Journal
CHEMICAL SCIENCE
Volume 10, Issue 10, Pages 3110-3117Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/c8sc05228j
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Funding
- National Natural Science Foundation of China [21327902, 21675083, 91753108, 21708018]
- Fundamental Research Funds for the Central Universities [020514380078, 020514380120, 020514380142, 14380088, 14380134]
- State Key Laboratory of Analytical Chemistry for Life Science [5431ZZXM1707, 5431ZZXM1804]
- 1000 Plan Youth Talent Program of China
- Program for High-Level Entrepreneurial and Innovative Talents Introduction of Jiangsu Province
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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.
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