Department of Physics and Astronomy, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Nat Nanotechnol. 2010 Nov;5(11):807-14. doi: 10.1038/nnano.2010.202. Epub 2010 Oct 24.
Small RNA molecules have an important role in gene regulation and RNA silencing therapy, but it is challenging to detect these molecules without the use of time-consuming radioactive labelling assays or error-prone amplification methods. Here, we present a platform for the rapid electronic detection of probe-hybridized microRNAs from cellular RNA. In this platform, a target microRNA is first hybridized to a probe. This probe:microRNA duplex is then enriched through binding to the viral protein p19. Finally, the abundance of the duplex is quantified using a nanopore. Reducing the thickness of the membrane containing the nanopore to 6 nm leads to increased signal amplitudes from biomolecules, and reducing the diameter of the nanopore to 3 nm allows the detection and discrimination of small nucleic acids based on differences in their physical dimensions. We demonstrate the potential of this approach by detecting picogram levels of a liver-specific miRNA from rat liver RNA.
小 RNA 分子在基因调控和 RNA 沉默治疗中具有重要作用，但如果不使用耗时的放射性标记测定或易错的扩增方法，就很难检测到这些分子。在这里，我们提出了一个用于快速电子检测细胞 RNA 中探针杂交 microRNA 的平台。在该平台中，首先将靶 microRNA 与探针杂交。然后，通过与病毒蛋白 p19 结合，将探针：microRNA 双链体进行富集。最后，使用纳米孔对双链体的丰度进行定量。将含有纳米孔的膜的厚度减小到 6nm 会导致生物分子的信号幅度增加，而将纳米孔的直径减小到 3nm 可以根据其物理尺寸的差异来检测和区分小核酸。我们通过从大鼠肝 RNA 中检测到皮克级水平的肝特异性 miRNA 来证明该方法的潜力。
