MicroRNAs (miRNAs) have been regarded as potential biomarkers in early diagnosis of cancer. Since the high sequence similarity among miRNA family members, biosensing miRNAs with single-base resolution is still a challenge, particularly when the different base is located at the terminal of miRNA. Herein, we developed two real-time fluorescence monitoring methods for miRNA detection utilizing efficient PBCV-1 DNA ligase mediated target miRNA dependent DNA ligation, followed by rolling circle signal amplification. Compared to duplex-specific nuclease (DSN) enhanced rolling circle transcription (RCT) system, nicking endonuclease (NEase) assisted rolling circle amplification (PRCA-NESA) can provide higher amplification efficiency, and achieve a limit-of-detection of 0.5 amol for miR-17 in 10 μL sample. More importantly, benefiting from the unique characteristics of PBCV-1 DNA ligase, we designed an asymmetric PRCA-NESA method, which can greatly discriminate the single-base difference at either 5′- or 3′-terminals of miRNAs. MiR-17 from various tumor cells also can be reliably detected. In conclusion, our strategy exploited the application potential of PBCV-1 DNA ligase in biosensing, and provided a new idea to highly specific miRNA detection, thereby would possess a promising potential for further application in biomedical research and early cancer diagnosis.

MicroRNA（miRNA）被认为是癌症早期诊断的潜在生物标记。由于miRNA家族成员之间具有高度的序列相似性，因此具有单碱基分辨率的生物传感miRNA仍然是一个挑战，特别是当不同碱基位于miRNA的末端时。在这里，我们开发了两种实时荧光监测方法，用于利用有效的PBCV-1 DNA连接酶介导的靶miRNA依赖性DNA连接进行miRNA检测，然后进行滚环信号放大。与双链特异性核酸酶（DSN）增强的滚环转录（RCT）系统相比，切口内切核酸酶（NEase）辅助的滚环扩增（PRCA-NESA）可以提供更高的扩增效率，并实现0.5 amol的检测限10μL样品中的miR-17。更重要的是，受益于PBCV-1 DNA连接酶的独特特性，我们设计了一种不对称的PRCA-NESA方法，该方法可以极大地区分miRNA 5'-或3'-末端的单碱基差异。还可以可靠地检测到来自各种肿瘤细胞的MiR-17。总之，我们的策略利用了PBCV-1 DNA连接酶在生物传感中的应用潜力，并为高度特异性的miRNA检测提供了新思路，从而在生物医学研究和早期癌症诊断中具有广阔的应用前景。