Department of Chemistry and Centre for Research on Biomolecular Interactions , York University , Toronto , Ontario M3J 1P3 , Canada.
Sunnybrook Research Institute and Department of Laboratory Medicine and Pathobiology, Faculty of Medicine , University of Toronto , Toronto , Ontario M5S 1A8 , Canada.
Anal Chem. 2018 Dec 18;90(24):14610-14615. doi: 10.1021/acs.analchem.8b04793. Epub 2018 Dec 4.
Direct quantitative analysis of multiple miRNAs (DQAMmiR) is a hybridization-based assay, in which the excess of the DNA hybridization probes is separated from the miRNA-probe hybrids, and the hybrids are separated from each other in gel-free capillary electrophoresis (CE) using two types of mobility shifters: single-strand DNA binding protein (SSB) added to the CE running buffer and peptide drag tags conjugated with the probes. Here we introduce the second-generation DQAMmiR, which utilizes peptide nucleic acid (PNA) rather than DNA hybridization probes and requires no SSB in the CE running buffer. PNA probes are electrically neutral, while PNA-miRNA hybrids are negatively charged, and this difference in charge can be a basis for separation of the hybrids from the probes. In this proof-of-principle work, we first experimentally confirmed that the PNA-RNA hybrid was separable from the excess of the PNA probe without SSB in the running buffer, resulting in a near 10 min time window, which would allow, theoretically, separation of up to 30 hybrids. Then, we adapted to PNA-RNA hybrids our previously developed theoretical model for predicting hybrid mobilities. The calculation performed with the modified theoretical model indicated that PNA-RNA hybrids of slightly different lengths could be separated from each other without drag tags. Accordingly, we designed a simple experimental model capable of confirming: (i) separation of tag-free hybrids of different lengths and (ii) separation of same-length hybrids due to a drag tag on the PNA probe. The experimental model included three miRNAs: 20-nt miR-147a, 20-nt miR-378g, and 22-nt miR-21. The three complementary PNA probes had lengths matching those of the corresponding target miRNAs. The probe for miR-147a had a short five-amino-acid drag tag; the other two had no drag tags. We were able to achieve baseline separation of the three hybrids from each other. The LOQ of 14 pM along with the high accuracy (recovery >90%) and precision (RSD ≈ 10%) of the assay at picomolar target concentrations suggest that PNA-facilitated DQAMmiR could potentially support practical miRNA analysis of clinical samples.
直接定量分析多个 miRNA（DQAMiR）是一种基于杂交的分析方法，其中过量的 DNA 杂交探针与 miRNA-探针杂交物分离，并且在无胶毛细管电泳（CE）中通过两种类型的迁移率位移剂将杂交物彼此分离：添加到 CE 运行缓冲液中的单链 DNA 结合蛋白（SSB）和与探针偶联的肽拖曳标签。在这里，我们介绍第二代 DQAMiR，它利用肽核酸（PNA）而不是 DNA 杂交探针，并且在 CE 运行缓冲液中不需要 SSB。PNA 探针是电中性的，而 PNA-miRNA 杂交物是带负电荷的，这种电荷差异可以作为将杂交物与探针分离的基础。在这项原理验证工作中，我们首先通过实验证实，在没有 SSB 的运行缓冲液中，PNA-RNA 杂交物可以与过量的 PNA 探针分离，产生近 10 分钟的时间窗口，理论上可以分离多达 30 个杂交物。然后，我们将我们之前开发的用于预测杂交物迁移率的理论模型应用于 PNA-RNA 杂交物。使用修改后的理论模型进行的计算表明，略微不同长度的 PNA-RNA 杂交物可以彼此分离，而无需拖曳标签。因此，我们设计了一个简单的实验模型，能够证实：（i）不同长度的无标签杂交物的分离和（ii）由于 PNA 探针上的拖曳标签而导致相同长度的杂交物的分离。实验模型包括三个 miRNA：20nt miR-147a、20nt miR-378g 和 22nt miR-21。三个互补的 PNA 探针的长度与相应靶 miRNA 匹配。miR-147a 的探针带有一个短的五氨基酸拖曳标签；其他两个没有拖曳标签。我们能够实现这三种杂交物彼此之间的基线分离。在皮摩尔浓度的靶标浓度下，LOQ 为 14 pM，并且具有高准确性（回收率>90%）和精密度（RSD≈10%），这表明 PNA 促进的 DQAMiR 有可能支持临床样本中实用的 miRNA 分析。
