Abstract
Biological pores have been used to study the transport of DNA and other molecules, but most pores have channels that allow only the movement of small molecules and single-stranded DNA and RNA. The bacteriophage phi29 DNA-packaging motor, which allows double-stranded DNA to enter the virus during maturation and exit during an infection, contains a connector protein with a channel that is between 3.6 and 6 nm wide. Here we show that a modified version of this connector protein, when reconstituted into liposomes and inserted into planar lipid bilayers, allows the translocation of double-stranded DNA. The measured conductance of a single connector channel was 4.8 nS in 1 M KCl. This engineered and membrane-adapted phage connector is expected to have applications in microelectromechanical sensing, microreactors, gene delivery, drug loading and DNA sequencing.
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Acknowledgements
We thank R. Zhang for Q-PCR analysis, J. Schmidt and L. Gu for α-haemolysin proteins; F. Haque for BLM experiments; Y. Shu for DNA preparation, C. Brokamp for advice on BLM, N. Stonehouse, Y. Cai and F. Xiao for recombinant connectors; A. Butti for fluorescence recovery after photobleaching, J. Meller and A. Herr for connector amphiphilicity analysis; A. Vonderheide for manuscript modification. The research was supported by NIH Nanomedicine Development Center: Phi29 DNA Packaging Motor for Nanomedicine, through the NIH Roadmap for Medical Research (PN2 EY 018230) (P.G.) and NIH Grant GM59944 (P.G.). P.G. is a co-founder of Kylin Therapeutics.
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P.G. designed and led the project in collaboration with C.M. in single pore measurements. V.S. (when she was a postdoctoral researcher with P.G. at Purdue University), J.G. and P.J. developed protocols for incorporating the connector into the membrane of liposome for image characterization and BLM experiments. P.J contributed the data for conductance measurements and Q-PCR. P.J. and J.G. contributed the data for the dsDNA translocation experiment. P.J. and J.G. performed all the data analysis. D.W. provided fusion procedure and technical training for BLM experiment. T.L. contributed materials and participated in PCR analysis. P.G. prepared the first draft of the manuscript with the input of C.M. P.J., J.G., D.W. and T.L. assisted with the manuscript revision.
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Wendell, D., Jing, P., Geng, J. et al. Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores. Nature Nanotech 4, 765–772 (2009). https://doi.org/10.1038/nnano.2009.259
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DOI: https://doi.org/10.1038/nnano.2009.259
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