The effects of an AC field on the transport of peptides and other molecules through the α-Hemolysin pore

Abstract

This research is based on superimposing an alternating current (AC) onto a direct current (DC) during the nanopore analysis of various peptides, single-stranded DNA, α-synuclein (AS) protein, and AS-drug complexes. Standard nanopore analysis consists of a constant DC voltage used to electrophoretically drive small molecules towards a pore. The superposition of the AC voltage at constant amplitude and different frequencies causes small molecules to oscillate as they approach the pore vestibule which will lead to alterations of the event parameters, the blockade current and blockade time. The DC voltage was set at 100 mV. It was found that a 200 mV AC voltage imposed over the DC voltage at frequencies from 10 MHz – 1 GHz was optimal to conduct the experiments. Initially, four α-helical peptides with a Fmoc-capping group on the N-terminus with the general formula Fmoc-DDAxKK, were studied since their DC behaviour is well known, e.g. Fmoc-DDA10KK (A10), Fmoc-DDA14KK (A14), Fmoc-DDA18KK (A18) and retro-inverse Fmoc-KKA10DD (RI-A10). These peptides are of different length and consequently different dipole moments, which is hypothesised to alter their mobility in the AC field. It was shown that the ratio of translocations to bumping events could be manipulated by a combination of AC voltage and frequencies. In particular, A10 could be studied without interference from RI-A10. Similarly, a large intrinsically disordered protein of 140 amino acids, AS, which translocates the pore readily in a DC field could be prevented from doing so by application of an AC field of 200 mV at 100 MHz. Additionally, three fragments of AS, C-terminal, N-terminal and the ΔNAC, were studied under the same AC conditions. After an AC field was applied, the C- and N-terminal were prevented from entering the pore due to their charge. Conversely, ΔNAC behaved as expected for a peptide with a negative C-terminus and/or with a large dipole moment, and the ratio of bumping events to translocation events, increased with an increase of the AC frequency. Finally, nanopore analysis with an AC field was useful to probe the conformational states of AS which are induced by drugs. Seven drugs were chosen to be studied in an AC field since their DC blockade histograms and binding properties are known. Five neuroprotective drugs (S-1-aminoindan, 3-methoxytyramine, caffeine, (-)-nicotine, metformin) and two neurotoxic drugs (cocaine and (+)-amphetamine) were studied. Surprisingly, the neuroprotective AS-drug complexes, under the effect of the AC field, showed signs of drug stripping.