Membrane-Embedded Channel of Bacteriophage phi29 DNA Packaging Motor for Single Molecule Sensing and Nanomedicine A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Energy, Environmental, Biological and Medical Engineering of the College of Engineering & Applied Science April 2012 By Jia Geng Bachelor of Science, Nanjing University 2004 Committee Chair: Jing-Huei Lee, Ph.D. ii I. ABSTRACT Linear double-stranded DNA (dsDNA) viruses package its genome into a preformed procapsid fueled by the energy from ATP hydrolysis. The bacteriophage phi29 motor has a truncated cone shaped protein component, named connector, with a central channel of 3.6 nm at its narrowest part. The connector protein has been successfully inserted into an artificial lipid bilayer membrane, and the channel exhibited robust capability under various salt and pH conditions as revealed by single channel studies. This channel is suitable for extremely precise assessment of the transportation of small molecules, such as ions, DNA and RNA. There is an urgent need to development a highly sensitive detection system, for the applications in the area of pathogen detection, disease diagnosis, environmental monitoring, etc. The current challenges and limitations of these technologies are the sensitivity and accuracy issues arising from background noise and nonspecific reactions. The property of phi29 motor channel has been studied at various conditions, and was incorporated into lipid membrane. The motor channel exercised a one-way traffic property during the process of dsDNA translocation with a valve mechanism. In addition, the opening and closure of the channel also exhibit reversible and controllable. A modified version of the connector channel is founded to have a smaller channel size, which is able to detect the ssDNA and ssRNA. These findings have important implications since this artificial membrane-embedded channel would allow detailed investigations into the mechanisms of viral motor operation, as well as future applications for therapeutic molecule packaging, delivery, single molecule sensing and drug screening. iii iv ACKNOWLEDGEMENTS I would like to express my deepest gratitude and best regards to Professor Peixuan Guo, my PhD advisor at University of Cincinnati, for his support and guidance during my PhD study. His devotion to research also inspired me to explorer the frontier of science. I also greatly appreciate the Dr. Jing-Huei Lee, Ph.D. for chairing my committee during my stay at University of Kentucky. His advices and informative instructions are very indispensable to my study. It is my great honor to have Dr. Dr. Chong Ahn, Dr. Jarek Meller and Dr Marepalli Rao in my committee, and their insightful suggestions and help are greatly appreciated. The research project are also greatly supported by our supporters: Dr. Carlo Montemagno and Dr. David Wendell from UC College of Engineering for single channel recording, Dr. Jacob Schmidt from UCLA and Dr. Liqun Gu from the University of Missouri for α-haemolysin proteins; Dr. Rong Zhang from UC for Q-PCR analysis; Dr. Nicola Stonehouse from University of Leeds , UK for recombinant connectors; Dr. Jarek Meller from CCHMC and Andrew Herr from UC for connector amphiphilicity analysis; Dr. Chong Ahn and Dr. Joon Sub Shim from UC for MEMS fabrication, and Dr. Xing-Jie Liang, Dr. Jinghong Li and Dr. Haichen Wu from China for valuable discussion and suggestions. I would like to sincerely thank many previous and current members in Dr. Guo’s group for the support and help in various aspects. Dr. Feng Xiao, Dr. Taejin Lee, Dr. Oana Coban, Dr. Faqing Yuan, Dr. Wenjuan Wang, Dr. Peng Jing, Dr. Anne Vonderheide and v Dr. Jing Liu for their previous research and instructions; Current members, especially Dr. Dan Shu, Dr. Hui Zhang, Dr. Farzin Haque, Dr. Randall Reif, Dr. Zhanxi Hao, Yi Shu, Huaming Fang, Chad Schwartz, Le Zhang and Daniel Binzel for their continuing encouragement and valuable advices. I would also sincerely thank other current colleagues: Dr. Brent Hallahan, Fengmei Pi, Hui Li, Nayeem Hossain, Shaoying Wang and Zhengyi Zhao, as well as work-study students in Dr. Guo’s lab. It has been great pleasure to work in the team. I would like to acknowledge many people on the faculty and staff of the Biomedical Engineering program at University of Cincinnati, and College o Pharmacy at University of for their help, assistance in various ways during my course of studies. This work won’t be possible without the personal and practical support of numerous people. Thus my sincere gratitude goes to my parents, all my friends, and my companions for their love and support over the last few years. This work was supported by the National Institutes of Health grants R01 EB012135, R01 EB003730, GM059944, and NIH Nanomedicine Development Center: Phi29 DNA Packaging Motor for Nanomedicine through the NIH Roadmap for Medical Research to Peixuan Guo (PN2 EY 018230). vi Table of Contents page Abstract .......... .......... ......................................................................................................... iii Acknowledgements ... ......................................................................................................... v List of Tables . .......... ......................................................................................................... xii List of Figures .......... ......................................................................................................... xiii Chapter 1 GENERAL INTRODUCTION AND LITERATURE REVIEW ............................. 1 Introduction .. ......................................................................................................... 2 Structure of phi29 DNA Packaging Motor ............................................................... 3 Packaging and assembly of DNA viruses ......................................................... 3 Structural components: procapsid, scaffolding and connector .......................... 3 Nonstructural components: pRNA and packaging enzyme gp16 ...................... 7 Packaging substrate: Genomic DNA-gp3 ......................................................... 10 Fiber (gp8.5), neck and tail (gp9, gp11-12) proteins ......................................... 11 Mechanism of the phi29 DNA packaging motor ...................................................... 11 Single-molecule studies of DNA packaging motors ........................................... 11 Symmetry argument: pentamer or hexamer ...................................................... 14 ATP consumption translated to force generation by the DNA packaging motor 17 Applications of Phi29 DNA Packaging Motor .......................................................... 18 Applications of the phi29 DNA packaging motor to nanotechnology ................. 19 Applications of the phi29 connector to nanotechnology .................................... 19 Applications of the phi29 pRNA to nanotechnology........................................... 24 Perspectives .. ......................................................................................................... 29 vii Chapter 2. Reconstitution of connector into lipid bilayer for dsDNA translocation ............. 31 Introduction ......................................................................................................... 31 Materials And Methods ............................................................................................ 33 C-His tagged connector construction ................................................................ 33 FITC labeling of the connector protein .............................................................. 33 Incorporation of the connector protein into giant liposome................................ 34 Separation and filtration of the connector-containing proteoliposome .............. 34 Lipid bilayer electrophysiological measurements .............................................. 35 Insertion of the connector into lipid bylayer ....................................................... 35 Passive translocation of dsDNA driven by electric field ..................................... 36 Results and Discussion ........................................................................................... 37 Reconstitution of the connector into liposomes ................................................. 37 Connector channel insertion into planar lipid membranes ................................ 38 Conductivity of the channels ............................................................................. 38 Translocation of double-stranded DNA ............................................................. 39 Comparison of the phi29 connector channel with α-haemolysin ........................ 40 Applications ........................................................................................................ 40 Limitations ......................................................................................................... 41 Conclusion ......................................................................................................... 42 Chapter 3. Reversible and controllable gating of the phi29 connector channel ................. 46 Introduction ......................................................................................................... 47 Materials and Methods ...........................................................................................