The temperature dependence of the helical twist of DNA.

DNA is the carrier of all cellular genetic information and increasingly used in nanotechnology. Quantitative understanding and optimization of its functions requires precise experimental characterization and accurate modeling of DNA properties. A defining feature of DNA is its helicity. DNA unwinds with increasing temperature, even for temperatures well below the melting temperature. However, accurate quantitation of DNA unwinding under external forces and a microscopic understanding of the corresponding structural changes are currently lacking. Here we combine single-molecule magnetic tweezers measurements with atomistic molecular dynamics and coarse-grained simulations to obtain a comprehensive view of the temperature dependence of DNA twist. Experimentally, we find that DNA twist changes by ΔTw(T) = (-11.0 ± 1.2)°/(°C·kbp), independent of applied force, in the range of forces where torque-induced melting is negligible. Our atomistic simulations predict ΔTw(T) = (-11.1 ± 0.3)°/(°C·kbp), in quantitative agreement with experiments, and suggest that the untwisting of DNA with temperature is predominantly due to changes in DNA structure for defined backbone substates, while the effects of changes in substate populations are minor. Coarse-grained simulations using the oxDNA framework yield a value of ΔTw(T) = (-6.4 ± 0.2)°/(°C·kbp) in semi-quantitative agreement with experiments.

Probing the salt dependence of the torsional stiffness of DNA by multiplexed magnetic torque tweezers.

The mechanical properties of DNA fundamentally constrain and enable the storage and transmission of genetic information and its use in DNA nanotechnology. Many properties of DNA depend on the ionic environment due to its highly charged backbone. In particular, both theoretical analyses and direct single-molecule experiments have shown its bending stiffness to depend on salt concentration. In contrast, the salt-dependence of the twist stiffness of DNA is much less explored. Here, we employ optimized multiplexed magnetic torque tweezers to study the torsional stiffness of DNA under varying salt conditions as a function of stretching force. At low forces (<3 pN), the effective torsional stiffness is ∼10% smaller for high salt conditions (500 mM NaCl or 10 mM MgCl2) compared to lower salt concentrations (20 mM NaCl and 100 mM NaCl). These differences, however, can be accounted for by taking into account the known salt dependence of the bending stiffness. In addition, the measured high-force (6.5 pN) torsional stiffness values of C = 103 ± 4 nm are identical, within experimental errors, for all tested salt concentration, suggesting that the intrinsic torsional stiffness of DNA does not depend on salt.

Probing the mechanical properties, conformational changes, and interactions of nucleic acids with magnetic tweezers.

Nucleic acids are central to the storage and transmission of genetic information. Mechanical properties, along with their sequence, both enable and fundamentally constrain the biological functions of DNA and RNA. For small deformations from the equilibrium conformations, nucleic acids are well described by an isotropic elastic rod model. However, external forces and torsional strains can induce conformational changes, giving rise to a complex force-torque phase diagram. This review focuses on magnetic tweezers as a powerful tool to precisely determine both the elastic parameters and conformational transitions of nucleic acids under external forces and torques at the single-molecule level. We review several variations of magnetic tweezers, in particular conventional magnetic tweezers, freely orbiting magnetic tweezers and magnetic torque tweezers, and discuss their characteristic capabilities. We then describe the elastic rod model for DNA and RNA and discuss conformational changes induced by mechanical stress. The focus lies on the responses to torque and twist, which are crucial in the mechanics and interactions of nucleic acids and can directly be measured using magnetic tweezers. We conclude by highlighting several recent studies of nucleic acid-protein and nucleic acid-small-molecule interactions as further applications of magnetic tweezers and give an outlook of some exciting developments to come.

Probing the Cooperativity of Binding Networks with High-Throughput Thermophoresis.

The formation of supramolecular complexes is found in many natural systems and is the basis for cooperative behavior. Here, we report on the development of a high-throughput platform to measure the complex binding behavior in 500 nL volumes and 1 536-well plates. The platform enabled us to elucidate the thermodynamic properties of a heterotrimeric DNA complex that portrays the structure of a biological relevant three-way junction. In a complementing set of cooperative networks, binding constants from ∼0.1 nM to ∼10 µM were measured by sampling a high-dimensional concentration space. Each intermediate binding state was probed simultaneously with only a single fluorescent label. Through systematic base pair variations, we observed the influence of the cooperative effect on single base pair mismatches. We further found coupled binding between seemingly independent binding sites through the complex structure of the three-way junction that could not have been observed without the measurement of the entire network. These results promote automated high-throughput thermophoresis to characterize arbitrary binding networks.

Twist-Bend Coupling and the Torsional Response of Double-Stranded DNA.

Recent magnetic tweezers experiments have reported systematic deviations of the twist response of double-stranded DNA from the predictions of the twistable wormlike chain model. Here we show, by means of analytical results and computer simulations, that these discrepancies can be resolved if a coupling between twist and bend is introduced. We obtain an estimate of 40±10 nm for the twist-bend coupling constant. Our simulations are in good agreement with high-resolution, magnetic-tweezers torque data. Although the existence of twist-bend coupling was predicted long ago [J. Marko and E. Siggia, Macromolecules 27, 981 (1994)MAMOBX0024-929710.1021/ma00082a015], its effects on the mechanical properties of DNA have been so far largely unexplored. We expect that this coupling plays an important role in several aspects of DNA statics and dynamics.

Steep pH Gradients and Directed Colloid Transport in a Microfluidic Alkaline Hydrothermal Pore.

All life on earth depends on the generation and exploitation of ionic and pH gradients across membranes. One theory for the origin of life proposes that geological pH gradients were the prebiotic ancestors of these cellular disequilibria. With an alkaline interior and acidic exterior, alkaline vents match the topology of modern cells, but it remains unknown whether the steep pH gradients persist at the microscopic scale. Herein, we demonstrate the existence of 6 pH-unit gradients across micrometer scales in a microfluidic vent replicate. Precipitation of metal sulfides at the interface strengthens the gradients, but even in the absence of precipitates laminar flow sustains the disequilibria. The gradients drive directed transport at the fluid interface, leading to colloid accumulation or depletion. Our results confirm that alkaline vents can provide an exploitable pH gradient, supporting their potential role at the emergence of chemiosmosis and the origin of life.

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.

Measuring Single-Molecule Twist and Torque in Multiplexed Magnetic Tweezers.

Magnetic tweezers permit application of precisely calibrated stretching forces to nucleic acid molecules tethered between a surface and superparamagnetic beads. In addition, magnetic tweezers can control the tethers' twist. Here, we focus on recent extensions of the technique that expand the capabilities of conventional magnetic tweezers by enabling direct measurements of single-molecule torque and twist. Magnetic torque tweezers (MTT) still control the DNA or RNA tether's twist, but directly measure molecular torque by monitoring changes in the equilibrium rotation angle upon overwinding and underwinding of the tether. In freely orbiting magnetic tweezers (FOMT), one end of the tether is allowed to rotate freely, while still applying stretching forces and monitoring rotation angle. Both MTT and FOMT have provided unique insights into the mechanical properties, structural transitions, and interactions of DNA and RNA. Here, we provide step-by-step protocols to carry out FOMT and MTT measurements. In particular, we focus on multiplexed measurements, i.e., measurements that record data for multiple nucleic acid tethers at the same time, to improve statistics and to facilitate the observation of rare events.