Nanomechanics of self-assembled DNA building blocks

DNA has become a powerful platform to design functional nanodevices. DNA nanodevices are often composed of self-assembled DNA building blocks that differ significantly from the structure of native DNA. In this study, we present Flow Force Microscopy as a massively parallel approach to study the nanomechanics of DNA self-assemblies on the single-molecular level. The high-throughput experiments performed in a simple microfluidic channel enable statistically meaningful studies with nanometer scale precision in a time frame of several minutes. A surprisingly high flexibility was observed for a typical construct used in DNA origami, reflected in a persistence length of 10.2 nm, a factor of five smaller than for native DNA. The enhanced flexibility is attributed to the discontinuous backbone of DNA self-assemblies that facilitate base pair opening by thermal fluctuations at the end of hybridized oligomers. We believe that the results will contribute to the fundamental understanding of DNA nanomechanics and help to improve the design of DNA nanodevices with applications in biological analysis and clinical research

Single-Polymer Friction Force Microscopy of dsDNA Interacting with a Nanoporous Membrane

Surface-grafted polymers can reduce friction between solids in liquids by compensating the normal load with osmotic pressure, but they can also contribute to friction when fluctuating polymers entangle with the sliding counter face. We have measured forces acting on a single fluctuating double-stranded DNA polymer, which is attached to the tip of an atomic force microscope and interacts intermittently with nanometer-scale methylated pores of a self-assembled polystyrene-block-poly(4-vinylpyridine) membrane. Rare binding of the polymer into the pores is followed by a stretching of the polymer between the laterally moving tip and the surface and by a force-induced detachment. We present results for the velocity dependence of detachment forces and of attachment frequency and discuss them in terms of rare excursions of the polymer beyond its equilibrium configuration

Molecular stiffness cues of an interpenetrating network hydrogel for cell adhesion

Understanding cells' response to the macroscopic and nanoscale properties of biomaterials requires studies in model systems with the possibility to tailor their mechanical properties and different length scales. Here, we describe an interpenetrating network (IPN) design based on a stiff PEGDA host network interlaced within a soft 4-arm PEG-Maleimide/thiol (guest) network. We quantify the nano- and bulk mechanical behavior of the IPN and the single network hydrogels by single-molecule force spectroscopy and rheological measurements. The IPN presents different mechanical cues at the molecular scale, depending on which network is linked to the probe, but the same mechanical properties at the macroscopic length scale as the individual host network. Cells attached to the interpenetrating (guest) network of the IPN or to the single network (SN) PEGDA hydrogel modified with RGD adhesive ligands showed comparable attachment and spreading areas, but cells attached to the guest network of the IPN, with lower molecular stiffness, showed a larger number and size of focal adhesion complexes and a higher concentration of the Hippo pathway effector Yes-associated protein (YAP) than cells linked to the PEGDA single network. The observations indicate that cell adhesion to the IPN hydrogel through the network with lower molecular stiffness proceeds effectively as if a higher ligand density is offered. We claim that IPNs can be used to decipher how changes in ECM design and connectivity at the local scale affect the fate of cells cultured on biomaterials

Dynamische Adhäsion und Reibung vermittelt durch supramolekulare Bindungen

Understanding and controlling adhesive interactions on the molecular scale is one of the main challenges in the field of nanotechnology. A new surface functionalization was developed in this thesis for investigating the molecular origin of adhesive interactions from single molecular level to assemblies of multiple bonds. The surface functionalization is based on supramolecular bonds established by the inclusion of ditopic connector molecules into two cyclodextrin (CD) molecules, one attached to a tip of an atomic force microscope and the other attached to a flat silicon surface. By using different connector molecules, the dynamics in friction and adhesion can be tuned. The dynamics of the molecular system were studied with respect to single bond kinetics and the flexibility of the attachment. The control of adhesion and friction was achieved by using photosensitive connector molecules which are sensitive to an external light stimuli. In order to enhance the applicability of the surface functionalization, the CD molecules were attached onto stiff polymers which can bridge the surface roughness of real contacts. The results of this thesis provide a deeper understanding of the molecular mechanisms underlying adhesive friction and open a new pathway for actively controlling friction and adhesion.Die Kontrolle von adhäsiven Wechselwirkungen auf molekularere Ebene ist von beson- derem Interesse im Forschungsfeld der Nanotechnologie. Im Rahmen dieser Arbeit wurde eine Oberflächenfunktionalisierung entwickelt, um die adhäsive Interaktion von Einzel- molekülen bis hin zu molekularen Essembles zu untersuchen. Die Funktionalisierung basiert auf supramolekularen Bindungen, bestehend aus einem ditopen Konnektormolekül, das an zwei Cyclodextrin (CD) Moleküle bindet, wobei das eine CD-Molekül an die Spitze eines Rasterkraftmikroskops und das andere an eine glatte Siliziumoberfläche gebunden ist. Durch die Auswahl verschiedener Konnektormoleküle kann das dynamische Verhalten von Reibung und Ahäsion angepasst werden. Die Dynamik des Kontakts wird im Hinblick auf die Kinetik einer einzelnen Bindung sowie auf die Flexibilität der Anbindung untersucht. Die Kontrolle von Adhäsion und Reibung wird mit Hilfe photosensitiver Konnektormoleküle erreicht, die sich durch einen externen Lichtstimulus schalten lassen. Um die Rauigkeit realer Kontakte zu überbrücken, wurden CD-Moleküle an Polymere angebunden, die ihrerseits an die Oberfläche und die AFM Spitze angebunden wurden. Die Ergebnisse dieser Arbeit tragen zu einem tieferen Verständnis der molekularen Prozesse von adhäsiver Reibung bei und zeigen eine Möglichkeit zur aktiven Kontrolle von Reibung und Adhäsion auf

BetaCasomorphin causes hypoalgesia in 10-day-old-rats:evidence for central mediation

Two experiments determined behavioral effectiveness of \u3b2-casomorphins(\u3b2-CM) in 10-d-old rats by evaluating changes in heat escape latency from a 48\ub0C stimulus applied to a forepaw. In one study rats were injected systemically with \u3b2-CM4, -5, or -7 at a dose range of 0.1-2.5 mg/kg. Only\u3b2-CM5 was effective, and the dose-response relationship was graded. The second study evaluated the locus of action of \u3b2-CM5 through two experimental manipulations: first, by injecting it (0.25 \u3bcg) into the lateral ventricles and by attempting to block its effects with systemic injections of naloxone. Second, rats received intracerebroventricular injections of naloxone (0.25 \u3bcg) and systemic injections of \u3b2-CM.\u3b2-CM was effective centrally, suggesting central detection of the drug. Naloxone injected into the lateral ventricles blocked the effects of systemic administration of \u3b2-CM, implying that circulating \u3b2-CM or their precursors cause behavioral change through central mechanisms

Single-Polymer Friction Force Microscopy of dsDNA Interacting with a Nanoporous Membrane

Surface-grafted polymers can reduce friction between solids in liquids by compensating the normal load with osmotic pressure, but they can also contribute to friction when fluctuating polymers entangle with the sliding counter face. We have measured forces acting on a single fluctuating doublestranded DNA polymer, which is attached to the tip of an atomic force microscope and interacts intermittently with nanometer-scale methylated pores of a self-assembled polystyrene-block-poly(4- vinylpyridine) membrane. Rare binding of the polymer into the pores is followed by a stretching of the polymer between the laterally moving tip and the surface and by a force-induced detachment. We present results for the velocity dependence of detachment forces and of attachment frequency and discuss them in terms of rare excursions of the polymer beyond its equilibrium configuration

Dynamic effects in friction and adhesion through cooperative rupture and formation of supramolecular bonds

We introduce a molecular toolkit for studying the dynamics in friction and adhesion from the single molecule level to effects of multivalency. As experimental model system we use supramolecular bonds established by the inclusion of ditopic adamantane connector molecules into two surface-bound cyclodextrin molecules, attached to a tip of an atomic force microscope (AFM) and to a flat silicon surface. The rupture force of a single bond does not depend on the pulling rate, indicating that the fast complexation kinetics of adamantane and cyclodextrin are probed in thermal equilibrium. In contrast, the pull-off force for a group of supramolecular bonds depends on the unloading rate revealing a non-equilibrium situation, an effect discussed as the combined action of multivalency and cantilever inertia effects. Friction forces exhibit a stick-slip characteristic which is explained by the cooperative rupture of groups of host-guest bonds and their rebinding. No dependence of friction on the sliding velocity has been observed in the accessible range of velocities due to fast rebinding and the negligible delay of cantilever response in AFM lateral force measurements

Switching adhesion and friction by light using photosensitive guest - host interactions

Friction and adhesion between two β-cyclodextrin functionalized surfaces can be switched reversibly by external light stimuli. The interaction between the cyclodextrin molecules attached to the tip of an atomic force microscope and a silicon wafer surface is mediated by complexation of ditopic azobenzene guest molecules. At the single molecule level, the rupture force of an individual complex is 61 ± 10 pN

Interactions between shape-persistent macromolecules as probed by AFM

Water-soluble shape-persistent cyclodextrin (CD) polymers with amino-functionalized end groups were prepared starting from diacetylene-modified cyclodextrin monomers by a combined Glaser coupling/click chemistry approach. Structural perfection of the neutral CD polymers and inclusion complex formation with ditopic and monotopic guest molecules were proven by MALDI–TOF and UV–vis measurements. Small-angle neutron and X-ray (SANS/SAXS) scattering experiments confirm the stiffness of the polymer chains with an apparent contour length of about 130 Å. Surface modification of planar silicon wafers as well as AFM tips was realized by covalent bound formation between the terminal amino groups of the CD polymer and a reactive isothiocyanate–silane monolayer. Atomic force measurements of CD polymer decorated surfaces show enhanced supramolecular interaction energies which can be attributed to multiple inclusion complexes based on the rigidity of the polymer backbone and the regular configuration of the CD moieties. Depending on the geometrical configuration of attachment anisotropic adhesion characteristics of the polymer system can be distinguished between a peeling and a shearing mechanism

Optoregulated force application to cellular receptors using molecular motors

Progress in our understanding of mechanotransduction events requires noninvasive methods for the manipulation of forces at molecular scale in physiological environments. Inspired by cellular mechanisms for force application (i.e. motor proteins pulling on cytoskeletal fibers), we present a unique molecular machine that can apply forces at cell-matrix and cell-cell junctions using light as an energy source. The key actuator is a light-driven rotatory molecular motor linked to polymer chains, which is intercalated between a membrane receptor and an engineered biointerface. The light-driven actuation of the molecular motor is converted in mechanical twisting of the entangled polymer chains, which will in turn effectively “pull” on engaged cell membrane receptors (e.g., integrins, T cell receptors) within the illuminated area. Applied forces have physiologically-relevant magnitude and occur at time scales within the relevant ranges for mechanotransduction at cell-friendly exposure conditions, as demonstrated in force-dependent focal adhesion maturation and T cell activation experiments. Our results reveal the potential of nanomotors for the manipulation of living cells at the molecular scale and demonstrate a functionality which at the moment cannot be achieved by other technologies for force application