Single-molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong, 518055, China.
Anal Chem. 2012 Aug 21;84(16):7037-42. doi: 10.1021/ac3012544. Epub 2012 Aug 6.
MicroRNAs (miRNAs) play important regulatory roles in a wide range of biological processes, and their aberrant expression is associated with cancer development and a variety of diseases. Here, we develop a simple, sensitive, and specific miRNA assay on the basis of circular exponential amplification in combination with the hairpin probes. The binding of target miRNA with a linear DNA template initiates the first strand displacement amplification (SDA) and generates the universal triggers which are complementary to the 3' protruding end of a hairpin probe. These universal triggers function not only as the primers to unfold the hairpin probes through an extension reaction, generating distinct fluorescence signals, but also as the amplification templates to initiate the second SDA reaction. Moreover, the second SDA reaction can release new triggers to initiate the above two consecutive SDA reactions, thus constituting a circular exponential amplification which enables the conversion of a small amount of miRNAs to a large number of universal triggers to unfold abundant hairpin probes. This hairpin probe-based circular exponential amplification assay exhibits high sensitivity with a detection limit of 3.80 × 10(-13) M and a detection range of 4 orders of magnitude. It can even discriminate single-nucleotide difference between miRNA family members and perform well in real sample analysis. Notably, in this assay, the long-stem hairpin probes are unfolded through an extension reaction rather than through a conventional hybridization reaction controlled by the thermodynamic equilibrium in the case of molecular beacons, making the design of hairpin probes very simple. This hairpin probe-based circular exponential amplification assay holds a great promise for further application in biomedical research and early clinical diagnosis.
微小 RNA（miRNAs）在广泛的生物过程中发挥着重要的调节作用，其异常表达与癌症的发生和多种疾病有关。在这里，我们基于循环指数扩增结合发夹探针开发了一种简单、灵敏、特异的 miRNA 检测方法。靶 miRNA 与线性 DNA 模板的结合启动了第一条链的置换扩增（SDA），并产生与发夹探针 3'突出端互补的通用触发物。这些通用触发物不仅作为引物通过延伸反应展开发夹探针，产生独特的荧光信号，而且作为扩增模板启动第二次 SDA 反应。此外，第二次 SDA 反应可以释放新的触发物，启动上述两个连续的 SDA 反应，从而构成一个循环指数扩增，将少量 miRNA 转化为大量通用触发物，以展开大量的发夹探针。这种基于发夹探针的循环指数扩增检测方法具有很高的灵敏度，检测限为 3.80×10^(-13)M，检测范围为 4 个数量级。它甚至可以区分 miRNA 家族成员之间的单核苷酸差异，并在实际样品分析中表现良好。值得注意的是，在这种检测方法中，长茎发夹探针通过延伸反应而不是通过分子信标中由热力学平衡控制的传统杂交反应展开，这使得发夹探针的设计非常简单。这种基于发夹探针的循环指数扩增检测方法在生物医学研究和早期临床诊断中有很大的应用前景。
