Active Transport by a Membrane Embedded Biomotor Nanopore

Abstract

Helicase plays a vital role in all living organisms to unpackage genes. Its structure and function have been studied by various methodology. Molecular transport through nanoscale channels including membrane channels, biological and artificial nanopores are of great importance in several key biological processes. Both biological and artificial nanopores have been constructed and reconstituted into planar lipid bilayer for biosensing and DNA sequencing. However, most of the DNA and molecule translocation through those pores are passive transport aided by external electrical field force. Here we report that a helicase from single-stranded DNA bacteriophage with ring shape can be reengineered and inserted into planar lipid bilayers. We designed this novel biological nanopore with the capability of active DNA transport fueled by the chemical energy from ATP hydrolysis. The membrane-embedded helicase nanopore acts as a conductive channel to allow the translocation of single-stranded DNA, while still retain the helicase activity to unwind double-stranded DNA on membrane. The helicase activity could be inhibited by removal of Mg2+ or ATP in vitro. DNA with abasic furans can be distinguished during transmembrane-unwinding process. This 1.3 nm-diameter channel can also form pores in live cell membranes. This helicase nanopore is expected to provide an interesting tool to study motor activity at single-molecule level, and have potential applications in nanotechnology and nanomedicine.