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      Active DNA unwinding and transport by a membrane-adapted helicase nanopore

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          Abstract

          Nanoscale transport through nanopores and live-cell membranes plays a vital role in both key biological processes as well as biosensing and DNA sequencing. Active translocation of DNA through these nanopores usually needs enzyme assistance. Here we present a nanopore derived from truncated helicase E1 of bovine papillomavirus (BPV) with a lumen diameter of c.a. 1.3 nm. Cryogenic electron microscopy (cryo-EM) imaging and single channel recording confirm its insertion into planar lipid bilayer (BLM). The helicase nanopore in BLM allows the passive single-stranded DNA (ssDNA) transport and retains the helicase activity in vitro. Furthermore, we incorporate this helicase nanopore into the live cell membrane of HEK293T cells, and monitor the ssDNA delivery into the cell real-time at single molecule level. This type of nanopore is expected to provide an interesting tool to study the biophysics of biomotors in vitro, with potential applications in biosensing, drug delivery and real-time single cell analysis.

          Abstract

          Active translocation of DNA through nanopores usually needs enzyme assistance. Here authors present a nanopore derived from helicase E1 of bovine papillomavirus (BPV) which acts as a conductive pore embedded in lipid membrane to allow the translocation of ssDNA and unwinding of dsDNA.

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          Most cited references 42

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          In vitro toxicity evaluation of graphene oxide on A549 cells.

          Graphene and its derivatives have attracted great research interest for their potential applications in electronics, energy, materials and biomedical areas. However, little information of their toxicity and biocompatibility is available. Herein, we performed a comprehensive study on the toxicity of graphene oxide (GO) by examining the influences of GO on the morphology, viability, mortality and membrane integrity of A549 cells. The results suggest that GO does not enter A549 cell and has no obvious cytotoxicity. But GO can cause a dose-dependent oxidative stress in cell and induce a slight loss of cell viability at high concentration. These effects are dose and size related, and should be considered in the development of bio-applications of GO. Overall, GO is a pretty safe material at cellular level, which is confirmed by the favorable cell growth on GO film. © 2010 Elsevier Ireland Ltd. All rights reserved.
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            Synthetic lipid membrane channels formed by designed DNA nanostructures.

            We created nanometer-scale transmembrane channels in lipid bilayers by means of self-assembled DNA-based nanostructures. Scaffolded DNA origami was used to create a stem that penetrated and spanned a lipid membrane, as well as a barrel-shaped cap that adhered to the membrane, in part via 26 cholesterol moieties. In single-channel electrophysiological measurements, we found similarities to the response of natural ion channels, such as conductances on the order of 1 nanosiemens and channel gating. More pronounced gating was seen for mutations in which a single DNA strand of the stem protruded into the channel. Single-molecule translocation experiments show that the synthetic channels can be used to discriminate single DNA molecules.
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              Mechanism of DNA translocation in a replicative hexameric helicase.

              The E1 protein of papillomavirus is a hexameric ring helicase belonging to the AAA + family. The mechanism that couples the ATP cycle to DNA translocation has been unclear. Here we present the crystal structure of the E1 hexamer with single-stranded DNA discretely bound within the hexamer channel and nucleotides at the subunit interfaces. This structure demonstrates that only one strand of DNA passes through the hexamer channel and that the DNA-binding hairpins of each subunit form a spiral 'staircase' that sequentially tracks the oligonucleotide backbone. Consecutively grouped ATP, ADP and apo configurations correlate with the height of the hairpin, suggesting a straightforward DNA translocation mechanism. Each subunit sequentially progresses through ATP, ADP and apo states while the associated DNA-binding hairpin travels from the top staircase position to the bottom, escorting one nucleotide of single-stranded DNA through the channel. These events permute sequentially around the ring from one subunit to the next.
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                Author and article information

                Contributors
                zhangleio@mail.xjtu.edu.cn
                lugw@scu.edu.cn
                geng.jia@scu.edu.cn
                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group UK (London )
                2041-1723
                8 November 2019
                8 November 2019
                2019
                : 10
                Affiliations
                [1 ]ISNI 0000 0001 0807 1581, GRID grid.13291.38, Department of Laboratory Medicine, State Key Laboratory of Biotherapy, National Clinical Research Center for Geriatrics, West China Hospital, , Sichuan University and Collaborative Innovation Center for Biotherapy, ; Chengdu, 610041 China
                [2 ]ISNI 0000 0004 0368 8293, GRID grid.16821.3c, Institute for Personalized Medicine, State Key Laboratory of Oncogenes and Related Genes, School of Biomedical Engineering, , Shanghai Jiao Tong University, ; Shanghai, 200030 China
                [3 ]ISNI 0000 0001 0599 1243, GRID grid.43169.39, MOE Key Laboratory for Nonequilibrium Synthesis and Modulation of Condensed Matter, School of Science, , Xi’an Jiaotong University, ; Xi’an, 710049 China
                [4 ]ISNI 0000 0001 0807 1581, GRID grid.13291.38, College of Chemistry, , Sichuan University, ; Chengdu, 610041 China
                [5 ]ISNI 0000 0000 9139 560X, GRID grid.256922.8, Henan-Macquarie Uni Joint Center for Biomedical Innovation, School of Life Sciences, , Henan University, ; Kaifeng, Henan 475004 China
                [6 ]ISNI 0000 0001 0807 1581, GRID grid.13291.38, Laboratory of Anesthesia and Critical Care Medicine, Department of Anesthesiology, West China Hospital, , Sichuan University, ; Chengdu, Sichuan 610000 China
                [7 ]ISNI 0000 0001 0807 1581, GRID grid.13291.38, West China Hospital Emergency Department (WCHED), State Key Laboratory of Biotherapy, West China Hospital, , Sichuan University, ; Chengdu, Sichuan 610041 China
                Article
                13047
                10.1038/s41467-019-13047-y
                6841704
                31704937
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                Funding
                Funded by: FundRef https://doi.org/10.13039/501100001809, National Natural Science Foundation of China (National Science Foundation of China);
                Award ID: 81541133
                Award Recipient :
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                © The Author(s) 2019

                Uncategorized

                biosensors, biomaterials - proteins, single-molecule biophysics, nanopores

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