Incorporation of a viral DNA-packaging motor channel in lipid bilayers for real-time, single-molecule sensing of chemicals and double-stranded DNA.
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.
College of Pharmacy, University of Kentucky, Lexington, Kentucky, USA.
Farzin
F
Haque
Jia
J
Geng
Carlo
C
Montemagno
Peixuan
P
Guo
D017103:Bacillus Phages / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000302:isolation & purification*; D042003:DNA Packaging / Q000502:physiology*; D008051:Lipid Bilayers / Q000378:metabolism*; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D058608:Nanopores; D036103:Nanotechnology / Q000379:methods*
D008051:Lipid Bilayers; D020409:Molecular Motor Proteins; D004247:DNA