A benchmark data set for the mechanical properties of double-stranded DNA and RNA under torsional constraint.

Nucleic acids are central to the storage and transmission of genetic information and play essential roles in many cellular processes. Quantitative understanding and modeling of their functions and properties requires quantitative experimental characterization. We use magnetic tweezers (MT) to apply precisely calibrated stretching forces and linking number changes to DNA and RNA molecules tethered between a surface and superparamagnetic beads. Magnetic torque tweezers (MTT) allow to control the linking number of double-stranded DNA or RNA tethers, while directly measuring molecular torque by monitoring changes in the equilibrium rotation angle upon over- or underwinding of the helical molecules. Here, we provide a comprehensive data set of double-stranded DNA and RNA under controlled stretching as a function of the linking number. We present data for extension and torque as a function of linking number in equilibrium. We report data for the critical torque of buckling and of the torsional stiffness of DNA and RNA as a function of applied force. Finally, we provide dynamic data for the hopping behavior at the DNA buckling point.

The free energy landscape of retroviral integration.

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Yet, strand transfer is intrinsically iso-energetic and it is not clear how efficient integration can be achieved. Here we investigate the dynamics of strand transfer and demonstrate that consecutive nucleoprotein intermediates interacting with a supercoiled target are increasingly stable, resulting in a net forward rate. Multivalent target interactions at discrete auxiliary interfaces render target capture irreversible, while allowing dynamic site selection. Active site binding is transient but rapidly results in strand transfer, which in turn rearranges and stabilizes the intasome in an allosteric manner. We find the resulting strand transfer complex to be mechanically stable and extremely long-lived, suggesting that a resolving agent is required in vivo.

Multiplexed protein force spectroscopy reveals equilibrium protein folding dynamics and the low-force response of von Willebrand factor.

Single-molecule force spectroscopy has provided unprecedented insights into protein folding, force regulation, and function. So far, the field has relied primarily on atomic force microscope and optical tweezers assays that, while powerful, are limited in force resolution, throughput, and require feedback for constant force measurements. Here, we present a modular approach based on magnetic tweezers (MT) for highly multiplexed protein force spectroscopy. Our approach uses elastin-like polypeptide linkers for the specific attachment of proteins, requiring only short peptide tags on the protein of interest. The assay extends protein force spectroscopy into the low force (<1 pN) regime and enables parallel and ultra-stable measurements at constant forces. We present unfolding and refolding data for the small, single-domain protein ddFLN4, commonly used as a molecular fingerprint in force spectroscopy, and for the large, multidomain dimeric protein von Willebrand factor (VWF) that is critically involved in primary hemostasis. For both proteins, our measurements reveal exponential force dependencies of unfolding and refolding rates. We directly resolve the stabilization of the VWF A2 domain by Ca2+ and discover transitions in the VWF C domain stem at low forces that likely constitute the first steps of VWF's mechano-activation. Probing the force-dependent lifetime of biotin-streptavidin bonds, we find that monovalent streptavidin constructs with specific attachment geometry are significantly more force stable than commercial, multivalent streptavidin. We expect our modular approach to enable multiplexed force-spectroscopy measurements for a wide range of proteins, in particular in the physiologically relevant low-force regime.

Time-Resolved Small-Angle X-ray Scattering Reveals Millisecond Transitions of a DNA Origami Switch.

Self-assembled DNA structures enable creation of specific shapes at the nanometer-micrometer scale with molecular resolution. The construction of functional DNA assemblies will likely require dynamic structures that can undergo controllable conformational changes. DNA devices based on shape complementary stacking interactions have been demonstrated to undergo reversible conformational changes triggered by changes in ionic environment or temperature. An experimentally unexplored aspect is how quickly conformational transitions of large synthetic DNA origami structures can actually occur. Here, we use time-resolved small-angle X-ray scattering to monitor large-scale conformational transitions of a two-state DNA origami switch in free solution. We show that the DNA device switches from its open to its closed conformation upon addition of MgCl2 in milliseconds, which is close to the theoretical diffusive speed limit. In contrast, measurements of the dimerization of DNA origami bricks reveal much slower and concentration-dependent assembly kinetics. DNA brick dimerization occurs on a time scale of minutes to hours suggesting that the kinetics depend on local concentration and molecular alignment.

Conformational Changes and Flexibility of DNA Devices Observed by Small-Angle X-ray Scattering.

Self-assembled DNA origami nanostructures enable the creation of precisely defined shapes at the molecular scale. Dynamic DNA devices that are capable of switching between defined conformations could afford completely novel functionalities for diagnostic, therapeutic, or engineering applications. Developing such objects benefits strongly from experimental feedback about conformational changes and 3D structures, ideally in solution, free of potential biases from surface attachment or labeling. Here, we demonstrate that small-angle X-ray scattering (SAXS) can quantitatively resolve the conformational changes of a DNA origami two-state switch device as a function of the ionic strength of the solution. In addition, we show how SAXS data allow for refinement of the predicted idealized three-dimensional structure of the DNA object using a normal mode approach based on an elastic network model. The results reveal deviations from the idealized design geometries that are otherwise difficult to resolve. Our results establish SAXS as a powerful tool to investigate conformational changes and solution structures of DNA origami and we anticipate our methodology to be broadly applicable to increasingly complex DNA and RNA devices.