在多个MicroRNA（DQAMmiR）的直接定量分析中实现单核苷酸特异性,Analytical Chemistry

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在多个MicroRNA（DQAMmiR）的直接定量分析中实现单核苷酸特异性
Analytical Chemistry ( IF 6.7 ) Pub Date : 2016-01-26 00:00:00 , DOI: 10.1021/acs.analchem.5b04682
David W. Wegman ₁ , Farhad Ghasemi ₁ , Alexander S. Stasheuski ₁ , Anna Khorshidi ₂ , Burton B. Yang ₂ , Stanley K. Liu ₃ , George M. Yousef ₄ , Sergey N. Krylov ₁

Affiliation

多种miRNA（DQAMmiR）的直接定量分析利用带有荧光检测功能的CE进行多种miRNA的快速，准确和灵敏定量。在这里，我们报告实现单核苷酸特异性，从而克服了DQAMmiR成为实用miRNA分析工具的主要障碍。通常，通过将温度升高至具有错配的探针-miRNA杂交体融化而匹配保持完整的水平来达到序列特异性。该升高的温度用作杂交温度。在DQAMmiR中这种看似微不足道的方法的实际实现面临两个主要挑战。首先，所有错配的杂种的解链温度应彼此相似，并且不应达到任何匹配杂种的解链温度。第二，升高的杂交温度不应使杂种与过量探针的CE分离以及杂种彼此之间的CE分离变差。第二个问题由于DQAMmiR中的分离依赖于单链DNA结合蛋白（SSB）而变得更加复杂，该蛋白的天然结构和结合特性可能会受到高温的极大影响。这些问题通过两种方法解决。首先，将锁定核酸（LNA）碱基整合到探针中，以标准化所有靶标miRNA杂交体的解链温度，从而允许单个杂交温度。LSB碱基不影响SSB的结合。第二，开发了双温度CE，其中分离从适当杂交所需的高毛细管温度开始，并在低质量的毛细管温度下继续进行，以确保从过量探针和杂交体之间进行高质量电泳分离。所开发的方法具有足够的鲁棒性，可以通过等速电泳与样品预浓缩相结合，以实现低于10 pM的检测限。

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Achieving Single-Nucleotide Specificity in Direct Quantitative Analysis of Multiple MicroRNAs (DQAMmiR)

Direct quantitative analysis of multiple miRNAs (DQAMmiR) utilizes CE with fluorescence detection for fast, accurate, and sensitive quantitation of multiple miRNAs. Here we report on achieving single-nucleotide specificity and, thus, overcoming a principle obstacle on the way of DQAMmiR becoming a practical miRNA analysis tool. In general, sequence specificity is reached by raising the temperature to the level at which the probe-miRNA hybrids with mismatches melt while the matches remain intact. This elevated temperature is used as the hybridization temperature. Practical implementation of this apparently trivial approach in DQAMmiR has two major challenges. First, melting temperatures of all mismatched hybrids should be similar to each other and should not reach the melting temperature of any of the matched hybrids. Second, the elevated hybridization temperature should not deteriorate CE separation of the hybrids from the excess probes and the hybrids from each other. The second problem is further complicated by the reliance of separation in DQAMmiR on single-strand DNA binding protein (SSB) whose native structure and binding properties may be drastically affected by the elevated temperature. These problems were solved by two approaches. First, locked nucleic acid (LNA) bases were incorporated into the probes to normalize the melting temperatures of all target miRNA hybrids allowing for a single hybridization temperature; binding of SSB was not affected by LNA bases. Second, a dual-temperature CE was developed in which separation started with a high capillary temperature required for proper hybridization and continued at a low capillary temperature required for quality electrophoretic separation of the hybrids from excess probes and the hybrids from each other. The developed approach was sufficiently robust to allow its integration with sample preconcentration by isotachophoresis to achieve a limit of detection below 10 pM.

更新日期：2016-01-26

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