentration (EC50) of 2.78 μM, and a dynamic detection range of 0.17–36.22 μM. It was able to detect the concentration ofd-2-HG in body fluids and cell-related samples with high accuracy and precisionin vitro. The ligand-binding site of DhdR was predicted and several biosensors with low affinity ford-2-HG were also generated by site-directed mutagenesis of DhdR.