The bacterial replication origin BUS promotes nucleobase capture.

Genome duplication is essential for the proliferation of cellular life and this process is generally initiated by dedicated replication proteins at chromosome origins. In bacteria, DNA replication is initiated by the ubiquitous DnaA protein, which assembles into an oligomeric complex at the chromosome origin (oriC) that engages both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) to promote DNA duplex opening. However, the mechanism of DnaA specifically opening a replication origin was unknown. Here we show that Bacillus subtilis DnaAATP assembles into a continuous oligomer at the site of DNA melting, extending from a dsDNA anchor to engage a single DNA strand. Within this complex, two nucleobases of each ssDNA binding motif (DnaA-trio) are captured within a dinucleotide binding pocket created by adjacent DnaA proteins. These results provide a molecular basis for DnaA specifically engaging the conserved sequence elements within the bacterial chromosome origin basal unwinding system (BUS).

Validation of the plasmid study to relate DNA damaging effects of radionuclides to those from external beam radiotherapy.

INTRODUCTION: The biological consequences of absorbed radiation doses are ill-defined for radiopharmaceuticals, unlike for external beam radiotherapy (EBRT). A reliable assay that assesses the biological consequences of any radionuclide is much needed. Here, we evaluated the cell-free plasmid DNA assay to determine the relative biological effects of radionuclides such as Auger electron-emitting [67Ga]GaCl3 or [111In]InCl3 compared to EBRT. METHODS: Supercoiled pBR322 plasmid DNA (1.25 or 5 ng/µL) was incubated with 0.5 or 1 MBq [67Ga]GaCl3 or [111In]InCl3 for up to 73 h or was exposed to EBRT (137Cs; 5 Gy/min; 0-40 Gy). The induction of relaxed and linear plasmid DNA, representing single and double strand breaks, respectively, was assessed by gel electrophoresis. Chelated forms of 67Ga were also investigated using DOTA and THP. Topological conversion rates for supercoiled-to-relaxed (ksrx) or relaxed-to-linear (krlx) DNA were obtained by fitting a kinetic model. RESULTS: DNA damage increased both with EBRT dose and incubation time for [67Ga]GaCl3 and [111In]InCl3. Damage caused by [67Ga]GaCl3 decreased when chelated. [67Ga]GaCl3 proved more damaging than [111In]InCl3; 1.25 ng/µL DNA incubated with 0.5 MBq [67Ga]GaCl3 for 2 h led to a 70% decrease of intact plasmid DNA as opposed to only a 19% decrease for [111In]InCl3. For both EBRT and radionuclides, conversion rates were slower for 5 ng/µL than 1.25 ng/µL plasmid DNA. DNA damage caused by 1 Gy EBRT was the equivalent to damage caused by 0.5 MBq unchelated [67Ga]GaCl3 and [111In]InCl3 after 2.05 ± 0.36 and 9.3 ± 0.77 h of incubation, respectively. CONCLUSIONS: This work has highlighted the power of the plasmid DNA assay for a rapid determination of the relative biological effects of radionuclides compared to external beam radiotherapy. It is envisaged this approach will enable the systematic assessment of imaging and therapeutic radionuclides, including Auger electron-emitters, to further inform radiopharmaceutical design and application.

Three-dimensional super-resolution fluorescence imaging of DNA.

Recent advances in fluorescence super-resolution microscopy are providing important insights into details of cellular structures. To acquire three dimensional (3D) super-resolution images of DNA, we combined binding activated localization microscopy (BALM) using fluorescent double-stranded DNA intercalators and optical astigmatism. We quantitatively establish the advantage of bis- over mono-intercalators before demonstrating the approach by visualizing single DNA molecules stretched between microspheres at various heights. Finally, the approach is applied to the more complex environment of intact and damaged metaphase chromosomes, unravelling their structural features.

Editorial for the Special Issue on Optical Trapping and Manipulation: From Fundamentals to Applications.

This Special Issue of Micromachines is devoted to optical trapping, and the enormous range of uses the method has found in the decades since its first demonstration [...].

The mechanism of DNA unwinding by the eukaryotic replicative helicase.

Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication.

Annealing helicase HARP closes RPA-stabilized DNA bubbles non-processively.

We investigate the mechanistic nature of the Snf2 family protein HARP, mutations of which are responsible for Schimke immuno-osseous dysplasia. Using a single-molecule magnetic tweezers assay, we construct RPA-stabilized DNA bubbles within torsionally constrained DNA to investigate the annealing action of HARP on a physiologically relevant substrate. We find that HARP closes RPA-stabilized bubbles in a slow reaction, taking on the order of tens of minutes for ∼600 bp of DNA to be re-annealed. The data indicate that DNA re-anneals through the removal of RPA, which is observed as clear steps in the bubble-closing traces. The dependence of the closing rate on both ionic strength and HARP concentration indicates that removal of RPA occurs via an association-dissociation mechanism where HARP does not remain associated with the DNA. The enzyme exhibits classical Michaelis-Menten kinetics and acts cooperatively with a Hill coefficient of 3 ± 1. Our work also allows the determination of some important features of RPA-bubble structures at low supercoiling, including the existence of multiple bubbles and that RPA molecules are mis-registered on the two strands.

Copper-free click chemistry for attachment of biomolecules in magnetic tweezers.

BACKGROUND: Single-molecule techniques have proven to be an excellent approach for quantitatively studying DNA-protein interactions at the single-molecule level. In magnetic tweezers, a force is applied to a biopolymer that is anchored between a glass surface and a magnetic bead. Whereas the relevant force regime for many biological processes is above 20pN, problems arise at these higher forces, since the molecule of interest can detach from the attachment points at the surface or the bead. Whereas many recipes for attachment of biopolymers have been developed, most methods do not suffice, as the molecules break at high force, or the attachment chemistry leads to nonspecific cross reactions with proteins. RESULTS: Here, we demonstrate a novel attachment method using copper-free click chemistry, where a DBCO-tagged DNA molecule is bound to an azide-functionalized surface. We use this new technique to covalently attach DNA to a flow cell surface. We show that this technique results in covalently linked tethers that are torsionally constrained and withstand very high forces (>100pN) in magnetic tweezers. CONCLUSIONS: This novel anchoring strategy using copper-free click chemistry allows to specifically and covalently link biomolecules, and conduct high-force single-molecule experiments. Excitingly, this advance opens up the possibility for single-molecule experiments on DNA-protein complexes and molecules that are taken directly from cell lysate.

Skewed brownian fluctuations in single-molecule magnetic tweezers.

Measurements in magnetic tweezers rely upon precise determination of the position of a magnetic microsphere. Fluctuations in the position due to Brownian motion allows calculation of the applied force, enabling deduction of the force-extension response function for a single DNA molecule that is attached to the microsphere. The standard approach relies upon using the mean of position fluctuations, which is valid when the microsphere axial position fluctuations obey a normal distribution. However, here we demonstrate that nearby surfaces and the non-linear elasticity of DNA can skew the distribution. Through experiment and simulations, we show that such a skewing leads to inaccurate position measurements which significantly affect the extracted DNA extension and mechanical properties, leading to up to two-fold errors in measured DNA persistence length. We develop a simple, robust and easily implemented method to correct for such mismeasurements.

Toward optical-tweezers-based force microscopy for airborne microparticles.

Optical tweezers have found widespread application in biological and colloidal physics for the measurement of pN forces over nanometer to micrometer length scales. Similar aerosol-phase measurements of interparticle force have not been reported in spite of the potential to better resolve particle coagulation kinetics. Various refractive index mismatches in the beam path as well as the need to explicitly account for gravity and inertial particle motion provide a number of challenges that must be overcome to make such measurements tractable. In this regard, we demonstrate schemes by which the particle position and trap stiffness may be unambiguously measured using bright-field microscopy with resolution comparable with analogous condensed-phase measurements. Moreover, some of the challenges of working with highly dynamic aqueous particles are introduced and exploited to observe size-dependent phenomena in aerosol optical tweezers. Notably, when combined with cavity-enhanced Raman spectroscopy, this provides a unique opportunity to explore trapping forces over a continuum of particle size and refractive index. It is expected that the methods developed will provide a basis for the measurement of pairwise interaction forces in aerosol optical tweezers while providing a probe of fundamental airborne particle trapping dynamics.

Self-digitization of samples into a high-density microfluidic bottom-well array.

This paper describes a sample digitization method that generates tens of thousands of nanoliter-sized droplets in a high-density array in a matter of minutes. We show that the sample digitization depends on both the geometric design of the microfluidic device and the viscoelastic forces between the aqueous sample and a continuous oil phase. Our design avoids sample loss: Samples are split into tens of thousands of discrete volumes with close to 100% efficiency without the need for any expensive valving or pumping systems. We envision this technology will have broad applications that require simple sample digitization within minutes, such as digital polymerase chain reactions and single-cell studies.

Single aerosol trapping with an annular beam: improved particle localisation.

In this paper we explore the trapping of aerosol droplets using an annular beam, formed by blocking the central portion of a Gaussian beam, and quantify the improvements over conventional Gaussian beam traps. Recent work on the modelling of single aerosol dynamics within an optical tweezer trap [Burnham et al., Journal of the Optical Society of America B, 2011, 28, 2856-2864] has indicated that the use of annular beams can allow smaller droplets to be trapped, which we experimentally verify. We also demonstrate that annular beams allow droplets to be trapped at higher powers, and with reduced axial displacement with increasing power, than Gaussian beams. We confirm these results, due to a reduction in the axial scattering forces, using this theoretical model. Finally back focal plane interferometry is used to determine the axial and lateral trap stiffnesses for a series of droplets, showing a significant increase in the axial : lateral trap stiffness ratio from 0.79 ± 0.04 to 1.15 ± 0.04 when an annular beam is used.

Magnetic forces and DNA mechanics in multiplexed magnetic tweezers.

Magnetic tweezers (MT) are a powerful tool for the study of DNA-enzyme interactions. Both the magnet-based manipulation and the camera-based detection used in MT are well suited for multiplexed measurements. Here, we systematically address challenges related to scaling of multiplexed magnetic tweezers (MMT) towards high levels of parallelization where large numbers of molecules (say 10(3)) are addressed in the same amount of time required by a single-molecule measurement. We apply offline analysis of recorded images and show that this approach provides a scalable solution for parallel tracking of the xyz-positions of many beads simultaneously. We employ a large field-of-view imaging system to address many DNA-bead tethers in parallel. We model the 3D magnetic field generated by the magnets and derive the magnetic force experienced by DNA-bead tethers across the large field of view from first principles. We furthermore experimentally demonstrate that a DNA-bead tether subject to a rotating magnetic field describes a bicircular, Limaçon rotation pattern and that an analysis of this pattern simultaneously yields information about the force angle and the position of attachment of the DNA on the bead. Finally, we apply MMT in the high-throughput investigation of the distribution of the induced magnetic moment, the position of attachment of DNA on the beads, and DNA flexibility. The methods described herein pave the way to kilo-molecule level magnetic tweezers experiments.

Retrieval of the complex refractive index of aerosol droplets from optical tweezers measurements.

The cavity enhanced Raman scattering spectrum recorded from an aerosol droplet provides a unique fingerprint of droplet radius and refractive index, assuming that the droplet is homogeneous in composition. Aerosol optical tweezers are used in this study to capture a single droplet and a Raman fingerprint is recorded using the trapping laser as the source for the Raman excitation. We report here the retrieval of the real part of the refractive index with an uncertainty of ± 0.0012 (better than ± 0.11%), simultaneously measuring the size of the micrometre sized liquid droplet with a precision of better than 1 nm (< ± 0.05% error). In addition, the equilibrium size of the droplet is shown to depend on the laser irradiance due to optical absorption, which elevates the droplet temperature above that of the ambient gas phase. Modulation of the illuminating laser power leads to a modulation in droplet size as the temperature elevation is altered. By measuring induced size changes of <1 nm, we show that the imaginary part of the refractive index can be retrieved even when less than 10 × 10(-9) with an accuracy of better than ± 0.5 × 10(-9). The combination of these measurements allows the complex refractive index of a droplet to be retrieved with high accuracy, with the possibility of making extremely sensitive optical absorption measurements on aerosol samples and the testing of frequently used mixing rules for treating aerosol optical properties. More generally, this method provides an extremely sensitive approach for measuring refractive indices, particularly under solute supersaturation conditions that cannot be accessed by simple bulk-phase measurements.

Systematic investigation of droplet generation at T-junctions.

Droplet microfluidics has attracted much attention in recent years. For many droplet-based applications, researchers want to predict the size of the droplets in a certain experimental condition. To meet this need, van Steijn and colleagues proposed an elegant theoretical model that predicts the volume of droplets generated in a common channel configuration for forming a steady-state, continuous stream of droplets, the T-junction geometry. To determine the accuracy of this model in predicting droplet volume, we performed a systematic experimental study over two orders of magnitude in capillary number. We found that this model, albeit elegant, has a limited range of interfacial tension over which it can predict accurately the droplet volume. Our experimental results, together with fluid dynamic simulations, allowed us to highlight the importance of physical fluid properties when employing theoretical models.

Bioconjugation of ultrabright semiconducting polymer dots for specific cellular targeting.

Semiconducting polymer dots (Pdots) represent a new class of ultrabright fluorescent probes for biological imaging. They exhibit several important characteristics for experimentally demanding in vitro and in vivo fluorescence studies, such as their high brightness, fast emission rate, excellent photostability, nonblinking, and nontoxic feature. However, controlling the surface chemistry and bioconjugation of Pdots has been a challenging problem that prevented their widespread applications in biological studies. Here, we report a facile yet powerful conjugation method that overcomes this challenge. Our strategy for Pdot functionalization is based on entrapping heterogeneous polymer chains into a single dot, driven by hydrophobic interactions during nanoparticle formation. A small amount of amphiphilic polymer bearing functional groups is co-condensed with the majority of semiconducting polymers to modify and functionalize the nanoparticle surface for subsequent covalent conjugation to biomolecules, such as streptavidin and immunoglobulin G (IgG). The Pdot bioconjugates can effectively and specifically label cellular targets, such as cell surface marker in human breast cancer cells, without any detectable nonspecific binding. Single-particle imaging, cellular imaging, and flow cytometry experiments indicate a much higher fluorescence brightness of Pdots compared to those of Alexa dye and quantum dot probes. The successful bioconjugation of these ultrabright nanoparticles presents a novel opportunity to apply versatile semiconducting polymers to various fluorescence measurements in modern biology and biomedicine.

Optical Trapping Enabled Parallel Delivery of Biological Stimuli with High Spatial and Temporal Resolution.

We have developed a method that employs nanocapsules, optical trapping, and single-pulse laser photolysis for delivering bioactive molecules to cells with both high spatial and temporal resolutions. This method is particularly suitable for a cell-culture setting, in which a single nanocapsule can be optically trapped and positioned at a pre-defined location next to the cell, followed by single-pulse laser photolysis to release the contents of the nanocapsule onto the cell. To parallelize this method such that a large array of nanocapsules can be manipulated, positioned, and photolyzed simultaneously, we have turned to the use of spatial light modulators and holographic beam shaping techniques. This paper outlines the progress we have made so far and details the issues we had to address in order to achieve efficient parallel optical manipulations of nanocapsules and particles.

Spectroscopic characterisation and manipulation of arrays of sub-picolitre aerosol droplets.

Arrays of optically tweezed aerosol droplets, each of sub-picolitre volume, are manipulated by holographic optical tweezers and characterised by cavity enhanced Raman spectroscopy. A spatial light modulator is employed to generate arrays of optical traps from a single laser beam and to control the array dimensions and relative trap positions. Comparative hygroscopicity measurements are performed concurrently on five trapped droplets by monitoring the evolving size of each droplet. This is extended to the controlled coalescence of an array of droplets accompanied by spectroscopic measurements. These data represent the first ever simultaneous measurements of the evolving composition and size of an array of aerosol droplets. We consider the possibility of using aerosol arrays as a platform for studying chemical reactions in sub-picolitre volumes, exploiting the versatility of aerosol arrays for performing optical digital microfluidic operations accompanied by micro-total analysis.

Optical manipulation of airborne particles: techniques and applications.

In the following paper, we discuss new methods to trap and manipulate airborne liquid aerosol droplets. We discuss the single gradient force trapping of water aerosols in the 2-14 micron diameter range using both 532 nm and 1064 nm light, as well as the holographic optical trapping of arrays of aerosols. Using this holographic technique, we are able to show controlled aerosol coagulation. We also discuss two techniques based on the radiation pressure trapping of aerosols, namely the dual beam fibre trap and the controlled guiding of aerosols using Bessel beams. We conclude with a discussion of new topics for study based upon these techniques and some possible applications.