### 双链特异性核酸酶介导的高灵敏度microRNA分析

1. 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 Xiaolia, Wang Yuconga, Zhang Xuejinga, Zhao Yunjiea, Liu Chenghuia,b, Li Zhengpinga,b

1. 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).

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.