Acta Chimica Sinica ›› 2014, Vol. 72 ›› Issue (3): 395-400.DOI: 10.6023/A14010012 Previous Articles     Next Articles

Special Issue: 石墨烯



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   

  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,;王愈聪,;Tel.:0312-5079403;Fax:0312-5079403;
  • 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.

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