Fig. 5: Real-time direct observation of sensing of an oligonucleotide unplugging strand.
From: A large size-selective DNA nanopore with sensing applications
a Schematic of three-colored microscopy setup. SUVs loaded with ATTO 655 and dTMR-40k are tethered on passivated surfaces. ATTO 488-labeled and plugged DNA nanopores are first added to solution in a flow cell. Upon insertion and pore formation, efflux of ATTO 655 through the channel-plugged nanopore happens, while the plugs restrict the translocation of the larger dTMR-40k dye polymer. Upon sensing the unplugging strand, the plug is released allowing passage for dTMR-40k dye to translocate out. b Single-particle trace of dTMR-40k- and ATTO 655-filled SUVs (green and red) docked by plugged DNA nanopore (blue). Before addition of the unplugging strand, insertion of the DNA nanopore permits only translocation of the ATTO 655 dye from the SUV. The gray arrow indicates nanopore flow in solution. After addition of the unplugging strand (black arrow and dashed line) and the subsequent actual unplugging event, the dTMR-40k will be able to translocate out. The observed increase in dTMR-40k fluorescence upon ATTO 655 flow is a result of dye dequenching and a proof of intact SUVs after insertion. All registered events are listed in Supplementary Table 5 and additional traces can be found in Supplementary Figs. 27 and 28.
