Probing DNA Hybridization Equilibrium by Cationic Conjugated Polymer for Highly Selective Detection and Imaging of Single-Nucleotide Mutation
Probing DNA Hybridization Equilibrium by Cationic Conjugated Polymer for Highly Selective Detection and Imaging of Single-Nucleotide Mutation
复制标题
通过阳离子共轭聚合物探测 DNA 杂交平衡，实现单核苷酸突变的高选择性检测和成像
DOI：
10.1021/acs.analchem.8b00870
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发表时间：
2018-06-05
影响因子：
7.4
通讯作者：
Su, Xin
中科院分区：
文献类型：
--
作者：
Li, Zehao;Zhou, Xu;Su, Xin
基于杂交的探针是一种很有前途的核酸靶标检测和成像工具。然而，单核苷酸的选择性仍然是具有挑战性的，因为在室温下杂交反应的特异性通常较低。利用短双链的特异性杂交和阳离子共轭聚合物(CCP)的信号放大作用，我们揭示了一种高效、简便的单核苷酸突变(SNM)高选择性检测和原位成像的方法。实现了对只有单核苷酸差异的核酸链的良好分辨，使SNM的灵敏检测丰度低至0.1%。单分子荧光共振能量转移(SmFRET)研究表明，CCP的存在在不同程度上增强了完全匹配双链和失配双链，这可以解释单核苷酸选择性高的原因。由于该探针设计简单，CCP亮度稳定，在固定细胞中实现了高选择性的mRNA原位成像。具有BRAF V600E点突变的黑色素瘤细胞株A375表现出比目前未报道有突变的肝癌细胞株HegG2更高的FRET效率。
Hybridization-based probes emerge as a promising tool for nucleic acid target detection and imaging. However, the single-nucleotide selectivity is still challenging because the specificity of hybridization reaction is typically low at room temperature. We disclose an effective and simple method for highly selective detection and in situ imaging of single-nucleotide mutation (SNM) by taking the advantages of the specific hybridization of short duplex and the signal amplifying effect of cationic conjugated polymer (CCP). Excellent discrimination of the nucleic acid strands only differing by single nucleotide was achieved enabling the sensitive detection of SNM at the abundance as low as 0.1%. Single-molecule fluorescence resonance energy transfer (smFRET) study reveals that the presence of CCP enhances the perfect matched duplex and the mismatched duplex to a different extent, which can be an explanation for the high single-nucleotide selectivity. Due to the simple design of the probe and the stable brightness of CCP, highly selective mRNA in situ imaging was achieved in fixed cells. Melanoma cell line A375 with BRAF V600E point mutation exhibits higher FRET efficiency than liver cancer cell line HegG2 that was not reported having the mutation at this point.