Specific protein detection using designed DNA carriers and nanopores.

Nanopores are a versatile technique for the detection and characterization of single molecules in solution. An ongoing challenge in the field is to find methods to selectively detect specific biomolecules. In this work we describe a new technique for sensing specific proteins using unmodified solid-state nanopores. We engineered a double strand of DNA by hybridizing nearly two hundred oligonucleotides to a linearized version of the m13mp18 virus genome. This engineered double strand, which we call a DNA carrier, allows positioning of protein binding sites at nanometer accurate intervals along its contour via DNA conjugation chemistry. We measure the ionic current signal of translocating DNA carriers as a function of the number of binding sites and show detection down to the single protein level. Furthermore, we use DNA carriers to develop an assay for identifying a single protein species within a protein mixture.We thank Vivek Thacker and Nadanai Laohakunakorn for critical reading of this manuscript. N.A.W.B. was supported by an EPSRC Doctoral Prize Award. U.F.K. acknowledges support by an ERC starting grant, PassMembrane 261101.This is the final published version. It first appeared at http://pubs.acs.org/doi/abs/10.1021/ja512521w

Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores.

The simultaneous detection of a large number of different analytes is important in bionanotechnology research and in diagnostic applications. Nanopore sensing is an attractive method in this regard as the approach can be integrated into small, portable device architectures, and there is significant potential for detecting multiple sub-populations in a sample. Here, we show that highly multiplexed sensing of single molecules can be achieved with solid-state nanopores by using digitally encoded DNA nanostructures. Based on the principles of DNA origami, we designed a library of DNA nanostructures in which each member contains a unique barcode; each bit in the barcode is signalled by the presence or absence of multiple DNA dumbbell hairpins. We show that a 3-bit barcode can be assigned with 94% accuracy by electrophoretically driving the DNA structures through a solid-state nanopore. Select members of the library were then functionalized to detect a single, specific antibody through antigen presentation at designed positions on the DNA. This allows us to simultaneously detect four different antibodies of the same isotype at nanomolar concentration levels.N.A.W.B. and U.F.K. acknowledge funding from an ERC starting grant (Passmembrane 261101) and an ERC consolidator grant (Designerpores 647144). N.A.W.B. also acknowledges funding from an EPSRC doctoral prize award.This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/nnano.2016.5

Particle transport across a channel via an oscillating potential

Membrane protein transporters alternate their substrate-binding sites between the extracellular and cytosolic side of the membrane according to the alternating access mechanism. Inspired by this intriguing mechanism devised by nature, we study particle transport through a channel coupled with an energy well that oscillates its position between the two entrances of the channel. We optimize particle transport across the channel by adjusting the oscillation frequency. At the optimal oscillation frequency, the translocation rate through the channel is a hundred times higher with respect to free diffusion across the channel. Our findings reveal the effect of time dependent potentials on particle transport across a channel and will be relevant for membrane transport and microfluidics application

Nanopores formed by DNA origami: a review.

Nanopores have emerged over the past two decades to become an important technique in single molecule experimental physics and biomolecule sensing. Recently DNA nanotechnology, in particular DNA origami, has been used for the formation of nanopores in insulating materials. DNA origami is a very attractive technique for the formation of nanopores since it enables the construction of 3D shapes with precise control over geometry and surface functionality. DNA origami has been applied to nanopore research by forming hybrid architectures with solid state nanopores and by direct insertion into lipid bilayers. This review discusses recent experimental work in this area and provides an outlook for future avenues and challenges.N.A.W.B. acknowledges funding from the EPSRC NanoDTC program and an EPSRC doctoral prize award, U.F.K. acknowledges funding from an ERC starting Grant.This is the accepted manuscript of a paper published in FEBS Letters (Bell NAW, Keyser UF, FEBS Letters 2014, 588, 3564–3570, doi:10.1016/j.febslet.2014.06.013)

Dependence of norfloxacin diffusion across bilayers on lipid composition.

Antibiotic resistance is a growing concern in medicine and raises the need to develop and design new drug molecules that can efficiently inhibit bacterial replication. Spurring the passive uptake of the drug molecules is an obvious solution. However our limited understanding of drug-membrane interactions due to the presence of an overwhelming variety of lipids constituting cellular membranes and the lack of facile tools to probe the bio-physical interactions between drugs and lipids imposes a major challenge towards developing new drug molecules that can enter the cell via passive diffusion. Here, we used a label-free micro-fluidic platform combined with giant unilamellar lipid vesicles to investigate the permeability of membranes containing mixtures of DOPE and DOPG in DOPC, leading to a label-free measurement of passive membrane-permeability of autofluorescent antibiotics. A fluoroquinolone drug, norfloxacin was used as a case study. Our results indicate that the diffusion of norfloxacin is strongly dependent on the lipid composition which is not expected from the traditional octanol-lipid partition co-efficient assay. The anionic lipid, DOPG, slows the diffusion process whereas the diffusion across liposomes containing DOPE increases with higher DOPE concentration. Our findings emphasise the need to investigate drug-membrane interactions with focus on the specificity of drugs to lipids for efficient drug delivery, drug encapsulation and targeted drug-delivery.SP and UFK acknowledge funding from an ERC starting grant, Passmembrane 261101 and an EPSRC grant GRAPHTED, EP/ K016636/1, and JC acknowledges the support from an Internal Graduate Studentship, Trinity College, Cambridge and a Research Studentship from the Cambridge Philosophical Society.This is the final version of the article. It first appeared from the Royal Society of Chemistry via http://dx.doi.org/10.1039/C5SM02371

A Surfactant Enables Efficient Membrane Spanning by Non-Aggregating DNA-Based Ion Channels.

Funder: Friedrich Naumann FoundationFunder: Cambridge Philosophical SocietyFunder: Winton Programme for the Physics of SustainabilityFunder: Jane Bourque-Driscoll FundDNA nanotechnology makes use of hydrophobically modified constructs to create synthetic membrane protein mimics. However, nucleic acid structures exhibit poor insertion efficiency, leading to a low activity of membrane-spanning DNA protein mimics. It is suggested that non-ionic surfactants improve insertion efficiency, partly by disrupting hydrophobicity-mediated clusters. Here, we employed confocal microscopy and single-molecule transmembrane current measurements to assess the effects of the non-ionic surfactant octylpolyoxyethylene (oPOE) on the clustering behavior and membrane activity of cholesterol-modified DNA nanostructures. Our findings uncover the role of aggregation in preventing bilayer interactions of hydrophobically decorated constructs, and we highlight that premixing DNA structures with the surfactant does not disrupt the cholesterol-mediated aggregates. However, we observed the surfactant's strong insertion-facilitating effect, particularly when introduced to the sample separately from DNA. Critically, we report a highly efficient membrane-spanning DNA construct from combining a non-aggregating design with the addition of the oPOE surfactant.DM acknowledges funding from the Winton Programme for the Physics of Sustainability and the Engineering and Physical Sciences Research Council (EPSRC, project ref. 1948702). MS acknowledges funding from the Friedrich Naumann Foundation, the Jane Bourque-Driscoll Fund and the Cambridge Philosophical Society. UFK acknowledges the ERC Consolidator Grant (De-signer-Pores 647144)