Tuning Polymer-Protein Interaction with Salt

Abstract

Biological nanopores are known to interact with synthetic and biological polymers, enabling their use in label-free single-molecule analytical tasks such as sequencing and/or mass discrimination. The latter, called nanopore-based single molecule mass spectrometry (Np-SMMS) has, to date, only been shown for one synthetic polymer, poly(ethyleneglycol) (PEG). It is based on the fact that binding of PEG inside the pore gives rise to blocks of ionic current, with the degree of block being detectably different for PEG molecules differing in size by only one monomer1,2.In order to extend the range of application of Np-SMMS, and to obtain information on the mechanism underying mass sensitivity of polymer-induced pore block, we have begun to test the interaction of other neutral polymers with biological nanoores. Here, we report on poly(dimethylacrylamide) (PDMA), a water-soluble neutral polymer. Under conditions used for Np-SMMS of PEG with alpha-hemolysine (4M KCl, +40 mV), we obtained current blocks with polydisperse PDMA (Mn 1500 g/mol by MALDI) that were low in frequency (<0.1 Hz/ µM) short (tau <100 µs) and noisy, resulting in little mass resolution compared to PEG. We reasoned that we might take advantage of a specific salt effect of fluoride anion reported for the aHL pore3 in order to increase blocking frequency and dwell time. Using electrolyte solutions consiting of 4M K+ as cation and various proportions of Cl- and F- as anions (1:1, 2:1, 3:1, 4:1) we were able to obtain longer events (tau up to 400 µs) and higher frequencies (up to 0.15 Hz/µM), allowing significantly better mass resolution for PDMA than in 4 M KCl. However, the large noise component in the blocked current levels for PDMA as opposed to PEG remained, still compromising the peak-to-valley ratio of histograms. These findings suggest that the specific salt effect of F- on polymer-protein interaction is independent of the polymer and may be useful in tuning polymer pore interaction for a range of analytes.(1) Robertson et al. Single-Molecule Mass Spectrometry in Solution Using a Solitary Nanopore. Proc. Natl. Acad. Sci. U S A 2007, 104, 8207-8211.(2) Baaken et al. High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore. ACS Nano 2015, 9, 6443-6449.(3) Rodrigues et al. Hofmeister Effect in Confined Spaces: Halogen Ions and Single Molecule Detection. Biophys. J. 2011, 100, 2929-2935.