Enhanced translocation of single DNA molecules through alpha-hemolysin nanopores by manipulation of internal charge.
Both protein and solid-state nanopores are under intense investigation for the analysis of nucleic acids. A crucial advantage of protein nanopores is that site-directed mutagenesis permits precise tuning of their properties. Here, by augmenting the internal positive charge within the alpha-hemolysin pore and varying its distribution, we increase the frequency of translocation of a 92-nt single-stranded DNA through the pore at +120 mV by approximately 10-fold over the wild-type protein and dramatically lower the voltage threshold at which translocation occurs, e.g., by 50 mV for 1 event.s(-1).muM(-1). Further, events in which DNA enters the pore, but is not immediately translocated, are almost eliminated. These experiments provide a basis for improved nucleic acid analysis with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification.
Maglia
Giovanni
G
Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom.
Restrepo
Marcela Rincon
MR
Mikhailova
Ellina
E
Bayley
Hagan
H
D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D001692:Biological Transport; D004277:DNA, Single-Stranded / Q000737:chemistry*; D005818:Genetic Engineering; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics; D049329:Nanostructures / Q000737:chemistry*; D036103:Nanotechnology; D017354:Point Mutation; D016062:Porosity; D017510:Protein Folding; D013499:Surface Properties
D001427:Bacterial Toxins; D004277:DNA, Single-Stranded; D006460:Hemolysin Proteins; C021091:staphylococcal alpha-toxin