Membrane channels built from DNA

DNA 'origami' is a technique for shaping long strands of DNA into nearly any three-dimensional structure¹. With their recent publication in Science, Langecker et al.² reveal the potential of this approach for a new set of applications centered on membrane pores. The paper describes a transmembrane channel made entirely from DNA and DNA-bound cholesterol moieties. As DNA nanostructures can be readily engineered because of the relatively simple rules that govern their folding, membrane channels with customized properties could find uses as biosensors and nucleic acid or protein sequencers.

In DNA origami, a single-stranded DNA, often obtained from the M13 phage genome, is folded by hundreds of 'staples'. The staples are short, single-stranded oligonucleotides that are complementary to two or more sequences in the scaffold and can therefore crosslink distant sites. With the help of computer programs, the sequences of the staples needed to create even complex structures can be easily determined. The structures form spontaneously in a simple one-pot thermal annealing reaction when scaffold and staple DNAs are mixed.

Previously, it was thought DNA nanostructures could only be used in aqueous solutions or surfaces. “I think everybody was surprised that a highly charged molecule like DNA can be made to penetrate a lipid membrane,” says Ulrich Keyser of the University of Cambridge (UK), whose group has developed a DNA-based channel to functionalize solid-state nanopores³. The inspiration for the design of the DNA transmembrane channel came from the bacterial protein pore α-hemolysin. Similar to α-hemolysin, the structure designed by Langecker et al.² consists of a channel that spans the lipid bilayer and a barrel-shaped cap that binds to the surface of the membrane. The channel comprises five DNA helices that surround a central opening with a diameter of 2 nm. Membrane adhesion is mediated by 26 cholesterol moieties attached to the membrane-facing side of the cap.

The authors demonstrate that the channel conducts an electrical current proportional to the potential that is placed across the membrane. Interestingly, the channel shows stochastic gating—fluctuations between an open and a closed conformation—similar to what is observed in many naturally occurring protein pores. The gating dynamics are amenable to engineering, as the insertion of a small heptanucleotide at a defined position within the channel substantially increases the time spent in the off state.

Credit: Used with permission of Langecker, M. et al.².

Many different kinds of nanopores are currently under investigation for molecular detection applications, such as DNA sequencing. The data presented by Langecker et al.² hint that DNA pores might be suitable for such applications by showing that the kinetics of the unfolding of DNA hairpins passing through the pore can be monitored and that different lengths of DNA can be distinguished.

For practical uses, the properties of DNA pores will have to be fine-tuned for each specific application, but DNA nanostructures are especially easy to modify. “A key advantage of DNA-based nanostructures is the ease with which they can be engineered to improve on your basic design,” says Björn Högberg of the Karolinska Institute in Stockholm. “In addition to making defined changes to the DNA structure itself, one can place basically any functional group anywhere in the structure with high spatial precision.” Various hetero-elements, ranging from small molecules (e.g., cholesterol²) and peptides⁴ to enzymes⁵ and carbon nanotubes⁶, have been positioned at precise locations within DNA nanostructures.

In addition to in vitro applications, future uses might also be found in medical therapies. “One can imagine quite sophisticated drug delivery applications of DNA nanostructures. For the DNA channel presented here, one could think of coupling gating to the absence or presence of cellular proteins or RNAs in order to only target specific cell types,” suggests Keyser.

Although DNA nanotechnology holds promise to provide molecular machines in the future, more development is needed before it can reach its potential. “A major problem is the availability of cheap methods to synthesize sufficient amounts of customized long-scaffold DNA,” says Keyser. Engineering of heteroelements also requires optimization. As Högberg notes, “We need to improve the precision of the placement of the heteroelements; compared to what is needed to build an active site of an enzyme, for example, we are still quite imprecise.”