Choice of fluorophore affects dynamic DNA nanostructures

The ability to dynamically remodel DNA origami structures or functional nanodevices is highly desired in the field of DNA nanotechnology. Concomitantly, the use of fluorophores to track and validate the dynamics of such DNA-based architectures is commonplace and often unavoidable. It is therefore crucial to be aware of the side effects of popular fluorophores, which are often exchanged without considering the potential impact on the system. Here, we show that the choice of fluorophore can strongly affect the reconfiguration of DNA nanostructures. To this end, we encapsulate a triple-stranded DNA (tsDNA) into water-in-oil compartments and functionalize their periphery with a single-stranded DNA handle (ssDNA). Thus, the tsDNA can bind and unbind from the periphery by reversible opening of the triplex and subsequent strand displacement. Using a combination of experiments, molecular dynamics (MD) simulations, and reaction-diffusion modelling, we demonstrate for 12 different fluorophore combinations that it is possible to alter or even inhibit the DNA nanostructure formation—without changing the DNA sequence. Besides its immediate importance for the design of pH-responsive switches and fluorophore labelling, our work presents a strategy to precisely tune the energy landscape of dynamic DNA nanodevices

"Kein Alkohol ist auch keine Lösung"

Aus den Alltagsgeschichten ehemaliger Grundwehrdiener beim österreichischen Bundesheer, in denen Alkohol meist eine zentrale Rolle spielt, entsprang das Interesse über das Trinkverhalten von österreichischen Präsenzdienern zu forschen. Im deutschsprachigen Raum existieren nur sehr wenige, quantitative Forschungen zum Alkoholkonsum von Wehrdienern und Soldaten sowie verwandten Themen. Die vorliegende Arbeit hatte deshalb zum Ziel die Thematik mit qualitativer Methodik zu untersuchen. Im Vordergrund stand somit nicht allgemein gültige Ergebnisse zu erhalten, sondern die Analyse von Einzelfällen. Zum einen sollte im Rahmen der Forschung herausgefunden werden, welche Veränderungen im Alkoholkonsum von männlichen Rekruten ab Eintritt in den Präsenzdienst beim österreichischen Bundesheer festzustellen sind, und zum anderen, welche Motive diesen Veränderungen im Trinkverhalten aus Sicht ehemaliger Grundwehrdiener zugrunde liegen. Zur Beantwortung dieser Fragestellungen wurden qualitative Interviews mit elf 19- bis 21-jährigen Männern geführt, bei denen die Absolvierung des Präsenzdienstes in Österreich nicht länger als ein Jahr zurückliegt. In Bezug auf die Schulbildung handelt es sich bei den Befragten um eine homogene Gruppe, alle 11 ehemaligen Grundwehrdiener weisen die Matura als höchsten Schulabschluss auf. Hinsichtlich Waffengattungen und Kasernenstandorten während des Grundwehrdienstes unterscheiden sich die interviewten Männer allerdings stark. Die Analyse der Interviews mit ehemaligen Präsenzdienern zeigte, dass ab dem Eintritt in das österreichische Bundesheer Veränderungen im Trinkverhalten der befragten jungen Männer verzeichnet werden können. Von einem Anstieg des Alkoholkonsums berichteten allerdings nur 3 der 11 Interviewten. Zu einer Herausbildung spezieller Konsummuster kommt es den Gesprächen zufolge vor allem in zwei Phasen des Wehrdienstes, in den ersten Wochen, der Grundausbildung, und in den letzten Wochen vor der Abrüstung. In der Grundausbildung wird nach Aussagen der Interviewpartner entweder sehr viel oder sehr wenig Alkohol konsumiert, wohingegen der Alkoholkonsum in der Abrüstungszeit bei fast allen Grundwehrdienern ansteigt. Die von den befragten ehemaligen Grundwehrdienern angegeben Gründe für die Zu- bzw. Abnahme des Alkoholkonsums beim Heer sind vielfältig. Das Konsumverhalten hängt zum einen stark mit der Totalität der Institution Bundesheer zusammen, welche die Rekruten vornehmlich in der Grundausbildung zu spüren bekommen. Als Beispiele seien hier psychischer Stress, körperliche Anstrengung, Schlafmangel und Langeweile genannt, die sich sowohl hemmend als auch fördernd auf den Alkoholkonsum auswirken können. Aber auch andere Aspekte, die mit traditionellen Vorstellungen von Männlichkeit beim Bundesheer zusammenhängen, beeinflussen das Trinkverhalten von Rekruten nach Erfahrungsberichten der interviewten ehemaligen Präsenzdiener. Das Bedürfnis Männlichkeitsritualen gerecht zu werden, um in der Männergesellschaft Bundesheer Anerkennung zu finden, kann in der Gruppe zu erhöhtem Alkoholkonsum führen

Nondeterministic self-assembly with asymmetric interactions.

We investigate general properties of nondeterministic self-assembly with asymmetric interactions, using a computational model and DNA tile assembly experiments. By contrasting symmetric and asymmetric interactions we show that the latter can lead to self-limiting cluster growth. Furthermore, by adjusting the relative abundance of self-assembly particles in a two-particle mixture, we are able to tune the final sizes of these clusters. We show that this is a fundamental property of asymmetric interactions, which has potential applications in bioengineering, and provides insights into the study of diseases caused by protein aggregation.Winton Programme for the Physics of Sustainability, Gates Cambridge, Oppenheimer PhD studentship, NanoDTC Cambridge (Grant ID: EP/L015978/1), Engineering and Physical Sciences Research Council (Grant ID: EP/L504920/1), Royal SocietyThis is the author accepted manuscript. The final version is available from the American Physical Society via http://dx.doi.org/10.1103/PhysRevE.94.02240

Two-Photon 3D Laser Printing Inside Synthetic Cells

Toward the ambitious goal of manufacturing synthetic cells from the bottom up, various cellular components have already been reconstituted inside lipid vesicles. However, the deterministic positioning of these components inside the compartment has remained elusive. Here, by using two-photon 3D laser printing, 2D and 3D hydrogel architectures are manufactured with high precision and nearly arbitrary shape inside preformed giant unilamellar lipid vesicles (GUVs). The required water-soluble photoresist is brought into the GUVs by diffusion in a single mixing step. Crucially, femtosecond two-photon printing inside the compartment does not destroy the GUVs. Beyond this proof-of-principle demonstration, early functional architectures are realized. In particular, a transmembrane structure acting as a pore is 3D printed, thereby allowing for the transport of biological cargo, including DNA, into the synthetic compartment. These experiments show that two-photon 3D laser microprinting can be an important addition to the existing toolbox of synthetic biology

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]

A DNA Segregation Module for Synthetic Cells

The bottom-up construction of an artificial cell requires the realization of synthetic cell division. Significant progress has been made toward reliable compartment division, yet mechanisms to segregate the DNA-encoded informational content are still in their infancy. Herein, droplets of DNA Y-motifs are formed by liquid–liquid phase separation. DNA droplet segregation is obtained by cleaving the linking component between two populations of DNA Y-motifs. In addition to enzymatic cleavage, photolabile sites are introduced for spatio-temporally controlled DNA segregation in bulk as well as in cell-sized water-in-oil droplets and giant unilamellar lipid vesicles (GUVs). Notably, the segregation process is slower in confinement than in bulk. The ionic strength of the solution and the nucleobase sequences are employed to regulate the segregation dynamics. The experimental results are corroborated in a lattice-based theoretical model which mimics the interactions between the DNA Y-motif populations. Altogether, engineered DNA droplets, reconstituted in GUVs, can represent a strategy toward a DNA segregation module within bottom-up assembled synthetic cells

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

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