Surface-Patterned DNA Origami Rulers Reveal Nanoscale Distance Dependency of the Epidermal Growth Factor Receptor Activation.

The nanoscale arrangement of ligands can have a major effect on the activation of membrane receptor proteins and thus cellular communication mechanisms. Here we report on the technological development and use of tailored DNA origami-based molecular rulers to fabricate "Multiscale Origami Structures As Interface for Cells" (MOSAIC), to enable the systematic investigation of the effect of the nanoscale spacing of epidermal growth factor (EGF) ligands on the activation of the EGF receptor (EGFR). MOSAIC-based analyses revealed that EGF distances of about 30-40 nm led to the highest response in EGFR activation of adherent MCF7 and Hela cells. Our study emphasizes the significance of DNA-based platforms for the detailed investigation of the molecular mechanisms of cellular signaling cascades.

"Molecular Activity Painting": Switch-like, Light-Controlled Perturbations inside Living Cells.

Acute subcellular protein targeting is a powerful tool to study biological networks. However, signaling at the plasma membrane is highly dynamic, making it difficult to study in space and time. In particular, sustained local control of molecular function is challenging owing to the lateral diffusion of plasma membrane targeted molecules. Herein we present "molecular activity painting" (MAP), a novel technology which combines photoactivatable chemically induced dimerization (pCID) with immobilized artificial receptors. The immobilization of artificial receptors by surface-immobilized antibodies blocks lateral diffusion, enabling rapid and stable "painting" of signaling molecules and their activity at the plasma membrane with micrometer precision. Using this method, we show that painting of the RhoA-myosin activator GEF-H1 induces patterned acto-myosin contraction inside living cells.

Configurable low-cost plotter device for fabrication of multi-color sub-cellular scale microarrays.

The construction and operation of a low-cost plotter for fabrication of microarrays for multiplexed single-cell analyses is reported. The printing head consists of polymeric pyramidal pens mounted on a rotation stage installed on an aluminium frame. This construction enables printing of microarrays onto glass substrates mounted on a tilt stage, controlled by a Lab-View operated user interface. The plotter can be assembled by typical academic workshops from components of less than 15,000 Euro. The functionality of the instrument is demonstrated by printing DNA microarrays on the area of 0.5 cm2 using up to three different oligonucleotides. Typical feature sizes are 5 µm diameter with a pitch of 15 µm, leading to densities of up to 10(4)-10(5) spots/mm2. The fabricated DNA microarrays are used to produce sub-cellular scale arrays of bioactive epidermal growth factor peptides by means of DNA-directed immobilization. The suitability of these biochips for cell biological studies is demonstrated by specific recruitment, concentration, and activation of EGF receptors within the plasma membrane of adherent living cells. This work illustrates that the presented plotter gives access to bio-functionalized arrays usable for fundamental research in cell biology, such as the manipulation of signal pathways in living cells at subcellular resolution.

Temperature-dependent FRET spectroscopy for the high-throughput analysis of self-assembled DNA nanostructures in real time.

We describe a method for the real-time and high-throughput monitoring of the self-assembly and disassembly of complex DNA superstructures, using temperature-dependent Förster resonance energy transfer (FRET) spectroscopy. Compared with other spectroscopic approaches, such as UV-visible and circular dichroism, the method described has advantages in terms of sensitivity, feasibility for high-throughput analysis and applicability to virtually any kind of supramolecular structure. To this end, two oligonucleotides out of the entire set building up the superstructure are labeled with a fluorescein and a tetramethylrhodamine, as FRET donor and acceptor, respectively. Correct assembly of the superstructure induces maximum FRET efficiency, whereas complete dissociation leads to minimal FRET. Monitoring of temperature-dependent FRET efficiency yields a thermal profile that is used for thermodynamic analysis. In the case of reversible and cooperative assembly/disassembly of the DNA superstructure, application of the van't Hoff law allows for the determination of the thermodynamic parameters of the process. Owing to slow temperature ramping, the entire assay requires about 17 h. The protocol allows to simultaneously analyze up to 384 samples with only 30 microl sample volume each.

Monte Carlo simulation of the assembly of bis-biotinylated DNA and streptavidin.

We present Monte Carlo simulations of the self-assembly of bivalent bis-biotinylated DNA molecules with the tetravalent biotin-binding protein streptavidin (STV). By fitting the STV binding probabilities for the four possible valencies, the modelling correctly reproduces the dependencies of various network parameters experimentally observed in an earlier study. The combined results from the experimental and theoretical studies suggest that the binding probability for divalent STV formation is about 50 times larger than for the formation of trivalent and about 200 times larger than for tetravalent STV. In accordance with the experimental results, the modelling also indicates that the mixture of an equimolar ratio of DNA and STV leads to a maximum in size of the oligomeric DNA-STV clusters formed. Furthermore, we found a percolation transition in which the DNA cluster size increases rapidly with increasing DNA concentration resulting in the formation of a single supercluster at elevated concentrations. This behaviour coincides with the occurrence of an immobile band previously observed in electrophoretic experiments, indicating the formation of extremely large DNA-STV aggregate networks.