Unpicking DNA translocation in nanopores with simultaneous single-molecule fluorescence and optical single channel recording.

Abstract

Nanopore-based DNA sequencing offers high throughput and long reads by measuring blockades in current as a strand is translocated through the pore, providing a single-molecule readout with high time resolution. However, electrical measurements alone offer only information regarding the current block itself; to improve nanopore sensing, a molecular picture of the steps of analyte binding, capture and translocation is required. Optical single channel recording of calcium flux through the nanopore enables parallel single-molecule capture events to be distinguished. We combine optical single channel recording with a single molecule fluorescence readout of DNA unzipping to correlate the steps of individual translocations and image the location of the system components. By annealing short oligomers bearing fluorophore-quencher pairs to the translocating DNA strand, we map the process of nanopore capture, threading and unzipping.