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

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.

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.