Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China.
Biosens Bioelectron. 2013 Apr 15;42:131-5. doi: 10.1016/j.bios.2012.10.097. Epub 2012 Nov 7.
It is well-known that microRNAs (miRNAs) have become an ideal class of biomarker candidates for clinical diagnosis of cancers, thus sensitive and selective detection of microRNAs is of great significance in understanding biological functions of miRNAs, early-phase diagnosis of cancers, as well as discovery of new targets for drugs. In this work, we have developed a sensitive method for microRNAs detection based on isothermal exponential amplification-assisted generation of catalytic G-quadruplex DNAzyme, and demonstrated its practical application in biological sample of cell lysate. The assay involves a combination of polymerase strand extension, single-strand nicking and catalytic reaction of G-quadruplex/hemin complex. It is designed such that, the target miRNA initiates the efficient synthesis of two kinds of short oligonucleotide fragments in the continuous cycle of the polymerization, nicking and displacement reactions, by means of thermostable polymerase and nicking endonuclease. One fragment has the same sequence as the target miRNA, except that the deoxyribonucleotides and thymine replace the ribonucleotides and uridine in the miRNA, to activate new cyclic chain reactions of polymerization, nicking and displacement reactions as the target miRNA. The other is the signal molecule of horseradish peroxidase (HRP)-mimicking G-quadruplex DNAzyme. With such designed signal amplification processes, the proposed assay showed a quantitative analysis of sequence-specific miRNAs in a wide range from 1 fM to 100 nM with a low detection limit of 1 fM. Moreover, this assay demonstrated excellent differentiation ability for the mismatch miRNAs targets and good performance in biological samples.
众所周知,microRNAs(miRNAs)已成为癌症临床诊断的理想生物标志物候选物,因此,对 miRNAs 的灵敏和选择性检测对于理解 miRNAs 的生物学功能、癌症的早期诊断以及新药物靶点的发现具有重要意义。在这项工作中,我们开发了一种基于等温指数扩增辅助生成催化 G-四链体 DNA 酶的 miRNA 灵敏检测方法,并在细胞裂解物的生物样本中证明了其实际应用。该测定法涉及聚合酶链延伸、单链切口和 G-四链体/血红素复合物的催化反应的组合。该测定法的设计使得目标 miRNA 在聚合酶、切口内切酶的热稳定性的连续循环中,有效地启动两种短寡核苷酸片段的高效合成。一种片段与目标 miRNA 具有相同的序列,除了脱氧核糖核苷酸和胸腺嘧啶取代 miRNA 中的核糖核苷酸和尿嘧啶,以激活作为目标 miRNA 的新聚合、切口和置换反应的循环链反应。另一种是辣根过氧化物酶(HRP)模拟 G-四链体 DNA 酶的信号分子。通过这种设计的信号放大过程,所提出的测定法在 1 fM 至 100 nM 的宽范围内对序列特异性 miRNAs 进行了定量分析,检测限低至 1 fM。此外,该测定法对错配 miRNA 靶标具有出色的区分能力,并在生物样品中具有良好的性能。