Figure 1
From: Quantifying mammalian genomic DNA hydroxymethylcytosine content using solid-state nanopores
Nanopore detection of 5 hmC modification (a) Schematic of measurement setup depicting a short biotinylated dsDNA bound by MS translocating through a SS-nanopore (left) and an example current trace for synthetic monobiotinylated 156 bp dsDNA + MS measured at 400 mV (right). Scale bars are 1 nS (vertical) and 250 ms (horizontal). (b) 5 hmC labeling scheme, consisting of glucosylation by βGT with 6-N3-Glu to form 6-azide-b-glucosyl-5 hmC and biotin functionalization by copper-free click chemistry. Right: EMSA showing biotin-labeled mono-5 hmC (lane 1) and synthetic monobiotinylated dsDNA (lane 2), both with excess MS. (c) Event rate vs. applied voltage for 156 bp mono-5 hmC dsDNA (550 nM) biotin-labeled at 5 hmC sites both with (red) and without (black) bound MS measured through a pore with diameter 8.5 nm. Solid lines are exponential fits to the data. Inset shows example current traces from each (400 mV, colors matched). Scale bars are 1 nS and 250 ms. (d) Scatter plots of mean event dwell time vs. mean conductance change collected and accompanying histograms for biotin-labeled mono-5 hmC (red, diameter 8.5 nm) and synthetic monobiotinylated dsDNA (blue, diameter 7.8 nm) measured at 400 mV. Faded regions represent events below the resolution limit.
