A large class of proteins and peptides form channels through lipid bilayer membranes (1
) to facilitate the transport of water, ions, or other entities through the otherwise impermeable membranes. Here, we report on a synthetic membrane channel that is constructed entirely from DNA and anchored to a lipid membrane with cholesterol side chains. The shape of our synthetic channel is inspired by the natural channel protein α-hemolysin (2
), but it differs from it in its physical properties such as charge, hydrophobicity, and size. We constructed the channel using molecular self-assembly with scaffolded DNA origami (3
) (). The channel consists of two modules: a stem that penetrates and spans a lipid membrane, and a barrel-shaped cap that adheres to the (cis)-side of the membrane. Adhesion to the lipid bilayer is mediated by 26 cholesterol moieties that are attached to the cis-facing surface of the barrel (). The stem protrudes centrally from the barrel and consists of six double-helical DNA domains that form a hollow tube. The interior of this tube acts as a transmembrane channel with a diameter of ≈ 2 nm and a length of ≈ 42 nm and runs through both stem and barrel ().
Fig. 1 Synthetic DNA membrane channels. (A) Schematic illustration of the channel formed by 54 double-helical DNA domains packed on a honeycomb lattice. Cylinders indicate double-helical DNA domains. Red: transmembrane stem. Orange strands with orange ellipsoids (more ...)
Transmission electron microscopy (TEM) images taken from purified structures () confirmed that the intended shape is realized (SOM text S1
, Figs. S1–S2
). Experiments with unilamellar lipid vesicles show that the synthetic DNA channels bind to lipid bilayer membranes ( and SOM text S2
, Figs. S4–S8
) in the desired orientation in which the cholesterol-modified face of the barrel forms tight contact with the membrane and the stem appears to protrude into the lipid bilayer (, Fig. S9
These observations suggested that the synthetic DNA channels could form membrane pores as designed. As the energetic cost for insertion of the charged DNA structure into the hydrophobic core of the lipid membrane would be prohibitively high, membrane penetration is thought to involve reorganization of the lipid bilayer around the charged stem structure, with the hydrophilic lipid headgroups oriented towards the DNA structure (cf. discussion in SOM text S3
In order to demonstrate the electrical conductivity of the resulting membrane pores, we performed single-channel electrophysiological experiments (11
) using an integrated chip-based setup (Fig. S14
). We added synthetic DNA channels at low concentrations (≈ 200 pM) to the cis side of the setup and applied voltage pulses to facilitate incorporation into the membrane (13
) (fig. S11A
). As with biological channels, successful membrane incorporation of individual synthetic DNA channels manifested itself in a stepwise increase in transmembrane current () along with an increase in electrical noise (fig. S11B
). Depending on the preparation method, we also observed incorporation of multiple DNA channels into the same membrane (fig. S13
). The synthetic DNA channels displayed an average Ohmic conductance of G = 0.87 ± 0.15 nS (I = 174 pA at 200 mV) per channel in a solution containing 1 M KCl and 2 mM MgCl2
(). A simple geometrical model (1
) predicts G = 0.78 nS for a channel with diameter 2 nm and length 42 nm (SOM text S4
) which agrees favorably with what we observe.
Fig. 2 Electrical characterization performed on painted DPhPC (1,2-diphytanoyl-sn-glycero-3-phosphocholine) bilayers on a chip-based electrophysiology setup. (A) Stepwise increase in ionic current during an incorporation event at V=200 mV. (B) Histogram of channel (more ...)
Many natural ion channels display current gating caused by switching between distinct channel conformations with different conductances (1
). The synthetic DNA channels also displayed gating behavior (, trace i–iii), which may be caused by thermal fluctuations of the structure. We hypothesized that stochastic unzipping and rezipping of short double-helical DNA domains in the channel may also contribute to the observed current gating. To test this idea we designed three channel ‘mutants’ that differed from the ‘wild type’ channel only by a single-stranded heptanucleotide protruding from the central transmembrane tube (, SOM text S6
). The mutant channels showed more pronounced gating than wildtype channels (, trace iv-vi), and they also significantly differed in their gating time statistics (, fig. S16
). Every investigated mutant channel displayed gating, while some of the wildtype channels did not show gating at all (, trace i). The transmembrane current hence depended on fine structural details of the synthetic DNA channel.
In the past, nanoscale membrane pores have shown great potential for the use as single molecule biosensors (15
), whose operation principle is based on the transient blockage of ionic current by analyte molecules. The type of sensing task that can be accomplished with such ‘nanopores’ depends on their size and their chemical structure. Nanopore sensors based on naturally occurring membrane pores provide excellent electrical properties, but altering the geometry of biological pores and introducing chemical functions through genetic engineering or chemical conjugation is challenging. By contrast, the geometry of synthetic DNA objects (23
) and their chemical properties (24
) can be tailored for custom nanopore sensing applications. Here, we used our synthetic DNA lipid membrane channel for single-molecule studies of DNA hairpin unzipping and guanine quadruplex (25
) unfolding. Single-stranded DNA is expected to fit through the 2 nm wide central pore of the DNA channel, while larger DNA secondary structures such as hairpins or quadruplexes are not (26
). In order to translocate through the DNA channel, the structures need to unzip or unfold as in similar experiments with α-hemolysin, thus providing a characteristic time delay in the current blockades that reveals the kinetics of structural transitions (26
For one set of experiments, we used a DNA hairpin with a 9 base pair long stem flanked by 50 thymidines on the 3′ end and 6 thymidines on the 5′ end (). The hairpin molecules were initially added to the cis side of a lipid membrane containing a single synthetic DNA channel that displayed a stable current baseline without gating. Application of a positive voltage bias leads to capture, unzipping, and translocation of the hairpin structures resulting in transient current blockades (). Reversal of the bias after approximately ≈ 30 minutes again led to transient current blockades, this time caused by molecules that had accumulated in the trans compartment by previous translocation through the DNA channel. The blockade amplitudes for both translocation directions were ΔIcis-trans = 11.9 ± 2.7 pA and ΔItrans-cis = 20.3 ± 4.2 pA. The blockade dwell times were distributed exponentially with a characteristic lifetime of τcis-trans = 1.5 ms and τtrans-cis = 1 ms, respectively ().
Fig. 3 DNA Translocation studies. (A) Addition of DNA hairpins (T5-HP-T50) to a DNA channel at V=200 mV results in the appearance of current blockades, indicating unzipping and translocation of hairpin molecules from cis to trans. Hairpins accumulated on the (more ...)
In another set of experiments, we added quadruplex-forming oligonucleotides with a 60 thymidine long single-stranded tail (Q-T60) to the cis side of a membrane containing a single synthetic DNA channel (). Again, we observed transient current blockades, which correspond to the capture and threading of quadruplex DNA molecules into the channel, followed by unfolding and subsequent translocation through the pore. Removal of the analyte from the cis compartment restored a stable baseline current without blockades. Subsequent addition of quadruplex DNA with a longer (dT)125
tail (Q-T125) led to larger current blockades. The average current blockades were ΔIQ-T60
= 5.6 ± 1.0 pA and ΔIQ-T125
= 15.3 ± 2.3 pA, respectively. The larger current blockade for Q-T125 as compared to Q-T60 translocations can be explained by the larger volume occupied in the channel by the longer T125
tail as compared to the T60
tail (SOM text S7
). The blockade dwell times are distributed exponentially with characteristic lifetimes of τQ-T60
= 9.7 ms and tQτQ-T125
= 8.1 ms. Thus, similar to biological pores our synthetic DNA channels can be used as sensing devices to discriminate analyte molecules by studying their translocation characteristics.
In addition to single molecule sensing, the synthetic DNA channels introduced here open up broad perspectives for applications as antimicrobial agents and interference with cellular homeostasis. More generally, we believe that fully synthetic lipid membrane channels are a crucial first step towards harnessing ion flux for driving sophisticated nanodevices that are inspired by the rich functional diversity of natural membrane machines such as ion pumps, rotary motors, and transport proteins.