Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores

Nature Nanotechnology volume 4, pages 765–772 (2009)Cite this article

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Structure of phi29 connector and DNA packaging motor.
Figure 2: Images of a giant liposome containing the connector.
Figure 3: Conductance assays confirm the insertion of the connector into bilayer lipid membranes (BLM).
Figure 4: Translocation of dsDNA through connector channels in a BLM.
Figure 5: Quantitative PCR analysis of DNA translocation events.

Similar content being viewed by others

References

  1. Black, L. W. DNA packaging in dsDNA bacteriophages. Ann. Rev. Microbiol. 43, 267–292 (1989).

    Article  CAS  Google Scholar 

  2. Guo, P. Introduction: Principles, perspectives, and potential applications in viral assembly. Semin. Virol. 5, 1–3 (1994).

    Article  CAS  Google Scholar 

  3. Guo, P. X. & Lee, T. J. Viral nanomotors for packaging of dsDNA and dsRNA. Mol. Microbiol. 64, 886–903 (2007).

    Article  CAS  Google Scholar 

  4. Rao, V. B. & Feiss, M. The bacteriophage DNA packaging motor. Annu. Rev. Genet. 42, 647–681 (2008).

    Article  CAS  Google Scholar 

  5. Guo, P., Peterson, C. & Anderson, D. Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage φ29. J. Mol. Biol. 197, 229–236 (1987).

    Article  CAS  Google Scholar 

  6. Chemla, Y. R. et al. Mechanism of force generation of a viral DNA packaging motor. Cell 122, 683–692 (2005).

    Article  CAS  Google Scholar 

  7. Hwang, Y., Catalano, C. E. & Feiss, M. Kinetic and mutational dissection of the two ATPase activities of terminase, the DNA packaging enzyme of bacteriophage lambda. Biochemistry 35, 2796–2803 (1996).

    Article  CAS  Google Scholar 

  8. Sabanayagam, C. R., Oram, M., Lakowicz, J. R. & Black, L. W. Viral DNA packaging studied by fluorescence correlation spectroscopy. Biophys. J. 93, L17–L19 (2007).

    Article  CAS  Google Scholar 

  9. Moffitt, J. R. et al. Intersubunit coordination in a homomeric ring ATPase. Nature 457, 446–450 (2009).

    Article  CAS  Google Scholar 

  10. Kochan, J., Carrascosa, J. L. & Murialdo, H. Bacteriophage lambda preconnectors: purification and structure. J. Mol. Biol. 174, 433–447 (1984).

    Article  CAS  Google Scholar 

  11. Rishovd, S., Holzenburg, A., Johansen, B. V. & Lindqvist, B. H. Bacteriophage P2 and P4 morphogenesis: structure and function of the connector. Virology 245, 11–17 (1998).

    Article  CAS  Google Scholar 

  12. Simpson, A. A. et al. Structure determination of the head-tail connector of bacteriophage phi29. Acta Cryst. D57, 1260–1269 (2001).

    CAS  Google Scholar 

  13. Guasch, A. et al. Detailed architecture of a DNA translocating machine: the high- resolution structure of the bacteriophage phi29 connector particle. J. Mol. Biol. 315, 663–676 (2002).

    Article  CAS  Google Scholar 

  14. Agirrezabala, X. et al. Structure of the connector of bacteriophage T7 at 8A resolution: structural homologies of a basic component of a DNA translocating machinery. J. Mol Biol. 347, 895–902 (2005).

    Article  CAS  Google Scholar 

  15. Bazinet, C. & King, J. The DNA translocation vertex of dsDNA bacteriophages. Ann. Rev. Microbiol. 39, 109–129 (1985).

    Article  CAS  Google Scholar 

  16. Guo, P., Grimes, S. & Anderson, D. A defined system for in vitro packaging of DNA-gp3 of the Bacillus subtilis bacteriophage phi29. Proc. Natl Acad. Sci. USA 83, 3505–3509 (1986).

    Article  CAS  Google Scholar 

  17. Guo, P., Erickson, S. & Anderson, D. A small viral RNA is required for in vitro packaging of bacteriophage phi29 DNA. Science 236, 690–694 (1987).

    Article  CAS  Google Scholar 

  18. Guo, P. et al. Inter-RNA interaction of phage phi29 pRNA to form a hexameric complex for viral DNA transportation. Mol. Cell 2, 149–155 (1998).

    Article  CAS  Google Scholar 

  19. Zhang, F. et al. Function of hexameric RNA in packaging of bacteriophage phi29 DNA in vitro. Mol. Cell 2, 141–147 (1998).

    Article  CAS  Google Scholar 

  20. Shu, D., Zhang, H., Jin, J. & Guo, P. Counting of six pRNAs of phi29 DNA-packaging motor with customized single molecule dual-view system. EMBO J. 26, 527–537 (2007).

    Article  CAS  Google Scholar 

  21. Thieffry, M., Chich, J. F., Goldschmidt, D. & Henry, J. P. Incorporation in lipid bilayers of a large conductance cationic channel from mitochondrial-membranes. EMBO J. 7, 1449–1454 (1988).

    Article  CAS  Google Scholar 

  22. Hinnah, S. C. et al. The chloroplast protein import channel Toc75: Pore properties and interaction with transit peptides. Biophys. J. 83, 899–911 (2002).

    Article  CAS  Google Scholar 

  23. Alcayaga, C., Venegas, R., Carrasco, A. & Wolff, D. Ion channels from the Bacillus subtilis plasma-membrane incorporated into planar lipid bilayers. FEBS Lett. 311, 246–250 (1992).

    Article  CAS  Google Scholar 

  24. Benz, R., Schmid, A., Nakae, T. & Vosscherperkeuter, G. H. Pore formation by LamB of Escherichia coli in lipid bilayer membranes. J. Bacteriol. 165, 978–986 (1986).

    Article  CAS  Google Scholar 

  25. Movileanu, L., Howorka, S., Braha, O. & Bayley, H. Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore. Nature Biotechnol. 18, 1091–1095 (2000).

    Article  CAS  Google Scholar 

  26. Kasianowicz, J. J., Brandin, E., Branton, D. & Deamer, D. W. Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl Acad. Sci. USA 93, 13770–13773 (1996).

    Article  CAS  Google Scholar 

  27. Vercoutere, W. et al. Rapid discrimination among individual DNA hairpin molecules at single-nucleotide resolution using an ion channel. Nat. Biotechnol. 19, 248–252 (2001).

    Article  CAS  Google Scholar 

  28. Vercoutere, W. A. et al. Discrimination among individual Watson-Crick base pairs at the termini of single DNA hairpin molecules. Nucleic Acids Res. 31, 1311–1318 (2003).

    Article  CAS  Google Scholar 

  29. Howorka, S., Cheley, S. & Bayley, H. Sequence-specific detection of individual DNA strands using engineered nanopores. Nat. Biotechnol. 19, 636–639 (2001).

    Article  CAS  Google Scholar 

  30. Song, L. et al. Structure of Staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274, 1859–1865 (1996).

    Article  CAS  Google Scholar 

  31. Butler, T. Z. et al. Single-molecule DNA detection with an engineered MspA protein nanopore. Proc. Natl Acad. Sci. USA 105, 20647–20652 (2008).

    Article  CAS  Google Scholar 

  32. Szabo, I., Tombola, F., Martinucci, S. & Zoratti, M. DNA interacts with Bacillus subtilis mechano-sensitive channels in native membrane patches. Cell Physiol. Biochem. 12, 127–134 (2002).

    Article  CAS  Google Scholar 

  33. Mobasheri, H. & Lea, E. J. Biophysics of gating phenomena in voltage-dependent OmpC mutant porin channels (R74C and R37C) of Escherichia coli outer membranes. Eur. Biophys. J. 31, 389–399 (2002).

    Article  CAS  Google Scholar 

  34. Carneiro, C. M. et al. Probing the volume changes during voltage gating of Porin 31BM channel with nonelectrolyte polymers. Biochim. Biophys. Acta 1612, 144–153 (2003).

    Article  CAS  Google Scholar 

  35. Wang, H. & Branton, D. Nanopores with a spark for single-molecule detection. Nature Biotechnol. 19, 622–623 (2001).

    Article  CAS  Google Scholar 

  36. Iqbal, S. M., Akin, D. & Bashir, R. Solid-state nanopore channels with DNA selectivity. Nature Nanotech. 2, 243–248 (2007).

    Article  CAS  Google Scholar 

  37. Cai, Y., Xiao, F. & Guo, P. N- or C-terminal alterations of motor protein gp10 of bacterial virus phi29 on procapsid assembly, pRNA binding and DNA packaging. Nanomedicine 4, 8–18 (2008).

    Article  CAS  Google Scholar 

  38. Guo, Y., Blocker, F., Xiao, F. & Guo, P. Construction and 3-D computer modeling of connector arrays with tetragonal to decagonal transition induced by pRNA of phi29 DNA-packaging motor. J. Nanosci. Nanotechnol. 5, 856–863 (2005).

    Article  CAS  Google Scholar 

  39. Ibanez, C., Garcia, J. A., Carrascosa, J. L. & Salas, M. Overproduction and purification of the connector protein of Bacillus subtilis phage φ29. Nucleic Acids Res. 12, 2351–2365 (1984).

    Article  CAS  Google Scholar 

  40. Robinson, M. A. et al. Affinity of molecular interactions in the bacteriophage phi29 DNA packaging motor. Nucleic Acids Res. 34, 2698–2709 (2006).

    Article  CAS  Google Scholar 

  41. Xiao, F. et al. Fabrication of massive sheets of single layer patterned arrays using reengineered Phi29 motor dodecamer. ACS Nano 3, 100–107 (2009).

    Article  CAS  Google Scholar 

  42. Lasic, D. D. Liposomes in Gene Delivery (CRC Press, 1997).

    Google Scholar 

  43. Gilbert, R. J. C. et al. Two structural transitions in membrane pore formation by Pneumolysin, the pore-forming toxin of Streptococcus pneumoniae. Cell 97, 647–655 (1999).

    Article  CAS  Google Scholar 

  44. Lopatin, A. N. & Nichols, C. G. [K + ] dependence of open-channel conductance in cloned inward rectifier potassium channels (IRK1, Kir2.1). Biophys. J. 71, 682–694 (1996).

    Article  CAS  Google Scholar 

  45. Szabo, I. et al. Double-stranded DNA can be translocated across a planar membrane containing purified mitochondrial porin. FASEB J. 12, 495–502 (1998).

    Article  CAS  Google Scholar 

  46. Braha, O. et al. Designed protein pores as components for biosensors. Chem. Biol. 4, 497–505 (1997).

    Article  CAS  Google Scholar 

  47. Wong, D., Jeon, T. J. & Schmidt, J. Single molecule measurements of channel proteins incorporated into biomimetic polymer membranes. Nanotechnology 17, 3710–3717 (2006).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Author notes

  1. Varuni Subramaniam

    Present address: Present address: Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, USA,

  2. David Wendell and Peng Jing: Both authors contributed equally to this work

Authors and Affiliations

  1. Department of Biomedical Engineering, College of Engineering and College of Medicine, University of Cincinnati, Cincinnati, 45267, Ohio, USA

    David Wendell, Peng Jing, Jia Geng, Varuni Subramaniam, Tae Jin Lee, Carlo Montemagno & Peixuan Guo

  2. Vontz Center Rm 1301, 3125 Eden Avenue, Cincinnati, 45267-0508, Ohio, USA

    Peixuan Guo

Authors
  1. David Wendell
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peng Jing
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jia Geng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Varuni Subramaniam
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tae Jin Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Carlo Montemagno
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Peixuan Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Peixuan Guo.

Ethics declarations

Competing interests

We are going to apply for a patent of this invention.

Supplementary information

Supplementary information

Supplementary information (PDF 13904 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nnano.2009.259

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing