Characterization of lipid composition and diffusivity in OLA generated vesicles.

Giant Unilamellar Vesicles (GUVs) are a versatile tool in many branches of science, including biophysics and synthetic biology. Octanol-Assisted Liposome Assembly (OLA), a recently developed microfluidic technique enables the production and testing of GUVs within a single device under highly controlled experimental conditions. It is therefore gaining significant interest as a platform for use in drug discovery, the production of artificial cells and more generally for controlled studies of the properties of lipid membranes. In this work, we expand the capabilities of the OLA technique by forming GUVs of tunable binary lipid mixtures of DOPC, DOPG and DOPE. Using fluorescence recovery after photobleaching we investigated the lateral diffusion coefficients of lipids in OLA liposomes and found the expected values in the range of 1 μm2/s for the lipid systems tested. We studied the OLA derived GUVs under a range of conditions and compared the results with electroformed vesicles. Overall, we found the lateral diffusion coefficients of lipids in vesicles obtained with OLA to be quantitatively similar to those in vesicles obtained via traditional electroformation. Our results provide a quantitative biophysical validation of the quality of OLA derived GUVs, which will facilitate the wider use of this versatile platform.Friedrich Naumann Foundation; Jane Bourque-Driscoll Fund; Cambridge Philosophical Society; Winton Programme for the Physics of Sustainability; Engineering and Physical Sciences Research Council; Wellcome Trust; ER

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

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]

Da fotografia como arte à arte como fotografia: a experiência do Museu de Arte Contemporânea da USP na década de 1970

Este ensaio visa sistematizar os primeiros resultados de uma pesquisa, ainda em curso, sobre o processo de legitimação da fotografia pelo sistema de arte no Brasil, cujo foco principal é o museu. Os museus de arte da cidade de São Paulo foram escolhidos para dar início a essa investigação. Primeiramente, será abordada, em linhas gerais, a presença da fotografia no Museu de Arte Moderna de São Paulo e na Bienal de São Paulo, dada a vinculação de origem do Museu de Arte Contemporânea com essas duas instituições paulistanas. Na seqüência será analisada a formação do acervo fotográfico do Museu de Arte Contemporânea da Universidade de São Paulo durante a década de 1970. Por fim, esse percurso permitirá observar que a atuação de Walter Zanini, o primeiro diretor do Museu, e as particularidades da posição do MAC-USP no sistema de arte no Brasil naquele período resultaram no entendimento da fotografia prioritariamente no âmbito da arte contemporânea de caráter experimental e não como obra de arte autônoma, segundo os princípios da chamada fotografia artística.This article presents the first findings of a research still under development about the process of legitimation of photography as a kind of art by the artistic scene in Brazil. The art museums of the city of São Paulo were chosen for starting that research. Initially, we will be investigating the presence of photography at the Contemporary Art Museum of São Paulo and at the Biennial of São Paulo, as the origin of the Contemporary Art Museum is tided to those two institutions. Following, the arrangement of the photographic technical reserve of the Contemporary Art Museum in the 1970s will be analyzed. This study will be focusing on the work of Walter Zanini, as the first director of the museum, and on the particularities of MAC-USP position in the art system in Brazil which resulted in the understanding of photography as belonging to the sphere of contemporary art in an experimental way and not as an autonomous work of art, according to the principals of the so called artistic photography

A Surfactant Enables Efficient Membrane Spanning By Non-aggregating DNA-based Ion Channels

DNA 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 behaviour, as well as membrane activity of cholesterol-modified DNA nanostruc-tures. Our findings uncover the role of aggregation in preventing bilayer interactions of hydro-phobically-decorated constructs, and we highlight that premixing DNA structures with the sur-factant does not disrupt the cholesterol-mediated aggregates. Yet, we observe 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)

Research data in support of: Tailoring interleaflet lipid transfer with a DNA-based synthetic enzyme

Data for "Tailoring interleaflet lipid transfer with a DNA-based synthetic enzyme" Nanoletters (2020). Further details can be found in the publication or its supporting information available free of charge at https://pubs.acs.org/doi/full/10.1021/acs.nanolett.0c00990 Files dithionite assays.zip contain micrographs of vesicles upon performing NBD reduction assay, from which time traces presented in Figure 3 of the paper (fluorescence vs time) were plotted. The images were collected on the Olympus confocal microscope, recorded every 10 s. FIJI was used to measure the pixel intensity, attributed to fluorescnce values. Files in current traces.zip contain transmembrane current traces for three published structures, as presented in Figure 2 (current vs time). These are ABF files collected with a pCLAMP software suite (using axopatch amplifier) upon applying 50 V across the DPhPC lipid membrane in the presence of bilayer-spanning DNA structures. LaserBleaching.zip contains datapoints collected during control experiment assessing laser bleaching (fluorescence vs time), presented in SI sup fig. 22. The experiment was performed in the same way (same microscope settings) as NBD reduction assay and presents marginal effects of laser bleaching during the said experiment. Two images gel_20_0 mM Mg.TIF and gel_20_1 mM Mg.PNG are the PAGE images obtained from a gel imaging setup, used for calculating the values presented in SI, sup fig. 15 and sup table 4. The bands correspond to: ladder (GeneRuler low range) and DNA duplex samples: 0D, 0fD, 1D, 2D, as described in detail in the associated publication. The gels were run at 100 V for 90 min, with DNA duplex samples folded at different concentrations of Mg. The strong effect of Mg on stability of the structures with internal modifications was deduced upon analysis. gel_20_0 mM Mg.TIF was run in the presence of 11 mM Mg in both the gel and the running buffer, while no Mg was present in the experiment resulting in gel_20_1 mM Mg.PNG. melingProfiles.xlsx is a file obtained from the spectrophotometer when collecting melting profiles for DNA structures (absorbance vs temperature) presented in SI, sup fig. 14, sup table 3. Absorbance at 260 nm of DNA duplexes at 1 uM concentration was collected in a 10-90 C range, with a rate of 1C/min

Research data in support of: Membrane activity of a DNA-based ion channel depends on the stability of its double-stranded structure.

The folders named: 8nm-0x, 8nm-2x, 4nm-2x and Control: current measurements, performed under the voltage of 50V, with a frame rate of 5 kHz, upon addition of 10nM of DNA structures: 8nm-2x, 8nm-0x, 4nm-2x, all labelled with two cholesterol molecules which enable their membrane insertion. As well as analogous data for the single-cholesterol control measurements. Files in an .abf format, obtained using pClamp suie. Additionlly, numerical values for traces collected during FRAP measurements, used to plot Supplementary Figure SD2.2. The dataset also includes the unprocessed PAGE gel from Supplementary Figure D1.1. as well as data collected during melting experiments (absorbance at 260nm vs temperature), presented in Supplementary Figure D1.2

Research data in support of: Cations regulate membrane-attachment and functionality of DNA nanostructures

Confocal micrographs of Giant Unilamellar Vesicles bearing different types of DNA nanostructures in several ionic conditions; experimental data corresponding to Uv-visible spectrophotometry, Differential Scanning Calorimetry, and Dynamic Light Scattering measurements. All-atom Molecular Dynamics simulations movies and extracted data.R.R.S also acknowledges funding support from the Mexican National Council for Science and Technology (CONACYT, Grant No. 472427) and the Cambridge Trust. L.D.M. also acknowledges funding from the European Research Council (ERC) under the Horizon 2020 Research and Innovation Programme (ERC-STG No 851667 NANOCELL). A.A. and H.J. acknowledge support from the National Science Foundation USA (DMR-1827346), the Human Frontier Science Project (RGP0047/2020), and also the supercomputer time provided through the XSEDE allocation grant (MCA05S028) and the Leadership Resource Allocation MCB20012 on Frontera of the Texas Advanced Computing Center

Clathrin-Inspired Coating and Stabilization of Liposomes by DNA Self-Assembly

The self-assembly of the protein clathrin on biological membranes facilitates essential processes of endocytosis in biological systems and has provided a source of inspiration for materials design by the highly ordered structural appearance. By mimicking the architecture of clathrin self-assemblies to coat liposomes with biomaterials, new classes of hybrid carriers can be derived. Here we present a method for fabricating DNA-coated liposomes by hydrophobically anchoring and subsequently growing a DNA network on the liposome surface which structurally mimics clathrin assemblies. Dynamic light scattering (DLS), ζ-potential and cryo-electron microscopy (cryo-EM) measurements independently demonstrate successful DNA coating. Nanomechanical measurements conducted with atomic force microscopy (AFM) show that the DNA coating enhances the mechanical stability of the liposomes relative to uncoated ones. Furthermore, we provide the possibility to reverse the coating process by triggering the disassembly of the DNA coating through a toehold-mediated displacement reaction. Our results describe a straightforward, versatile, and reversible approach for coating and stabilizing lipid vesicles by an interlaced DNA network. This method has potential for further development towards the ordered arrangement of tailored functionalities on the surfaces of liposomes and for applications as hybrid nanocarrier