Employing LiCl Salt Gradient in the Wild-type α-Hemolysin Nanopore to Slow Down DNA Translocation and Detect Methylated Cytosine
In this research, we demonstrate a label-free detection, biological nanopore-based method to distinguish methylated cytosine (mC) from naked cytosine (C) in sample mixtures containing both C and mC at prolonged translocation duration. Using 15-fold increase in LiCl salt concentration going from cis to trans chamber, we increased the translocation dwell time of ssDNA by over 56-fold and the event capture rate by 68-fold in comparison with symmetric concentration of 1M1.0M KCl (control). This is a consequence of counter-ions binding and effectively lowering the overall charge ofn DNA, which in turn lessens the electrophoretic drive of the system and slows the translocation velocity. Moreover, salt gradients can create a large electric field that will funnel ions and polymers towards the pore, increasing the capture rate and translocation dwell time of DNA. As a result, in 0.2M – 3M3.0M LiCl solution, ssDNA achieved a prolonged dwell time of 52 μs/nucleotide and a capture rate of 60 ssDNA per second. Importantly, lowering the translocation speed of ssDNA enhances the resulting resolution, allowing 5’-mC to be distinguished from C without using methyl-specific labels. We successfully distinguished 5’-mC from C when mixed together at ratios at 1:1, 3:7, and 7:3. Distribution of current blockade amplitudes of all mixtures adopted bimodal shapes, with peak-to-peak ratios coarsely corresponding to the mixture composition (e.g. the density and distribution of events shifted in correspondence with changes in 18b-0mC and 18-2mC concentration ratios in the mixture).