A synthetic lipid scramblase built from DNA

The programmable self-assembly facilitated by DNA nanotechnology provides unparalleled capabilities to construct functional devices at the nanoscale. Recently, DNA nanostructures have been developed to interact with biological membranes and serve as artificial counterparts of natural ion channels, or membrane bending, scaffolding, or fusion proteins. In this thesis, we design a synthetic DNA-built enzyme that facilitates rapid lipid mixing between the two leaflets of a lipid bilayer. It thereby mimics the function of yet another class of membrane proteins $--$ lipid scramblases. Characterising this DNA nanostructure with gel electrophoresis, dynamic light scattering, atomic force and transmission electron microscopy, we find that the cholesterol groups required for membrane insertion, also induce clustering. Hence, we establish an easy-to-implement strategy to control hydrophobically mediated aggregation thereby introducing a solution to a common problem of amphiphilic DNA constructs. With a combination of fluorescence microscopy experiments and molecular dynamics simulations, we identify the mechanism behind the scrambling activity. The spontaneous membrane insertion of our DNA scramblase induces a toroidal pore that is lined by the lipid headgroups. This DNA-stabilised pore connects the inner and outer bilayer leaflet thereby facilitating diffusive lipid transport that rapidly equilibrates the membrane’s lipid composition. In good agreement between experiments and simulations, we find the scrambling rate catalysed by our DNA-made enzyme to exceed 10$^7$ lipids per second, orders of magnitude faster than natural scramblase proteins. We thereby pioneer the use of self-assembled DNA nanostructures for controlling the lipid composition of biological membranes, opening new avenues for applications of membrane-interacting DNA systems in biophysical research and medicine.Engineering and Physical Sciences Research Council (EPSRC), Cambridge Trust Vice Chancellor's Awar

Tailoring Interleaflet Lipid Transfer with a DNA-based Synthetic Enzyme.

Lipid membranes, enveloping all living systems, are of crucial importance, and control over their structure and composition is a highly desirable functionality of artificial structures. However, the rational design of protein-inspired systems is still challenging. Here we have developed a highly functional nucleic acid construct that self-assembles and inserts into membranes, enabling lipid transfer between inner and outer leaflets. By designing the structure to account for interactions between the DNA, its hydrophobic modifications, and the lipids, we successfully exerted control over the rate of interleaflet lipid transfer induced by our DNA-based enzyme. Furthermore, we can regulate the level of lipid transfer by altering the concentration of divalent ions, similar to stimuli-responsive lipid-flipping proteins

Amplified Plasmonic Forces from DNA Origami-Scaffolded Single Dyes in Nanogaps

Developing highly enhanced plasmonic nanocavities allows direct observation of light–matter interactions at the nanoscale. With DNA origami, the ability to precisely nanoposition single-quantum emitters in ultranarrow plasmonic gaps enables detailed study of their modified light emission. By developing protocols for creating nanoparticle-on-mirror constructs in which DNA nanostructures act as reliable and customizable spacers for nanoparticle binding, we reveal that the simple picture of Purcell-enhanced molecular dye emission is misleading. Instead, we show that the enhanced dipolar dye polarizability greatly amplifies optical forces acting on the facet Au atoms, leading to their rapid destabilization. Using different dyes, we find that emission spectra are dominated by inelastic (Raman) scattering from molecules and metals, instead of fluorescence, with molecular bleaching also not evident despite the large structural rearrangements. This implies that the competition between recombination pathways demands a rethink of routes to quantum optics using plasmonics

Image potential states as quantum probe of graphene interfaces

Image potential states (IPSs) are electronic states localized in front of a surface in a potential well formed by the surface projected bulk band gap on one side and the image potential barrier on the other. In the limit of a two-dimensional solid a double Rydberg series of IPSs has been predicted which is in contrast to a single series present in three-dimensional solids. Here, we confirm this prediction experimentally for mono- and bilayer graphene. The IPSs of epitaxial graphene on SiC are measured by scanning tunnelling spectroscopy and the results are compared to ab-initio band structure calculations. Despite the presence of the substrate, both calculations and experimental measurements show that the first pair of the double series of IPSs survives, and eventually evolves into a single series for graphite. Thus, IPSs provide an elegant quantum probe of the interfacial coupling in graphene systems.Comment: Accepted for publication in New Journal of Physic

Controlling aggregation of cholesterol-modified DNA nanostructures.

DNA nanotechnology allows for the design of programmable DNA-built nanodevices which controllably interact with biological membranes and even mimic the function of natural membrane proteins. Hydrophobic modifications, covalently linked to the DNA, are essential for targeted interfacing of DNA nanostructures with lipid membranes. However, these hydrophobic tags typically induce undesired aggregation eliminating structural control, the primary advantage of DNA nanotechnology. Here, we study the aggregation of cholesterol-modified DNA nanostructures using a combined approach of non-denaturing polyacrylamide gel electrophoresis, dynamic light scattering, confocal microscopy and atomistic molecular dynamics simulations. We show that the aggregation of cholesterol-tagged ssDNA is sequence-dependent, while for assembled DNA constructs, the number and position of the cholesterol tags are the dominating factors. Molecular dynamics simulations of cholesterol-modified ssDNA reveal that the nucleotides wrap around the hydrophobic moiety, shielding it from the environment. Utilizing this behavior, we demonstrate experimentally that the aggregation of cholesterol-modified DNA nanostructures can be controlled by the length of ssDNA overhangs positioned adjacent to the cholesterol. Our easy-to-implement method for tuning cholesterol-mediated aggregation allows for increased control and a closer structure-function relationship of membrane-interfacing DNA constructs - a fundamental prerequisite for employing DNA nanodevices in research and biomedicine.This work was supported by: European Research Council (ERC) consolidator grant [DesignerPores 647144 to U.F.K.]. Winton Programme for the Physics of Sustainability; Gates Cambridge; Oppenheimer Ph.D. studentship; and the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 792270 [to K.G.]. Engineering and Physical Sciences Research Council (EPSRC); and the Cambridge Trust Vice Chancellor’s Award [to A.O.]. National Science Foundation (USA) [DMR-1827346]; National Institutes of Health [P41-GM104601]; the supercomputer time provided through XSEDE allocation grant [MCA05S028]; and the Blue Waters petascale supercomputer system (UIUC) [to A.A. and H.J.]. Winton Programme for the Physics of Sustainability; and Engineering and Physical Sciences Research Council (EPSRC) [to D.S.]. The FEI Tecnai G2-Spirit was funded by the Wellcome Trust [090932/Z/09/Z]. Funding for open access charge: ERC [DesignerPores 647144]

Large-Conductance Transmembrane Porin Made from DNA Origami.

DNA nanotechnology allows for the creation of three-dimensional structures at nanometer scale. Here, we use DNA to build the largest synthetic pore in a lipid membrane to date, approaching the dimensions of the nuclear pore complex and increasing the pore-area and the conductance 10-fold compared to previous man-made channels. In our design, 19 cholesterol tags anchor a megadalton funnel-shaped DNA origami porin in a lipid bilayer membrane. Confocal imaging and ionic current recordings reveal spontaneous insertion of the DNA porin into the lipid membrane, creating a transmembrane pore of tens of nanosiemens conductance. All-atom molecular dynamics simulations characterize the conductance mechanism at the atomic level and independently confirm the DNA porins' large ionic conductance.K.G. acknowledges funding from the Winton Programme for the Physics of Sustainability, Gates Cambridge, and the Oppenheimer Ph.D. studentship; U.F.K. from an ERC Consolidator Grant (Designerpores 647144); and M.R. from the Early Postdoc Mobility fellowship of the Swiss National Science Foundation. A.A., J.Y., and C.Y.L. acknowledge support form the National Science Foundation under grants DMR-1507985, PHY-1430124, and EEC-1227034 and the supercomputer time provided through XSEDE Allocation grant MCA05S028 and the Blue Waters petascale supercomputer system (UIUC). M.W. and S.P.B. acknowledge support from Marie Skłodowska Curie Actions within the Initial Training Networks Translocation Network, project no. 607694.This is the final version of the article. It first appeared from the American Chemical Society at http://dx.doi.org/10.1021/acsnano.6b03759

A synthetic enzyme built from DNA flips 107 lipids per second in biological membranes.

Mimicking enzyme function and increasing performance of naturally evolved proteins is one of the most challenging and intriguing aims of nanoscience. Here, we employ DNA nanotechnology to design a synthetic enzyme that substantially outperforms its biological archetypes. Consisting of only eight strands, our DNA nanostructure spontaneously inserts into biological membranes by forming a toroidal pore that connects the membrane's inner and outer leaflets. The membrane insertion catalyzes spontaneous transport of lipid molecules between the bilayer leaflets, rapidly equilibrating the lipid composition. Through a combination of microscopic simulations and fluorescence microscopy we find the lipid transport rate catalyzed by the DNA nanostructure exceeds 107 molecules per second, which is three orders of magnitude higher than the rate of lipid transport catalyzed by biological enzymes. Furthermore, we show that our DNA-based enzyme can control the composition of human cell membranes, which opens new avenues for applications of membrane-interacting DNA systems in medicine

Deoxyribonucleic Acid Encoded and Size-Defined π-Stacking of Perylene Diimides

Natural photosystems use protein scaffolds to control intermolecular interactions that enable exciton flow, charge generation, and long-range charge separation. In contrast, there is limited structural control in current organic electronic devices such as OLEDs and solar cells. We report here the DNA-encoded assembly of π-conjugated perylene diimides (PDIs) with deterministic control over the number of electronically coupled molecules. The PDIs are integrated within DNA chains using phosphoramidite coupling chemistry, allowing selection of the DNA sequence to either side, and specification of intermolecular DNA hybridization. In this way, we have developed a “toolbox” for construction of any stacking sequence of these semiconducting molecules. We have discovered that we need to use a full hierarchy of interactions: DNA guides the semiconductors into specified close proximity, hydrophobic–hydrophilic differentiation drives aggregation of the semiconductor moieties, and local geometry and electrostatic interactions define intermolecular positioning. As a result, the PDIs pack to give substantial intermolecular π wave function overlap, leading to an evolution of singlet excited states from localized excitons in the PDI monomer to excimers with wave functions delocalized over all five PDIs in the pentamer. This is accompanied by a change in the dominant triplet forming mechanism from localized spin–orbit charge transfer mediated intersystem crossing for the monomer toward a delocalized excimer process for the pentamer. Our modular DNA-based assembly reveals real opportunities for the rapid development of bespoke semiconductor architectures with molecule-by-molecule precision

Ion Channels Made from a Single Membrane-Spanning DNA Duplex.

Because of their hollow interior, transmembrane channels are capable of opening up pathways for ions across lipid membranes of living cells. Here, we demonstrate ion conduction induced by a single DNA duplex that lacks a hollow central channel. Decorated with six porpyrin-tags, our duplex is designed to span lipid membranes. Combining electrophysiology measurements with all-atom molecular dynamics simulations, we elucidate the microscopic conductance pathway. Ions flow at the DNA-lipid interface as the lipid head groups tilt toward the amphiphilic duplex forming a toroidal pore filled with water and ions. Ionic current traces produced by the DNA-lipid channel show well-defined insertion steps, closures, and gating similar to those observed for traditional protein channels or synthetic pores. Ionic conductances obtained through simulations and experiments are in excellent quantitative agreement. The conductance mechanism realized here with the smallest possible DNA-based ion channel offers a route to design a new class of synthetic ion channels with maximum simplicity.K.G. acknowledges funding from the Winton Programme for the Physics of Sustainability, Gates Cambridge, and the Oppenheimer Ph.D. studentship, U.F.K. from an ERC starting Grant Passmembrane 261101 and Oxford Nanopore Technologies, and M.R. from the Early Postdoc Mobility fellowship of the Swiss National Science Foundation. A.A., J.Y., and C.Y.L. acknowledge support form the National Science Foundation under Grants DMR-1507985, PHY-1430124, and EEC-1227034 and the supercomputer time provided through XSEDE Allocation Grant MCA05S028 and the Blue Waters petascale supercomputer system (UIUC). M.W. and S.P.B. acknowledge support from Marie Skłodowska Curie Actions within the Initial Training Networks Translocation Network, project no. 607694 and I.M. from the Marie Skłodowska Curie Fellowship “Nano-DNA” (FP7-PEOPLE-2012-IEF, No 331952).This is the final version of the article. It first appeared from ACS at http://dx.doi.org/10.1021/acs.nanolett.6b02039

Laser-Supported CD133+ Cell Therapy in Patients with Ischemic Cardiomyopathy: Initial Results from a Prospective Phase I Multicenter Trial

Objectives: This study evaluates the safety, principal feasibility and restoration potential of laser-supported CD133+ intramyocardial cell transplantation in patients with ischemic cardiomyopathy. Methods: Forty-two patients with severe ischemic cardiomyopathy (left ventricular ejection fraction (LVEF) >15% and 3 myocardial segments) determined in the preoperative MRI was inversely correlated with a LVEF increase after laser-supported cell therapy (p = 0.024). Conclusions: This multicenter trial demonstrates that laser-supported CD133+ cell transplantation is safe and feasible in patients with ischemic cardiomyopathy undergoing CABG, and in most cases, it appears to significantly improve the myocardial function. Importantly, our data show that the beneficial effect was significantly related to the extent of transmural delayed enhancement, suggesting that MRI-guided selection of patients is mandatory to ensure the effectiveness of the therapy. Trial Registration: EudraCT 2005-004051-35) Controlled-Trials.com ISRCTN4999863