A biomimetic DNA-based channel for the ligand-controlled transport of charged molecular cargo across a biological membrane

Abstract

The re-engineering of biological protein pore scaffolds is a successful approach 12, 13 which has led, for example, to components for label-free biosensing [14][15][16][17] and portable genome sequencing 18, 19 . Creating completely new architectures with synthetic materials can offer greater design freedom and translate into more functions and applications [20][21][22][23][24][25][26] . A key challenge in the de-novo design of membrane channels is, however, to achieve an atomistically defined structure of predictable nanomechanical properties 3 because the traditional building blocks of polypeptides and organic polymers are highly flexible 2, 4 . DNA, by contrast, is known to fold into predetermined structures and is able to meet most the criteria required for creating synthetic channels [27][28][29][30][31][32][33] . Indeed, membrane-spanning DNA nanopores have been very 3" "recently built to feature a central hollow barrel which is open at both ends [5][6][7][8][9] . The barrel is composed of six hexagonally arranged, interconnected DNA duplexes that enclose a 2 nm-wide lumen with a length ranging from 17 to 42 nm. The innovative step was the inclusion of hydrophobic anchors 5, 7, 9 to insert the negatively charged pores into the hydrophobic bilayer membrane. While of novelty and considerable interest 34, 35 , the barrels do not exploit the full design flexibility offered by DNA nanotechnology and do not exhibit the higher-order functions of ion channels which can bind ligands, respond by nanomechanical opening, and select cargo for transport.We used the simple geometric shape of an open barrel as a starting point to rationally design a nanodevice that can regulate the flux of matter across a bilayer membrane.The aim of the first design step was reduce the pore height to approximate the bilayer thickness 36 and thereby avoid structural flexibility and potential leakiness 37 . A pore height of 7 nm (Fig. 1a, NP) was achieved using a six-helix-bundle architecture with six concatenated DNA strands, each of which connects two neighboring duplexes at their termini (Fig. 1b). This connectivity is drastically simpler than classical origami 38 based on cadnano software where oligonucleotides run through multiple duplexes and cause a minimum height of approx. 15 nm 5, 38 . Our design with connections at the duplex ends also avoids traditional internal cross-overs that cause structural deviations from parallel aligned duplexes 39 .The second step was to design a molecular gate that closes one barrel entrance but reopens the channel upon binding of a ligand. A origami plate has been previously used as a controllable lid for a DNA origami box 32 . But our molecular models supported by biophysical studies 37 suggest that the plate might be structurally too flexible and leaky 4" "to form a tight seal. As a solution, we designed a nanodevice that features in its closed state, NP-C (Fig. 1c), a simple "lock" strand which is bound closely to the entrance by hybridization to two docking sites. The sites are formed by th...

Biomimetic Hybrid Nanocontainers with Selective Permeability

“…The two DNA nanopores were successfully assembled as shown by agarose gel electrophoresis (Figure 2 B, left bottom panel; Figure S5). NP‐3C migrated higher than NP‐0C (Figure 2 B, lanes 2 and 1, respectively) due to hydrophobic interaction with the gel matrix which could, however, be reduced by adding detergent SDS (Figure 2 B, lanes 4 and 3) 6d…”

“…Like all rationally designed DNA nanostructures, DNA pores can be easily fabricated through the self‐assembly of component oligonucleotides. The modular construction principle has enabled customized pore diameters7 and installation of a controllable gate to regulate transport 6d. The negatively charged DNA pores carry hydrophobic membrane anchors for membrane insertion.…”

“…NP‐3C is composed of six interconnected DNA duplexes and carries at its outside perimeter three cholesterol tags for membrane insertion (Figure 1; 2D DNA map, sequences of six oligonucleotides; Figure S1 and Table S1) 6d. A second pore without cholesterol anchors, NP‐0C, served as a negative control.…”

Beyond the smiley face: applications of structural DNA nanotechnology

“…Similarly, a transmembrane molecular valve which controls gated transport of material across a phospholipid bilayer has been created [27]. The valve is created from seven linked DNA strands, and binds to a ligand [29]. Once bound, the valve opens up the channel, selectively allowing positively and negatively charged small organic molecules to pass.…”

“…Once bound, the valve opens up the channel, selectively allowing positively and negatively charged small organic molecules to pass. Another amphipathic DNA origami structure consisting of cholesterol anchors on top of a flat membrane binding interface, containing ordered arrays on the membrane due to oligonucleotide overhangs present laterally [29]. Ultimately, this structure is capable of deforming free-standing lipid membranes while showing similar biological activity to coat-forming proteins [30].…”