DNA Translocation through Nanopores at Physiological Ionic Strengths Requires Precise Nanoscale Engineering

Many important processes in biology involve the translocation of a biopolymer through a nanometer-scale pore. Moreover, the electrophoretic transport of DNA across nanopores is under intense investigation for single-molecule DNA sequencing and analysis. Here, we show that the precise patterning of the ClyA biological nanopore with positive charges is crucial to observe the electrophoretic translocation of DNA at physiological ionic strength. Surprisingly, the strongly electronegative 3.3 nm internal constriction of the nanopore did not require modifications. Further, DNA translocation could only be observed from the wide entry of the nanopore. Our results suggest that the engineered positive charges are important to align the DNA in order to overcome the entropic and electrostatic barriers for DNA translocation through the narrow constriction. Finally, the dependencies of nucleic acid translocations on the Debye length of the solution are consistent with a physical model where the capture of double-stranded DNA is diffusion-limited while the capture of single-stranded DNA is reaction-limited.