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Issue 10, 2019
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Direct sequencing of 2′-deoxy-2′-fluoroarabinonucleic acid (FANA) using nanopore-induced phase-shift sequencing (NIPSS)

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Abstract

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.

Graphical abstract: Direct sequencing of 2′-deoxy-2′-fluoroarabinonucleic acid (FANA) using nanopore-induced phase-shift sequencing (NIPSS)

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Publication details

The article was received on 23 Nov 2018, accepted on 23 Jan 2019 and first published on 23 Jan 2019


Article type: Edge Article
DOI: 10.1039/C8SC05228J
Citation: Chem. Sci., 2019,10, 3110-3117
  • Open access: Creative Commons BY-NC license
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    Direct sequencing of 2′-deoxy-2′-fluoroarabinonucleic acid (FANA) using nanopore-induced phase-shift sequencing (NIPSS)

    S. Yan, X. Li, P. Zhang, Y. Wang, H. Chen, S. Huang and H. Yu, Chem. Sci., 2019, 10, 3110
    DOI: 10.1039/C8SC05228J

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