基于发卡探针DNA/G-四链体模拟酶结构转化的高灵敏无标记荧光检测DNA

Highly Sensitive and Label-free Fluorescence Detection of DNA Based on Structure Switch of Hairpin Probe DNA to G-Quadruplex-based DNAzyme

建立了基于目标DNA诱导的发卡探针DNA转化为G-四链体DNA过氧化物模拟酶的非标记荧光增强型DNA检测新体系。发卡探针DNA即分子信标探针由两部分构成,环状部分与目标DNA互补,茎部一端由一条富G序列构成。无目标DNA存在时,分子信标处于闭合状态,形成发卡结构;富G序列由于部分处于杂交状态,无法形成G四链体。当目标DNA与发卡探针DNA杂交并打开发卡结构,富G序列解链并自发折叠成G-四链体结构。G-四链体结构与血红素结合形成DNA模拟酶,催化H2O2还原的同时将无荧光的底物10-乙酰基-3,7-二羟基吩恶嗪（ADHP）氧化成荧光产物。通过测量荧光信号,实现对目标DNA的定量检测。优化后的体系检测条件为p H 8.0,10 mmol/L K＋,0.2μmol/L Hemin,50μmol/L ADHP。在优化的条件下,目标DNA在0.005~1.0 nmol/L浓度范围内与体系荧光信号呈线性关系,检出限为3.0 pmol/L。本方法可以区分完全互补和单碱基错配的目标DNA。

A label-free fluorescent assay for highly sensitive detection of DNA was developed based on structure switch of hairpin DNA probe to a G-quadruplex-based DNAzyme triggered by target DNA. The hairpin DNA probe includes sequences that correspond to the base sequence of the G-quadruplex and to the sequence complementary to the target DNA, respectively. The hairpin structure of the probe DNA isenergetically favored over the G-quadruplex structure in the absence of target DNA, resulting in the protection of the G-quadruplex in an inactive hairpin configuration. The hybridization of target DNA to the loop domain opens the stem of the hairpin, resulting in the self-assembly of the uncaged G-quadruplex structure, which acts as a DNAzymatic label for the signal production and amplification in the presence of hemin. The peroxidase- like DNAzyme oxidizes non-fluoresecnt 10-acetyl-3,7-Dihydroxypenoxa-zin （ADHP） to the florescent product by H2O2, giving rise to fluorescence emission. This allowed the utilization of the H2O2-ADHP fluorescent system for quantitative analysis of DNA. The experimental conditions were optimized as ： pH 8.0, 10 mmol/L K＋, 0.2 μmoL/L Hemin, 50 μmol/L ADHP. The assay showed a linear relationship toward target DNA concentration in the range of 5.0 pmol/L-1.0 nmol/L, with a limit of detection of 3.0 pmol/L （S/N= 3 ）. The assay exhibited good selectivity against single-base mismatched DNA.