Direct sequencing of 2′-deoxy-2′-fluoroarabinonucleic acid (FANA) using nanopore-induced phase-shift sequencing (NIPSS)
Direct sequencing of 2′-deoxy-2′-fluoroarabinonucleic acid (FANA) using nanopore-induced phase-shift sequencing (NIPSS)
复制标题
使用纳米孔诱导相移测序 (NIPSS) 对 2'-脱氧-2'-氟阿拉伯核酸 (FANA) 进行直接测序
DOI：
10.1039/c8sc05228j
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发表时间：
2019-03-14
期刊：
影响因子：
8.4
通讯作者：
Yu, Hanyang
中科院分区：
文献类型：
--
作者：
Yan, Shuanghong;Li, Xintong;Yu, Hanyang
2‘-脱氧-2’-氟阿拉伯脱氧核糖核酸(FANA)是异种核酸的一种，因其具有良好的基因沉默和催化活性，在分子医学和合成生物学领域得到了广泛的研究。尽管迫切需要FANA，但任何现有平台都无法直接对其进行排序。纳米孔测序，直接根据其物理和化学性质识别单个分子分析物，显示了直接XNA测序的前景。作为概念的证明，不同的FANA均聚物在污垢分枝杆菌孔蛋白A(MSPA)纳米孔中显示出很好的孔道堵塞信号。通过将Fana与DNA驱动链连接，利用phi29 DNA聚合酶通过纳米孔诱导相移测序(NIPSS)证明了Fana直接测序。当与Fana模板结合时，phi29 DNA聚合酶在单分子检测中显示出意想不到的逆转录酶活性。经过对该集合的进一步研究，phi29 DNA聚合酶被证明是Fana的一种先前未知的逆转录酶，在室温下工作，可能是纳米孔测序的理想选择。这些结果代表了第一次对糖修饰的XNA进行直接测序，并表明phi29 DNA聚合酶可以作为一种有希望的酶来持续对多种XNA进行测序。
2'-deoxy-2'-fluoroarabinonucleic acid (FANA), which is one type of xeno-nucleic acid ( XNA), has been intensively studied in molecular medicine and synthetic biology because of its superior gene-silencing and catalytic activities. Although urgently required, FANA cannot be directly sequenced by any existing platform. Nanopore sequencing, which identifies a single molecule analyte directly from its physical and chemical properties, shows promise for direct XNA sequencing. As a proof of concept, different FANA homopolymers show well-distinguished pore blockage signals in a Mycobacterium smegmatis porin A ( MspA) nanopore. By ligating FANA with a DNA drive-strand, direct FANA sequencing has been demonstrated using phi29 DNA polymerase by Nanopore-Induced Phase Shift Sequencing ( NIPSS). When bound with an FANA template, the phi29 DNA polymerase shows unexpected reverse transcriptase activity when monitored in a single molecule assay. Following further investigations into the ensemble, phi29 DNA polymerase is shown to be a previously unknown reverse transcriptase for FANA that operates at room temperature, and is potentially ideal for nanopore sequencing. These results represent the first direct sequencing of a sugar-modified XNA and suggest that phi29 DNA polymerase could act as a promising enzyme for sustained sequencing of a wide variety of XNAs.