Incorporation of a viral DNA-packaging motor channel in lipid bilayers for real-time, single-molecule sensing of chemicals and double-stranded DNA.

Farzin Haque, Jia Geng, Carlo Montemagno, Peixuan Guo

Nature protocols 2013 Feb

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Over the past decade, nanopores have rapidly emerged as stochastic biosensors. This protocol describes the cloning, expression and purification of the channel of the bacteriophage phi29 DNA-packaging nanomotor and its subsequent incorporation into lipid membranes for single-pore sensing of double-stranded DNA (dsDNA) and chemicals. The membrane-embedded phi29 nanochannel remains functional and structurally intact under a range of conditions. When ions and macromolecules translocate through this nanochannel, reliable fingerprint changes in conductance are observed. Compared with other well-studied biological pores, the phi29 nanochannel has a larger cross-sectional area, which enables the translocation of dsDNA. Furthermore, specific amino acids can be introduced by site-directed mutagenesis within the large cavity of the channel to conjugate receptors that are able to bind specific ligands or analytes for desired applications. The lipid membrane-embedded nanochannel system has immense potential nanotechnological and biomedical applications in bioreactors, environmental sensing, drug monitoring, controlled drug delivery, early disease diagnosis and high-throughput DNA sequencing. The total time required for completing one round of this protocol is around 1 month.

Citation

Farzin Haque, Jia Geng, Carlo Montemagno, Peixuan Guo. Incorporation of a viral DNA-packaging motor channel in lipid bilayers for real-time, single-molecule sensing of chemicals and double-stranded DNA. Nature protocols. 2013 Feb;8(2):373-92