CAJ | 학술논문

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H2 O2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pH 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。
건립료기우목표DNA유도적발잡탐침DNA전화위G-사련체DNA과양화물모의매적비표기형광증강형DNA검측신체계。발잡탐침DNA즉분자신표탐침유량부분구성,배상부분여목표DNA호보,경부일단유일조부G서렬구성。무목표DNA존재시,분자신표처우폐합상태,형성발잡결구；부G서렬유우부분처우잡교상태,무법형성G사련체。당목표DNA여발잡탐침DNA잡교병타개발잡결구,부G서렬해련병자발절첩성G-사련체결구。 G-사련체결구여혈홍소결합형성DNA모의매,최화H2 O2환원적동시장무형광적저물10-을선기-3,7-이간기분악진(ADHP)양화성형광산물。통과측량형광신호,실현대목표DNA적정량검측。우화후적체계검측조건위pH 8.0,10 mmol/L K+,0.2μmol/L Hemin,50μmol/L ADHP。재우화적조건하,목표DNA재0.005~1.0 nmol/L농도범위내여체계형광신호정선성관계,검출한위3.0 pmol/L。본방법가이구분완전호보화단감기착배적목표DNA。
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 is <br> energetically 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 H2 O2 , giving rise to fluorescence emission. This allowed the utilization of the H2 O2-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.