Single pore translocation of folded,double-stranded,and tetra-stranded DNA through channel of bacteriophage phi29 DNA packaging motor |
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| Affiliation: | 1. Nanobiotechnology Center, University of Kentucky, Lexington, KY 40536, USA;2. Markey Cancer Center, University of Kentucky, Lexington, KY 40536, USA;3. Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40536, USA;4. Department of Biostatistics, University of Kentucky, Lexington, KY 40536, USA;5. Department of Biomedical Engineering, University of Cincinnati, Cincinnati, OH 45267, USA;1. Department of Neurosurgery, State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, and West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, PR China;2. Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing, China;3. Institute of Neurosurgery, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, PR China;1. Hainan Provincial Key Laboratory of Tropical Medicine, Hainan Medical College, Haikou 571199, China;2. Oncology Institute, Fourth Affiliated Hospital of Soochow University, Wuxi 214062, China;1. Department of Cardiology and Angiology I, University Heart Center Freiburg, Germany;2. Department of Clinical Chemistry, University Hospital Freiburg, Germany;3. Department of Clinical Research, Tumor Biology Center Freiburg, Germany;4. Andreas Hettich GmbH & Co KG, Tuttlingen, Gemany;5. Vascular Biotechnology Laboratory, Baker IDI, Melbourne, Australia;6. Department of Diagnostic Radiology Medical Physics, University Hospital Freiburg, Germany;7. Department of Pharmaceutical Technology and Biopharmacy, University of Freiburg, Germany;8. Atherothrombosis and Vascular Laboratory, Baker IDI, Melbourne, Australia;1. Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians-Universität München, Marchioninistr. 15, 81377 Munich, Germany;2. Department of Physics & Center for NanoScience, Ludwig-Maximilians-Universität München, Geschwister-Scholl-Platz 1, 80539 Munich, Germany;1. Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University, Chengdu 610064, PR China;2. State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, PR China;3. State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, PR China |
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| Abstract: | The elegant architecture of the channel of bacteriophage phi29 DNA packaging motor has inspired the development of biomimetics for biophysical and nanobiomedical applications. The reengineered channel inserted into a lipid membrane exhibits robust electrophysiological properties ideal for precise sensing and fingerprinting of dsDNA at the single-molecule level. Herein, we used single channel conduction assays to quantitatively evaluate the translocation dynamics of dsDNA as a function of the length and conformation of dsDNA. We extracted the speed of dsDNA translocation from the dwell time distribution and estimated the various forces involved in the translocation process. A ∼35-fold slower speed of translocation per base-pair was observed for long dsDNA, a significant contrast to the speed of dsDNA crossing synthetic pores. It was found that the channel could translocate both dsDNA with ∼32% of channel current blockage and with ∼64% for tetra-stranded DNA (two parallel dsDNA). The calculation of both cross-sectional areas of the dsDNA and tetra-stranded DNA suggested that the blockage was purely proportional to the physical space of the channel lumen and the size of the DNA substrate. Folded dsDNA configuration was clearly reflected in their characteristic current signatures. The finding of translocation of tetra-stranded DNA with 64% blockage is in consent with the recently elucidated mechanism of viral DNA packaging via a revolution mode that requires a channel larger than the dsDNA diameter of 2 nm to provide room for viral DNA revolving without rotation. The understanding of the dynamics of dsDNA translocation in the phi29 system will enable us to design more sophisticated single pore DNA translocation devices for future applications in nanotechnology and personal medicine. |
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| Keywords: | Phi29 connector Single channel conductance Nanobiotechnology Electrophysiology DNA conformation Nanopore |
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