基于“分段”-“完整”转化的G-四链体脱氧核糖核酸酶传感器用于多聚核苷酸激酶的检测  

G-Quadruplex DNAzyme Biosensor for Quantitative Detection of T4 Polynucleotide Kinase Activity by Using Split-to-Intact G-Quadruplex DNAzyme Conversion

作　　者：周璐[1] 程慧[1] 王金娥[1] 裴仁军[1] 

机构地区：[1]中国科学院苏州纳米技术与纳米仿生研究所,苏州215123

出　　处：《分析化学》2016年第1期13-18,共6页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(Nos.31301495;21275156;21305154)资助~~

摘　　要：采用"分段"转化为"完整"G-四链体脱氧核糖核酸酶的策略,构建了检测多聚核苷激酶(T4 PNK)的传感器。将形成G-四链体的富G序列PS5.M序列拆分成5'和3'端均为羟基的两条链:链S_1OH和链S_2OH即分别具有12个碱基的"分段"的PS5.M。在ATP存在下,T4 PNK酶可以将链S_2OH的5'端的羟基磷酸化,转化为S_2P;在S_1OH、S_2P以及Helper链S-H存在下,T4 DNA连接酶(T4 DNA Ligase)将链S_1OH和链S_2P连接成"完整"的PS5.M序列。在体系中再加入核酸外切酶Ⅲ(ExoⅢ),从S-H的3'端剪切S-H,释放出PS5.M。在K^+存在下,PS5.M与氯化血红素(Henfin)作用,形成具有类过氧化物酶活性的复合物,催化H_2O_2氧化2 2'-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)的反应,通过检测氧化产物在418 nm处的吸收值变化,实现对T4 PNK活性的定量检测。线性检测范围为0.02~3.0 U/mL舱出限为0.014 U/mL(S/N=3)。对Hela细胞和HEK293细胞实际样本的T4 PNK活性进行了检测,平均回收率为95.6%~105.7%。A biosensor for detection of T4 polynucleotide kinase（PNK） activity was fabricated by using a split-to-intact DNAzyme conversion strategy.G-quadrplex-forming G-rich sequence PS5.M was chosen as a model DNAzyme and splitted into two short parts of S1OH and S2OH,which both had hydroxyl groups at 3＇and 5＇terminals,and could hybridize to another sequence,helper DNA（S-H）,to form a duplex structure.In the presence of T4 PNK,5＇-end of S2OH was phophorylated to obtain S2P.Then S1OH and S2P could be linked by T4 DNA ligase to intact PS5.M.In the presence of exonuclease Ⅲ（Exo Ⅲ）,SH chain in the duplex of PS5.M-SH could be cleaved into nucleotides,releasing free PS5.M.The released PS5.M could form catalytically active G-quadruplex DNAzyme upon binding hemin in the presence of K^＋,which could catalyze the oxidation of 2 2＇-azino bis-（3-ethylbenzthiazoline-6-sulphonate）（ABTS） by H2O2.Quantitative detection of T4 PNK activity could be achieved by measuring the maximal absorbance of product at 418 nm.The absorbance of the sensing system at 418 nm exhibited a linear relationship with T4 PNK activity in the range of 0.02-0.3 U/mL with a detection limit of 0.014 U/mL（S/N=3）.The biosensor was also applied to the determination of PNK activity in Hela cell and HEK 293 cell,and good recoveries of 95.6%-105.7%were obtained at three spiked levels.

关 键 词：多聚核苷酸激酶 脱氧核糖核酸酶 G-四链体 生物传感器 

分 类 号：O657.3[理学—分析化学] TP212[理学—化学]