Detection of Individual Proteins Bound along DNA Using Solid-State Nanopores.
DNA in cells is heavily covered with all types of proteins that regulate its genetic activity. Detection of DNA-bound proteins is a challenge that is well suited to solid-state nanopores as they provide a linear readout of the DNA and DNA-protein volume in the pore constriction along the entire length of a molecule. Here, we demonstrate that we can realize the detection of even individual DNA-bound proteins at the single-DNA-molecule level using solid-state nanopores. We introduce and use a new model system of anti-DNA antibodies bound to lambda phage DNA. This system provides several advantages since the antibodies bind individually, tolerate high salt concentrations, and will, because of their positive charge, not translocate through the pore unless bound to the DNA. Translocation of DNA-antibody samples reveals the presence of short 12 μs current spikes within the DNA traces, with amplitudes that are about 4.5 times larger than that of dsDNA, which are associated with individual antibodies. We conclude that transient interactions between the pore and the antibodies are the primary mechanism by which bound antibodies are observed. This work provides a proof-of-concept for how nanopores could be used for future sensing applications.
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
Calin
C
Plesa
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
Justus W
JW
Ruitenberg
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
Menno J
MJ
Witteveen
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands.
Cees
C
Dekker
D000974:Antibodies, Antinuclear / Q000737:chemistry; D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D058608:Nanopores; D036103:Nanotechnology
D000974:Antibodies, Antinuclear; D004268:DNA-Binding Proteins; D004247:DNA
DNA; Protein; antibody; binding; nanopore; resolution