Translocation and Binding in the Recognition of Short Oligonucleotides by a Biological Nanopore

Abstract

Polymer interactions with pore-forming membrane proteins, as evidenced by the resulting block of ionic current, may be classified into two types: (1) the threading of an extended polymer chain of which only a small length contributes to the block and (2) a binding reaction, where the polymer enters the pore entirely and interacts as a particle. The former phenomenon typically occurs in dilute aqueous electrolyte solutions (e.g. KCl <1 M) and requires long and charged molecules and can allow sequencing (e.g. of DNA). In contrast, the binding reaction typically occurs with short, neutral polymer molecules, requires high salinity (e.g. KCl 3-4 M) and enables the high-resolution discrimination of polymer masses1. The aerolysin nanopore has recently been shown to strongly interact with short adenine oligonucleotides (A3-A10) and this interaction allows mass discrimination on the basis of the depth of block of ionic current induced by the binding of the analyte,2 suggesting a binding-type interaction. Using low-noise current recording, we have indentified strong dynamics of this interaction between DC and 50 kHz and indentified short visits to deeper blocked states preceding and following the principal, mass-dependent state (pre- and post-blocks). Statistical analysis indicates that the probability of post- but not pre-blocks decreases with (1) oligomer length and (2) transmembrane voltage. We interpret this finding in terms of a combined translocation and binding interaction, probably involving several binding sites for DNA in the pore. In line with this hypothesis, longer polynucleotides (A30) showed stable and long-lasting interactions with near-complete block of current.(1) Robertson et al. Proc. Natl. Acad. Sci. U S A 2007, 104, 8207-8211.(2) Cao et al. Nature Nanotechnology 2016, 1-7.