Freely orbiting magnetic tweezers to directly monitor changes in the twist of nucleic acids

The double-stranded nature of DNA links its replication, transcription and repair to rotational motion and torsional strain. Magnetic tweezers (MT) are a powerful single-molecule technique to apply both forces and torques to individual DNA or RNA molecules. However, conventional MT do not track rotational motion directly and constrain the free rotation of the nucleic acid tether. Here we present freely orbiting MT (FOMT) that allow the measurement of equilibrium fluctuations and changes in the twist of tethered nucleic acid molecules. Using a precisely aligned vertically oriented magnetic field, FOMT enable tracking of the rotation angle from straight forward (x,y)-position tracking and permits the application of calibrated stretching forces, without biasing the tether's free rotation. We utilize FOMT to measure the force-dependent torsional stiffness of DNA from equilibrium rotational fluctuations and to follow the assembly of recombination protein A filaments on DNA

Direct Imaging of the circular chromosome of a live bacterium

Although the physical properties of chromosomes, including their morphology, mechanics, and dynamics are crucial for their biological function, many basic questions remain unresolved. Here we directly image the circular chromosome in live E. coli with a broadened cell shape. We find that it exhibits a torus topology with, on average, a lower-density origin of replication and an ultrathin flexible string of DNA at the terminus of replication. At the single-cell level, the torus is strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. Our data show a domain organization underlying the chromosome structure of E. coli, where MatP proteins induce site-specific persistent domain boundaries at Ori/Ter, while transcription regulators HU and Fis induce weaker transient domain boundaries throughout the genome. These findings provide an architectural basis for the understanding of the dynamic spatial organization of bacterial genomes in live cells

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a “conventional” magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the “conventional” magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument

A Simple Self-Calibrating Method To Measure the Height of Fluorescent Molecules and Beads at Nanoscale Resolution

We describe a simple self-calibrating technique, incident-beam interference sweeping, for measuring the height of fluorescent labels. Using a tilted back-reflecting mirror and a scanning laser beam, a modulated fluorescence emission allows height determination of a label from a surface with a resolution of ∼3 nm. In addition, we show that the absolute distance of a label from the top-mounted mirror can be determined with a resolution of a few tens of nanometers over a micrometer range

Assembly dynamics of microtubules at molecular resolution

Microtubules are highly dynamic protein polymers that form a crucial part of the cytoskeleton in all eukaryotic cells. Although microtubules are known to self-assemble from tubulin dimers, information on the assembly dynamics of microtubules has been limited, both in vitro and in vivo, to measurements of average growth and shrinkage rates over several thousands of tubulin subunits. As a result there is a lack of information on the sequence of molecular events that leads to the growth and shrinkage of microtubule ends. Here we use optical tweezers to observe the assembly dynamics of individual microtubules at molecular resolution. We find that microtubules can increase their overall length almost instantaneously by amounts exceeding the size of individual dimers (8 nm). When the microtubule-associated protein XMAP215 (ref. 6) is added, this effect is markedly enhanced and fast increases in length of about 40-60 nm are observed. These observations suggest that small tubulin oligomers are able to add directly to growing microtubules and that XMAP215 speeds up microtubule growth by facilitating the addition of long oligomers. The achievement of molecular resolution on the microtubule assembly process opens the way to direct studies of the molecular mechanism by which the many recently discovered microtubule end-binding proteins regulate microtubule dynamics in living cells

Highly Parallel Magnetic Tweezers by Targeted DNA Tethering

Single-molecule force-spectroscopy methods such as magnetic and optical tweezers have emerged as powerful tools for the detailed study of biomechanical aspects of DNA-enzyme interactions. As typically only a single molecule of DNA is addressed in an individual experiment, these methods suffer from a low data throughput. Here, we report a novel method for targeted, nonrandom immobilization of DNA-tethered magnetic beads in regular arrays through microcontact printing of DNA end-binding labels. We show that the increase in density due to the arrangement of DNA-bead tethers in regular arrays can give rise to a one-order-of-magnitude improvement in data-throughput in magnetic tweezers experiments. We demonstrate the applicability of this technique in tweezers experiments where up to 450 beads are simultaneously tracked in parallel, yielding statistical data on the mechanics of DNA for 357 molecules from a single experimental run. Our technique paves the way for kilo-molecule force spectroscopy experiments, enabling the study of rare events in DNA–protein interactions and the acquisition of large statistical data sets from individual experimental runs