基于TALEs蛋白特异性识别的新型核酸甲基化电化学传感器研究

细菌16S rRNA的碱基甲基化是引起细菌耐药的重要机制之一，为探明细菌耐药机制，需要检测16S rRNA序列中甲基化碱基及位点。目前对5-mC的检测研究较多，对N4-mC、N6-mA和N7-mG的研究较少，主要是缺乏对甲基化碱基进行精准识别的分子。本项目针对这一问题，提出开展以转录激活子样效应因子蛋白（TALEs）为碱基甲基化识别分子的电化学传感器方法学研究。TALEs对碱基和甲基化碱基具有独特的识别能力，能克服核酸杂交中的碱基错配问题。课题拟以与耐药性相关的细菌16s rRNA 的A位点为检测靶标，制备相应的TALEs蛋白作为识别分子，构建电化学传感器。由于5-mC的氨基酸双残基已破译，可直接用来制备相应的TALEs蛋白；对N6-mA、N4-mC和N7-mG，先破译其氨基酸双残基，再制备相应的TALEs蛋白。利用所制备的TALEs蛋白构建传感器，实现细菌核酸碱基甲基化时空动态检测。

Methylation of nucleic acid bases in bacterial 16S rRNA is one of the important mechanisms causing bacterial resistance. To explore the mechanism of bacterial resistance, we need to know the bases and sites of methylation in 16S rRNA sequence. At present, there are many researches on the detection method of 5-mC and the detection studies about of N4-mC, N6-mA and N7-mG were few. The main reason is the lack of molecules that can accurately identify methylated bases. Aiming at this problem, this project proposes a rapid detection methodology for electrochemical biosensors with transcription activator-like effector protein (TALEs) as the base methylation recognition molecule. TALEs has a unique ability to identify bases and methylated bases, which can overcome the problem of base mismatch in nucleic acid hybridization. In this study, the A-site of bacterial 16S rRNA related to drug resistance was used as the detection target, and the corresponding TALEs protein is used as the recognition molecule to construct the electrochemical sensor. Since the amino acid double residues of 5-methylcytosine (5-mc) have been deciphered, they can be directly used to prepare the corresponding TALEs protein; for N6-mA, N4-mC, and N7-mG, it is necessary to decipher their amino acid double residues before preparing the corresponding tales protein. Using the prepared TALEs protein to construct the sensor, the spatiotemporal dynamic detection of the methylation of bacterial nucleic acid can be realized.