Current pulse signature of native kanamycin aptamer and its implication for molecular interactions on a single protein nanopore sensing interface

Due to the pore size limitation of single α-hemolysin (α-HL) nanopore sensing interface, ssDNA with secondary conformations can only pass through the nanopore after unzipping as linear ssDNA. For hairpin DNA, a tail with 15-50 bases was usually added to the stem terminal (5' or 3') to facilitate the capture rate and unzipping process, and the typical translocation signal behaves as a square wave with a short dip at the end of the pulse. In this work, the pulse signal of native kanamycin aptamer, a hairpin DNA without the added long tail, was investigated with the single nanopore sensing interface, and different current pulse pattern was observed. The pulse signal exhibited two precise current levels with significantly extended duration of the second, and both duration of the two levels correlate to the interaction of the aptamer to kanamycin. Moreover, the pulse signal not only reveals the selectivity of the aptamer to its target, but also sensitive to the loop sequence change of the aptamer. This work shows that a single nanopore sensing interface could be used as a unique alternative means for interaction investigation of hairpin DNA aptamer without labeling or adding the extra-long tail.