基于分子信标策略的CRISPR/Cas12a荧光传感器用于循环肿瘤DNA的放大检测  

CRISPR/Cas12a Fluorescence Sensor Based on Molecular Beacon for Amplification Detection of Circular Tumor DNA

作　　者：张函笑 代晓春 周亚楠 吕旭珍 弓韬 赵旭华 于保锋 ZHANG Han-Xiao;DAI Xiao-Chun;ZHOU Ya-Nan;LYU Xu-Zhen;GONG Tao;ZHAO Xu-Hua;YU Bao-Feng(Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学基础医学院,生物化学与分子生物学教研室,太原030001

出　　处：《分析化学》2023年第2期184-193,共10页Chinese Journal of Analytical Chemistry

基　　金：山西省自然科学基金项目(Nos.202103021224240,201901D211317,201901D111190);山西医科大学细胞生理学教育部重点实验室开放基金项目(No.KLMEC/SXMU-202011);山西省“1331工程”重点学科建设计划项目(No.1331KSC)资助。

摘　　要：构建了一种以分子信标(Molecular beacon,MB)为信号探针的CRISPR/Cas12a生物传感器,用于循环肿瘤DNA(Circular tumor DNA,ctDNA)的快速放大检测。MB具有良好的稳定性,其颈部末端分别标记有荧光素(FAM)和四甲基罗丹明(TAMRA)两种荧光基团。ctDNA不存在时,CRISPR/Cas12a体系无活性,无法切割MB,因此MB两端的荧光基团由于形成发夹结构的颈部相互靠近而发生荧光共振能量转移(Fluorescence resonance energy transfer,FRET),显示出TAMRA的荧光。当ctDNA存在时,ctDNA特异性识别Cas12a/crRNA二元复合物并激活Cas12a的反式切割活性。由于单链DNA是Cas12a最敏感的底物,因此MB的环部单链首先被切割,继而引起颈部双链的解离而导致两种荧光团彼此远离,无法发生FRET,最终显示出FAM的荧光信号。对MB环部的碱基数目、MB浓度以及crRNA与Cas12a的浓度比例等实验条件进行了优化。在最优条件下,1.7~500 pmol/L范围内,ctDNA浓度与传感器在518 nm处的荧光强度呈线性关系,检出限为0.6 pmol/L(3σ)。将此传感器用于血清样品中ctDNA的检测,回收率在93%~110%之间。A CRISPR/Cas12a-based biosensor using molecular beacon(MB)as the reporter was constructed for amplification detection of circular tumor DNA(ctDNA).The molecular beacon with good stability was labeled with FAM and TAMRA at its ends,respectively.In the absence of ctDNA,the CRISPR/Cas12a system was inactive and MB could not be cleaved.Therefore,two fluorophores were in close proximity to each other,resulting in fluorescence resonance energy transfer(FRET).In the presence of ctDNA,it could recognize the Cas12a/crRNA complex and activate the trans-cleavage activity of Cas12a.Because ssDNA was the most susceptible substrate of Cas12a,the loop of MB was rapidly cleaved first.After cleavage,two fluorophores were far from each other,leading to the disappearance of FRET phenomenon and an obvious fluorescent enhancement of FAM.The experimental conditions such as the base numbers of the hairpin loop,the concentration of MB,and the concentration ratio of crRNA to Cas12a were optimized.Under the optimal conditions,a linear relationship ranging from 1.7 pmol/L to 500 pmol/L for ct DNA quantitative detection was observed and its detection limit was 600 fmol/L.In addition,this sensor could be used to detect ctDNA in serum samples and the recoveries were in the range of 93%–110%.

关 键 词：CRISPR 生物传感器 荧光共振能量转移 循环肿瘤DNA 

分 类 号：O657.3[理学—分析化学] R730[理学—化学]