双链特异性核酸酶介导的高灵敏度microRNA分析

doi:10.6023/A14010012

化学学报 ›› 2014, Vol. 72 ›› Issue (3): 395-400.DOI: 10.6023/A14010012 上一篇下一篇

所属专题：石墨烯

研究论文

双链特异性核酸酶介导的高灵敏度microRNA分析

李晓利^a, 王愈聪^a, 张学晶^a, 赵云颉^a, 刘成辉^a,b, 李正平^a,b

a 河北大学化学与环境科学学院保定 071002;
b 陕西师范大学化学化工学院西安 710062

投稿日期:2014-01-04 发布日期:2014-02-25
通讯作者: 刘成辉，E-mail：liuch@snnu.edu.cn；王愈聪，E-mail：wangyucong@hbu.edu.cn；Tel.：0312-5079403；Fax：0312-5079403 E-mail:liuch@snnu.edu.cn；wangyucong@hbu.edu.cn
基金资助:
项目受国家自然科学基金（Nos. 21105020，20905018）及河北大学引进人才项目（No. 2009177）资助.

Double Strand-Specific Nuclease-Assisted Sensitive Detection of MicroRNA

Li Xiaoli^a, Wang Yucong^a, Zhang Xuejing^a, Zhao Yunjie^a, Liu Chenghui^a,b, Li Zhengping^a,b

a College of Chemistry and Environmental Science, Hebei University, Baoding 071002;
b School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062

Received:2014-01-04 Published:2014-02-25
Supported by:
Project supported by the National Natural Science Foundation of China (Nos. 21105020, 20905018) and financial support of Hebei University (No. 2009177).

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摘要/Abstract

基于氧化石墨烯（GO）对荧光标记单链DNA探针的荧光猝灭效应以及双链特异性核酸酶（DSN）选择性切割DNA/RNA杂合结构中DNA单链的特性，本文建立了一种新型恒温信号放大方法用于microRNA（miRNA）的高灵敏度检测. 靶标miRNA首先与荧光DNA探针杂交，DSN能够特异性地将杂合双链中的DNA探针水解为碎片但不会降解miRNA，GO对酶切产生的寡核苷酸碎片吸附能力显著降低，使得荧光基团远离GO表面而不被猝灭. 释放出的miRNA可再次发生与荧光DNA探针杂交、DSN酶切等反应，如此反复，可实现恒温条件下一个miRNA分子与多个探针杂交、酶切、释放荧光基团的循环过程，最终体系的荧光信号得到显著放大，通过记录体系的荧光信号即可实现对靶标miRNA的灵敏检测.

关键词: microRNA, 双链特异性核酸酶, 氧化石墨烯, 恒温信号扩增, 荧光

In this study, a new isothermal signal amplification method is developed for sensitive detection of microRNAs (miRNAs) by integrating the distinct advantages of graphene oxide (GO) for efficient fluorescence quenching of fluorophore-labeled single strand DNA (ssDNA) and double strand (ds)-specific nuclease (DSN) for highly selective digestion of DNA strand in DNA/RNA hybrids. DSN is a nuclease purified from hepatopancreas of Red King crab, which shows a strong preference for cleaving dsDNA and DNA in DNA/RNA hybrid duplexes. On contrast, DSN is practically inactive towards ssDNA or single- or double-stranded RNA. Herein, let-7a is selected as the proof-of-concept target miRNA and a fluorescein-labeled ssDNA probe is designed to be complementary to let-7a. The ssDNA probe, which will not be hydrolyzed by DSN in the absence of let-7a, will be adsorbed on GO via π-π stacking, resulting in efficient fluorescence quenching. When let-7a is introduced, it will hybridize with the ssDNA probe to form a double helix structure (dsDNA). DSN can selectively cleave the DNA oligonucleotides of the DNA/RNA hybrid to produce very small DNA fragments. Let-7a is thus released and will hybridize with another ssDNA probe again, which will be further cleaved by DSN. In this manner, each let-7a molecule can specifically trigger various cycles of hybridization and DSN cleavage of fluorescent ssDNA to yield numerous small fragments of DNA oligonucleotides. It should be noted that the π-π stacking interaction between GO and the very small DNA fragments bearing the fluorophores will be remarkably weakened, making the fluorescence maintained. Therefore, the DSN-mediated cycling of fluorescent ssDNA cleavage greatly amplifies the fluorescence signal for miRNA detection. Under the optimized experimental conditions, the fluorescence signal is proportional linearly to the concentration of let-7a in the range from 100 pmol/L to 5 nmol/L, and the detection limit is calculated to be 60 pmol/L (3σ). Furthermore, this proposed approach can also be applied to the simultaneous detection of multiplex miRNA targets.

Key words: microRNA, double strand-specific nuclease, graphene oxide, isothermal signal amplification, fluorescence

引用此文

李晓利, 王愈聪, 张学晶, 赵云颉, 刘成辉, 李正平. 双链特异性核酸酶介导的高灵敏度microRNA分析[J]. 化学学报, 2014, 72(3): 395-400.

Li Xiaoli, Wang Yucong, Zhang Xuejing, Zhao Yunjie, Liu Chenghui, Li Zhengping. Double Strand-Specific Nuclease-Assisted Sensitive Detection of MicroRNA[J]. Acta Chimica Sinica, 2014, 72(3): 395-400.

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双链特异性核酸酶介导的高灵敏度microRNA分析

Double Strand-Specific Nuclease-Assisted Sensitive Detection of MicroRNA

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