Department of Chemical Engineering and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213-3890, United States.
Biomacromolecules. 2013 Jul 8;14(7):2253-61. doi: 10.1021/bm400388a. Epub 2013 Jun 18.
Hybridization analysis of short DNA and RNA targets presents many challenges for detection. The commonly employed sandwich hybridization approach cannot be implemented for these short targets due to insufficient probe-target binding strengths for unmodified DNA probes. Here, we present a method capable of rapid and stable sandwich hybridization detection for 22 nucleotide DNA and RNA targets. Stable hybridization is achieved using an n-alkylated, polyethylene glycol γ-carbon modified peptide nucleic acid (γPNA) amphiphile. The γPNA's exceptionally high affinity enables stable hybridization of a second DNA-based probe to the remaining bases of the short target. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the n-alkane modification on the γPNA with capillary electrophoresis running buffer containing nonionic surfactant micelles. We find that sandwich hybridization of both probes is stable under multiple binding configurations and demonstrate single base mismatch discrimination. The binding strength of both probes is also stabilized via coaxial stacking on adjacent hybridization to targets. We conclude with a discussion on the implementation of the proposed sandwich hybridization assay as a high-throughput microRNA detection method.
杂交分析的短 DNA 和 RNA 靶标提出了许多挑战的检测。常用的夹心杂交方法不能实现这些短的目标，由于探针-靶标结合强度不足未修饰的 DNA 探针。在这里，我们提出了一种能够快速稳定的夹心杂交检测 22 个核苷酸的 DNA 和 RNA 靶标。稳定杂交是采用 n-烷基化，聚乙二醇 γ-碳修饰的肽核酸（γPNA）两亲分子。γPNA 的异常高的亲和力使稳定杂交的第二个 DNA 为基础的探针的短目标的其余碱基。在杂交的两种探针，电泳迁移率的变化是通过 n-烷烃修饰的γPNA 与含有非离子型表面活性剂胶束的毛细管电泳运行缓冲液的相互作用来测量的。我们发现，夹心杂交的两种探针是稳定的多个结合配置，并证明单碱基错配的歧视。两个探针的结合强度也稳定通过同轴堆叠在相邻杂交的目标。我们的讨论结束时，作为一种高通量 microRNA 检测方法的夹心杂交试验的实施。
