Real-time deformability cytometry: on-the-fly cell mechanical phenotyping.

We introduce real-time deformability cytometry (RT-DC) for continuous cell mechanical characterization of large populations (>100,000 cells) with analysis rates greater than 100 cells/s. RT-DC is sensitive to cytoskeletal alterations and can distinguish cell-cycle phases, track stem cell differentiation into distinct lineages and identify cell populations in whole blood by their mechanical fingerprints. This technique adds a new marker-free dimension to flow cytometry with diverse applications in biology, biotechnology and medicine.

Camera-based three-dimensional real-time particle tracking at kHz rates and Ångström accuracy.

Optical and magnetic tweezers are widely employed to probe the mechanics and activity of individual biomolecular complexes. They rely on micrometre-sized particles to detect molecular conformational changes from the particle position. Real-time particle tracking with Ångström accuracy has so far been only achieved using laser detection through photodiodes. Here we demonstrate that camera-based imaging can provide a similar performance for all three dimensions. Particle imaging at kHz rates is combined, with real-time data processing being accelerated by a graphics-processing unit. For particles that are fixed in the sample cell we can detect 3-Å-sized steps that are introduced by cell translations at rates of 10 Hz, while for DNA-tethered particles 5 Å steps at 1 Hz can be resolved. Moreover, 20 particles can be tracked in parallel with comparable accuracy. Our approach provides a simple and robust way for high-resolution tweezer experiments using multiple particles at a time.

Fork sensing and strand switching control antagonistic activities of RecQ helicases.

RecQ helicases have essential roles in maintaining genome stability during replication and in controlling double-strand break repair by homologous recombination. Little is known about how the different RecQ helicases found in higher eukaryotes achieve their specialized and partially opposing functions. Here, we investigate the DNA unwinding of RecQ helicases from Arabidopsis thaliana, AtRECQ2 and AtRECQ3 at the single-molecule level using magnetic tweezers. Although AtRECQ2 predominantly unwinds forked DNA substrates in a highly repetitive fashion, AtRECQ3 prefers to rewind, that is, to close preopened DNA forks. For both enzymes, this process is controlled by frequent strand switches and active sensing of the unwinding fork. The relative extent of the strand switches towards unwinding or towards rewinding determines the predominant direction of the enzyme. Our results provide a simple explanation for how different biological activities can be achieved by rather similar members of the RecQ family.

Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity.

Dna2 is a nuclease-helicase involved in several key pathways of eukaryotic DNA metabolism. The potent nuclease activity of Saccharomyces cerevisiae Dna2 was reported to be required for all its in vivo functions tested to date. In contrast, its helicase activity was shown to be weak, and its inactivation affected only a subset of Dna2 functions. We describe here a complex interplay of the two enzymatic activities. We show that the nuclease of Dna2 inhibits its helicase by cleaving 5' flaps that are required by the helicase domain for loading onto its substrate. Mutational inactivation of Dna2 nuclease unleashes unexpectedly vigorous DNA unwinding activity, comparable with that of the most potent eukaryotic helicases. Thus, the ssDNA-specific nuclease activity of Dna2 limits and controls the enzyme's capacity to unwind dsDNA. We postulate that regulation of this interplay could modulate the biochemical properties of Dna2 and thus license it to carry out its distinct cellular functions.

Efficient preparation of internally modified single-molecule constructs using nicking enzymes.

Investigations of enzymes involved in DNA metabolism have strongly benefited from the establishment of single molecule techniques. These experiments frequently require elaborate DNA substrates, which carry chemical labels or nucleic acid tertiary structures. Preparing such constructs often represents a technical challenge: long modified DNA molecules are usually produced via multi-step processes, involving low efficiency intermolecular ligations of several fragments. Here, we show how long stretches of DNA (>50 bp) can be modified using nicking enzymes to produce complex DNA constructs. Multiple different chemical and structural modifications can be placed internally along DNA, in a specific and precise manner. Furthermore, the nicks created can be resealed efficiently yielding intact molecules, whose mechanical properties are preserved. Additionally, the same strategy is applied to obtain long single-strand overhangs subsequently used for efficient ligation of ss- to dsDNA molecules. This technique offers promise for a wide range of applications, in particular single-molecule experiments, where frequently multiple internal DNA modifications are required.

Energetics at the DNA supercoiling transition.

Twisting a DNA molecule held under constant tension is accompanied by a transition from a linear to a plectonemic DNA configuration, in which part of the applied twist is absorbed in a superhelical structure. Recent experiments revealed the occurrence of an abrupt extension change at the onset of this transition. To elucidate its origin we study this abrupt DNA shortening using magnetic tweezers. We find that it strongly depends on the length of the DNA molecule and the ionic strength of the solution. This behavior can be well understood in the framework of a model in which the energy per writhe for the initial plectonemic loop is larger than for subsequent turns of the superhelix. By quantitative data analysis, relevant plectoneme energies and other parameters were extracted, providing good agreement with a simple theory. As a direct confirmation of the initial-loop model, we find that for a kinked DNA molecule the abrupt extension change occurs at significantly lower twist than the subsequent superhelix formation. This should allow pinning of the plectoneme position within supercoiled DNA if a kinked substrate is used, and enable the detection of enzymes and proteins which, themselves, bend or kink DNA.

Torsional stiffness of single superparamagnetic microspheres in an external magnetic field.

We used magnetic tweezers to measure the torsional stiffness of single micrometer-sized superparamagnetic spheres as a function of the applied magnetic field. By investigating the axial fluctuations of DNA-bound microspheres, we found that considerable rotational microsphere fluctuations can occur. Quantitative noise analysis allowed us to determine the rotational stiffness of individual microspheres, which was found to saturate at high magnetic fields. The saturation can be qualitatively explained considering the properties of the magnetic nanoparticles within the microsphere. Consequences for spatial resolution limits in single-molecule magnetic tweezer experiments and usage of DNA mechanics as a sensitive probe in magnetometry are discussed.