Single DNA rotaxanes of a transmembrane pore protein

Abstract

Supramolecular chemistry at the single-molecule level. An α-hemolysin transmembrane pore can be threaded in a desired orientation by DNA-PEG hybrid strands to yield functional rotaxanes. The single-molecule rotaxanes display stable and reversible two-state switching capacity depending on the applied potential, orientation, and the method of threading and capture used.

Figure 1

Schematic representation of the systems employed for transmembrane protein rotaxane. a) System employed for rotaxane formation by capture on the cis side by streptavidin and, b) thread molecule with a DNA hairpin for rotaxane formation by capture on the trans side with streptavidin.

Figure 2

Blocking events caused by thread molecules. Typical events are shown from traces recorded at +140 mV when the following thread molecules were added to the cis chamber: a) 1 μM 1, c) 1μM 1 and 10 μM streptavidin and, d) hairpin thread 1 μM 2. Events were also observed at −140 mV with thread molecules added to the trans side: b) 1μM 1, e) 1μM 1 and 10μM streptavidin, and f) 1μM 1 and 5μM complementary oligonucleotide strand. g) Rotaxane formation at −120/+120 mV transmembrane potential with 1μM 1 and 5μM complementary oligonucleotide strand on the trans side and 10 μM streptavidin on the cis side. Traces were recorded in symmetric KCl 500mM, MOPS 5mM pH 7.5, bessel filtered at 5 kHz and sampled at 6 μs (a), and 100 μs (b-f) and bessel filtered at 2 kHz and sampled at 200 μs (g).

Figure 3

a) Current versus voltage relationships obtained under asymmetric conditions (300 and 500 mM KCl in the cis and trans chambers, respectively, both buffered with 5mM MOPS to pH 7.5) for α-HL (•),α-HL threaded with the streptavidin complex of 1 added to the trans side (▪) and thread 1 hybridized with the complementary oligonucleotide added to the trans side (○). b) Current versus voltage representations for α-HL (•),α-HL rotaxane with thread 1 (○) and rotaxane with strand 2 (□) in KCl 500mM, MOPS 5mM pH 7.5.