Detection of Individual Proteins Bound along DNA Using Solid-State Nanopores

Abstract

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.

Direct observation of DNA knots using a solid-state nanopore

“…Additionally, the knot translocation duration can be used to estimate the knot size, which is observed to be less than 100 nm for the majority of knots, although a further understanding of the translocation process is required to accurately relate this to the equilibrium knot size. From a nanopore applications perspective, efforts to sequence or detect DNA-bound proteins with nanopores will have to take into account the presence and effects of these knots 46 . These results present a major step towards the ability to directly interrogate polymer knots and thus increase our understanding of this ubiquitous phenomenon.…”

Single-Molecule Studies of Unlabeled Full-Length p53 Protein Binding to DNA

“…as a short pulse superimposed on the main DNA translocation event), 23,31 at least beyond reasonable doubt compared to control experiments. This may be due to a lack of time resolution and the relatively large variation of observed protein positions along the DNA (see AFM results above).…”

Recent progress in dissecting molecular recognition by DNA polymerases with non-native substrates

“…In one strategy, nanopores with internally bound proteins [49] could evaluate a single enzyme over long time scales by measuring small changes in the protein’s conformation. In a second strategy, translocation of a protein-P/T complex through a nanopore could sequentially evaluate various single-molecules and permit determination of an activity distribution from single-molecule populations [50,51]. …”