Active DNA Unwinding and Transport by a Membrane-Adapted Helicase Nanopore

Nanoscale transport through nanopore and live-cell membrane plays a vital role in both key biological processes as well as biosensing, DNA sequencing and single-cell analysis in-situ. Active translocation of DNA through these nanopores usually need enzyme assistance. Here we present a new class of nanopore derived from truncated helicase E1 of Bovine papillomavirus (BPV) with a lumen diameter of c.a. 1.3 nm, named helicase nanopore. Engineering of the pore with GST tag improves its membrane compatibility, and Cryo-Electron Microscopy (Cryo-EM) imaging of their proteoliposome vesicles reveal the interaction of both surface attachment and membrane insertion. The helicase nanopore reconstituted in planar lipid bilayer could not only act as a conductive pore to allow the translocation of ssDNA, but also retain the ability to unwind double-stranded DNA (dsDNA) in vitro. The measured conductance of the helicase nanopore was c.a. 1.34 nS in 1 M KCl, pH ,7 and the mean unwinding speed for dsDNA was 65.54 ± 48.06 bp per second under +120 mV. The helicase nanopore activity could be inhibited by the removal of Mg²⁺ or adenosine triphosphate (ATP). Furthermore, we incorporate this helicase nanopore into the live cell membrane of HEK293T, and monitor the ssDNA delivery into the cell real-time at single molecule level. This new type of nanopore is expected to provide an interesting tool to study the biophysics of biomotors in vitro, with potential applications in biosensing, drug delivery and real-time single cell analysis.