Forced translocation of DNA hairpins through a tight molecular nanopore studied by atomic force microscopy in force-spectroscopy mode

The transit of Deoxyribonucleic acid (DNA) through a molecular nanopore has been studied by pulling a beta-cyclodextrin ring over single stranded DNA using an atomic force microscope. After an exhaustive search, it was determined that n-propyl silane surface provides the most reliable background surface for performing force spectroscopy of DNA. A versatile rotaxane complex was formed between a hydrophobic molecule and cyclodextrin. DNA was then added to this complex to form a DNA rotaxane. The cyclodextrin is covalently linked by a molecular tether to the force probe, which both pulls on the cyclodextrin and reads off the forces required for passage of the DNA. The bases do not provide an obstacle to the passage of the cyclodextrin, but hairpins represent large barriers to transit, and analysis of the pulling kinetics show the opening rates of the hairpins. The utility of the system was determined by investigating the kinetics of the analogous biological system with two unusual hairpins: Unusually stable hairpins (consisting of the bases CTTG in the loop) and sequences with CTG repeats motifs. CTTG hairpins prove to be completely impassable both to reverse transcriptase and the cyclodextrin, while CTG repeats pass with an ease appropriate to their bulk measurements, but still enough force to stall a polymerase enzyme proving that the kinetic analysis is alone responsible for the stalling.