Abstract
We present a novel single-molecule method for rapidly evaluating small-molecule binding to individual DNA molecules using nanopores fabricated in ultrathin silicon membranes. A measurable shift in the residual ion current through a approximately 3.5 nm pore results from threading of a dye-intercalated DNA molecule, as compared to the typical residual current of native DNA. The average level of the residual current can be used to directly quantify the fraction of bound molecules to DNA, providing a new way to determine binding isotherms. Spatial sensitivity is also demonstrated by designing a two-segment DNA molecule that contains small-molecule binding sites in one of its two segments. Translocations of such molecules exhibit two current levels upon incubation with a DNA-binding dye, caused by selectively bound dye in one of the DNA segments. Our results, as shown here with four different dyes, coincide well with bulk fluorescence measurements performed under identical conditions. The nanopore approach for "reading-out" molecular binding along a DNA molecule, combined with the miniscule amounts of DNA required and the potential for scalability using nanopore arrays, provide a novel platform for future applications in analytical drug screening.