Sequence-Specific Incorporation of Enzyme-Nucleotide Chimera by DNA Polymerases
Sequence-Specific Incorporation of Enzyme-Nucleotide Chimera by DNA Polymerases
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DOI：
10.1002/anie.201604641
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发表时间：
2016-08-16
影响因子：
16.6
通讯作者：
Marx, Andreas
中科院分区：
文献类型：
--
作者：
Welter, Moritz;Verga, Daniela;Marx, Andreas
DNA聚合酶根据新生核苷酸对的形状和几何形状为不断增长的多核苷酸链选择正确的核苷酸，从而确保高DNA复制选择性。高保真的DNA聚合酶被认为具有紧密的活性位点，几乎不会偏离规范结构。然而，众所周知，DNA聚合酶使用带有微小修饰的核苷酸作为底物，这是许多核心生物技术应用的关键。我们表明，即使是高保真的DNA聚合酶也能够有效地利用被辣根过氧化物酶等大蛋白修饰的核苷酸嵌合体作为模板依赖的DNA合成的底物，尽管这种“货物”比自然底物大100倍以上。我们利用这一能力开发了能够以单核苷酸分辨率肉眼检测DNA和RNA的系统。
DNA polymerases select the right nucleotide for the growing polynucleotide chain based on the shape and geometry of the nascent nucleotide pairs and thereby ensure high DNA replication selectivity. High-fidelity DNA polymerases are believed to possess tight active sites that allow little deviation from the canonical structures. However, DNA polymerases are known to use nucleotides with small modifications as substrates, which is key for numerous core biotechnology applications. We show that even high-fidelity DNA polymerases are capable of efficiently using nucleotide chimera modified with a large protein like horseradish peroxidase as substrates for template-dependent DNA synthesis, despite this "cargo" being more than 100-fold larger than the natural substrates. We exploited this capability for the development of systems that enable naked-eye detection of DNA and RNA at single nucleotide resolution.