Enhanced Golden Gate Assembly: evaluating overhang strength for improved ligation efficiency.

Molecular cloning, a routine yet essential technique, relies heavily on efficient ligation, which can be significantly improved using Golden Gate Assembly (GGA). A key component of GGA is the use of type IIS enzymes, which uniquely cleave downstream of their recognition sequences to generate various overhangs, including non-palindromic ones. Recent advancements in GGA include the development of newly engineered enzymes with enhanced activity. Additionally, high-throughput GGA assays, which allow for the simultaneous study of all possible overhangs, have identified optimal GGA substrates with high efficiencies and fidelities, greatly facilitating the design of complex assemblies. Interestingly, these assays reveal unexpected correlations between ligation efficiencies and overhang stabilities. One hypothesis for this observation is that newly hydrolyzed DNA fragments with strong overhangs can readily re-ligate, thereby slowing down the overall process. In this paper, we employ a combination of gel electrophoresis and numerical calculations to test this hypothesis, ultimately determining that it does not hold true under the conditions established by conventional GGA assays. Using an assembly of 10 fragments, we demonstrate that strong overhangs yield higher GGA efficiency, while weak overhangs result in lower efficiency. These findings enable us to propose optimal overhangs for efficient GGA assays, significantly increasing yield.

Robust, high-yield, rapid fabrication of DNA constructs for Magnetic Tweezers.

Single-molecule techniques are highly sensitive tools that can reveal reaction intermediates often obscured in experiments involving large ensembles of molecules. Therefore, they provide unprecedented information on the mechanisms that control biomolecular reactions. Currently, one of the most significant single-molecule assays is Magnetic Tweezers (MT), which probes enzymatic reactions at high spatio-temporal resolutions on tens, if not hundreds, of molecules simultaneously. For high-resolution MT experiments, a short double-stranded DNA molecule (less than 2000 base pairs) is typically attached between a micron-sized superparamagnetic bead and a surface. The fabrication of such a substrate is key for successful single-molecule assays, and several papers have discussed the possibility of improving the fabrication of short DNA constructs. However, reported yields are usually low and require additional time-consuming purification steps (e.g., gel purification). In this paper, we propose the use of a Golden Gate Assembly assay that allows for the production of DNA constructs within minutes (starting from PCR products). We discuss how relevant parameters may affect the yield and offer single-molecule experimentalists a simple yet robust approach to fabricate DNA constructs.

Aggregation kinetics of a concentrated colloidal suspension under oscillatory flow.

The aggregation behavior of an attractive colloidal silica suspension under oscillatory flow is studied using rheological measurement. We show that the competition between the aggregation of the particles and the aggregate breakup under external stress leads to a non-monotonous evolution of the elastic modulus with time. Remarkably, under certain conditions, the elasticity is not an increasing function of time but exhibits a maximum. The value of the maximum of the elastic modulus depends on the applied shear amplitude and the ionic strength of the suspension. Scaling laws that describes the evolutions of the elastic modulus as a function of the salinity and of the deformation amplitude are proposed and discussed.

Magnetic Control over the Fractal Dimension of Supramolecular Rod Networks.

Controlling supramolecular polymerization is of fundamental importance to create advanced materials and devices. Here we show that the thermodynamic equilibrium of Gd3+-bearing supramolecular rod networks is shifted reversibly at room temperature in a static magnetic field of up to 2 T. Our approach opens opportunities to control the structure formation of other supramolecular or coordination polymers that contain paramagnetic ions.

Jet instability of a shear-thickening concentrated suspension.

We investigate the flow of a concentrated suspension of colloidal particles at deformation rates higher than the discontinuous shear-thickening transition shear rate. We show that, under its own weight, a jet of a concentrated enough colloidal suspension, simultaneously flows while it sustains tensile stress and transmits transverse waves. This results in a new flow instability of jets of shear-thickening suspensions: the jet is submitted to rapid transverse oscillations, that we characterize.

Geometrical instability in the imbibition of a sphere.

We study the imbibition of a spherical porous aggregate. When the difference in pressure between the inside and the outside of the aggregate is large enough, the imbibition front becomes unstable. This instability leads to the acceleration of the imbibition process. In more complex geometries with non-constant curvatures, the imbibition becomes locally unstable in the regions with the highest curvatures, leading to spatially heterogeneous front velocities.

Complexation of DNA with ruthenium organometallic compounds: the high complexation ratio limit.

Interactions between DNA and ruthenium organometallic compounds are studied by using visible light absorption and circular dichroism measurements. A titration technique allowing for the absolute determination of the advancement degree of the complexation, without any assumption about the number of complexation modes is developed. When DNA is in excess, complexation involves intercalation of one of the organometallic compound ligands between DNA base pairs. But, in the high complexation ratio limit, where organometallic compounds are in excess relative to the DNA base pairs, a new mode of interaction is observed, in which the organometallic compound interacts weakly with DNA. The weak interaction mode, moreover, develops when all the DNA intercalation sites are occupied. A regime is reached in which one DNA base pair is linked to more than one organometallic compound.

Large-Scale Statistical Analysis of Defect Emission in hBN: Revealing Spectral Families and Influence of Flake Morphology.

Quantum emitters in two-dimensional layered hexagonal boron nitride are quickly emerging as a highly promising platform for next-generation quantum technologies. However, the precise identification and control of defects are key parameters to achieve the next step in their development. We conducted a comprehensive study by analyzing over 10,000 photoluminescence emission lines from liquid exfoliated hBN nanoflake samples, revealing 11 narrow sets of defect families within the 1.6 to 2.2 eV energy range. This challenges hypotheses of a random energy distribution. We also reported averaged defect parameters, including emission line widths, spatial density, phonon side bands, and Franck-Condon-related factors. These findings provide valuable insights into deciphering the microscopic origin of emitters in hBN hosts. We also explored the influence of the hBN host morphology on defect family formation, demonstrating its crucial impact. By tuning the flake size and arrangement, we achieve selective control of defect types while maintaining high spatial density. This offers a scalable approach to defect emission control, diverging from costly engineering methods. It emphasizes the significance of the morphological aspects of hBN hosts for gaining insights into defect origins and expanding their spectral control.

Spectral properties of polypyridyl ruthenium complexes intercalated in DNA: theoretical insights into the surrounding effects of [Ru(dppz)(bpy)2]2+.

The UV/Visible absorption properties of a polypyridyl ruthenium complex upon intercalation on DNA are studied at the mixed quantum mechanics molecular mechanics level of theory. Vertical excitation transitions are computed by time dependent density functional theory. Particular emphasis is put on the different levels at which the macromolecular environment is treated, and in particular on the analysis of the effect of mechanical, electrostatic and polarizable embedding. We show that with the highest level of theory the experimental absorption wavelengths are reproduced with a difference of only 2 or 3 nm for the low energy bands. The systematic analysis of the individual vertical transitions allows us to get much more insights into the role played by the environment, in particular, in metal to ligand and intra ligand charge transfer transitions that can lead to the production of DNA oxidative lesions exploitable in phototherapy.

Stress overshoot, hysteresis, and the Bauschinger effect in sheared dense colloidal suspensions.

The mechanical nonlinear response of dense Brownian suspensions of polymer gel particles is studied experimentally and by means of numerical simulations. It is shown that the response to the application of a constant shear rate depends on the previous history of the suspension. When the flow starts from a suspension at rest, it exhibits an elastic response followed by a stress overshoot and then a plastic flow regime. Conversely, after flow reversal, the stress overshoot does not occur, and the apparent elastic modulus is reduced while numerical simulations reveal that the anisotropy of the local microstructure is delayed relative to the macroscopic stress.

Formation and self-organization kinetics of alpha-CD/PEO-based pseudo-polyrotaxanes in water. A specific behavior at 30 degrees C.

alpha-Cyclodextrins (alpha-CDs) have the ability to form inclusion complexes with poly(ethylene oxide) (PEO) polymer chains. These pseudo-polyrotaxanes (PPRs) can be obtained by quenching an alpha-CD/PEO mixture in water from 70 degrees C down to a lower temperature (typically in the range from 5 to 30 degrees C) thanks to favorable interactions between alpha-CD cavities and PEO chains. Moreover, starting from a liquid alpha-CD/PEO mixture at a total mass fraction of 15% w/w at 70 degrees C, the formation of PPRs with time at a lower temperature induces a white physical gel with time, and phase separation is observed. We established that PPR molecules are exclusively found in the precipitated phase although unthreaded alpha-CD molecules and unthreaded PEO chains are in the liquid phase. At 30 degrees C, the physical gel formation is much slower than at 5 degrees C. At 30 degrees C, we established that, in a first step, alpha-CDs thread onto PEO chains, forming PPR molecules which are not in good solvent conditions in water. At a higher length scale, rapid aggregation of the PPR molecules occurs, and threaded alpha-CD-based nanocylinders form (cylinder length L = 5.7 nm and cylinder radius R = 4.7 nm). At a higher length scale, alpha-CD-based nanocylinders associate in a Gaussian way, engendering the formation of precipitated domains which are responsible for the high turbidity of the studied system. At the end of this first step (i.e., after 20 min), the system still remains liquid and the PPRs are totally formed. Then, in a second step (i.e., after 150 min), the system undergoes its reorganization characterized by a compacity increase of the precipitated domains and forms a physical gel. We found that PPRs are totally formed after 20 min at 30 degrees C and that the system stays in a nongel state up to 150 min. This opens new perspectives regarding the PPR chemical modification: between these two characteristic times, we can easily envisage an efficient chemical modification of the PPR molecules in water, as for instance an end-capping reaction leading to the synthesis of polyrotaxanes.

Flexibility and thermal dynamic stability increase of dsDNA induced by Ru(bpy)2dppz2+ based on AFM and HRM technique.

Ru(bpy)2dppz2+ has been widely used as a probe for exploring the structure of double-stranded DNA (dsDNA). The flexibility change of DNA helix is important in many of its biological functions but not well understood. Here, flexibility change of dsDNA helix caused by intercalation with Ru(bpy)2dppz2+ was investigated using the atomic force microscopy. At first, the interactions between ruthenium complex and dsDNA helix were characterized and the binding site size (p = 2.87 bp) and binding constant (Ka = 5.9 * 107 M-1) were determined by the relative extension of DNA helix using the equation of McGhee and von Hippel. By measuring intercalator-induced DNA elongation and the mean square of end-to-end distance at different molar ratios of Ru(bpy)2dppz2+ to dsDNA, the changes of persistence length under different ruthenium concentrations were determined by the worm-like chain model. We found that the persistence length of dsDNA decreased with increasing Ru(bpy)2dppz2+ concentration, demonstrating that the flexibility of dsDNA obviously enhanced due to the intercalation. Especially, the persistence length changed greatly from 54 to 34 nm on changing the molar ratio of ruthenium to dsDNA from 0 to 0.2. We speculated that the intercalation of dsDNA with Ru(bpy)2dppz2+ resulted in local deformation or bending of the DNA duplex. In addition, the thermal dynamic stability of DNA helix was measured with high resolution melting method which revealed the increase in thermal dynamic stability of DNA helix due to the ruthenium intercalation.

Holographic Traction Force Microscopy.

Traction Force Microscopy (TFM) computes the forces exerted at the surface of an elastic material by measuring induced deformations in volume. It is used to determine the pattern of the adhesion forces exerted by cells or by cellular assemblies grown onto a soft deformable substrate. Typically, colloidal particles are dispersed in the substrate and their displacement is monitored by fluorescent microscopy. As with any other fluorescent techniques, the accuracy in measuring a particule's position is ultimately limited by the number of evaluated fluorescent photons. Here, we present a TFM technique based on the detection of probe particle displacements by holographic tracking microscopy. We show that nanometer scale resolutions of the particle displacements can be obtained and determine the maximum volume fraction of markers in the substrate. We demonstrate the feasibility of the technique experimentally and measure the three-dimensional force fields exerted by colorectal cancer cells cultivated onto a polyacrylamide gel substrate.

Polymer-free electrospinning of tannic acid and cross-linking in water for hybrid supramolecular nanofibres.

Electrospinning is the process of choice allowing the preparation of nanofibrous materials from a solution usually based on a high molar mass polymer. The solution must bring enough chain entanglements to avoid any breaking or Rayleigh instability of the electrospun jet resulting thus in the deposition of a continuous and regular solid nanofibre. It has been however shown that some few non-polymeric molecules can be electrospun without using a carrier polymer. We demonstrate here the case of tannic acid. Indeed, it was possible to electrospin this molecule solubilised in a mixture of water and ethanol as well as in pure water. Rheology, dynamic light scattering and cryo-TEM highlight the formation of tannic acid aggregates in solution. Above a critical concentration, these aggregates form a supramolecular interconnected network strong enough to allow the electrospinning of a continuous and regular nanofibre. The resulting nanoweb is mechanically stable and can be handled and wrapped. Furthermore, as opposed to the other small molecules for which polymer-free electrospinning was also demonstrated, tannic acid nanowebs can be efficiently cross-linked in water either by oxidative reaction with sodium periodate or, most interestingly, with FeIII by a combination of oxidative reaction and the formation of coordination complexes. The proposed electrospinning and cross-linking strategy is easy, of low cost, and scalable and uses non-toxic solvents as well as biocompatible and biofunctional molecules. Furthermore, thanks to the chelation capacity of tannic acid having the ability to coordinate with a wide variety of metals, hybrid smart nanowebs can be envisaged for diverse applications such as biomedical, catalysis as well as environment.

Investigation on the pitting of potato starch granules during high frequency ultrasound treatment.

In this paper, the pitting of potato starch granules in aqueous suspensions (1%) by high-frequency high-power ultrasound (850kHz at a power of 0.2W, 2W or 3.7W; and also 500kHz and 1MHz at a power of 2W) is reported. The number of pits per starch granules was found to be independent of the amylose content of starches, and the surface properties of starch granules as modified through SDS and ethanol washing. At 850kHz, the maximum number of pits per starch granule, for both normal and waxy starches, did not exceed 11. However, a close inspection of fractionated starch granules based on their sizes showed that there is an optimum granule size for which a maximum pit number is obtained. For example, starch granules with diameter size range of ∼15 to ∼30µm had a maximum pit number (between 10 and 20 pits per granule) when sonicated (2W, 850kHz and 30min); while sonication of small (<10µm) and very large (>45µm) granules resulted in a smaller number of pits per granule (∼5). Further, the maximum number of pits per granules is also found to be proportional to the ultrasound frequency, with values of approximately 7, 10 and 11 at 0.50, 0.85, and 1MHz, respectively. FTIR measurements did not show any breakup of starch molecules.

Relaxation of jammed colloidal suspensions after shear cessation.

The dynamics of heterogeneities in a shear thickening, concentrated colloidal suspension is investigated through speckle visibility spectroscopy, a dynamic light scattering technique recently introduced [P. K. Dixon and D. J. Durian, Phys. Rev. Lett. 90, 184302 (2003)]. Formation of shear-induced heterogeneities is observed in the jamming regime, and their relaxation after shear cessation is monitored as a function of the applied shear stress. The relaxation time of these heterogeneities increases when a higher stress is applied.

Crackling of a coagulating suspension.

A way of analyzing the intermittent dynamics of nonstationary systems is introduced. It is used to study the dynamics of a concentrated suspension while it is flocculating, probed by diffusing wave spectroscopy. When the repulsion interactions between the particles are slowly decreased their motion appears to be intermittent. It is characterized by rapid crackling periods, separated by arrested dynamics. Duration of the crackling periods exhibit a power-law distribution function.

Aging of a colloidal glass under a periodic shear.

The aging dynamics under a periodic shear of a concentrated suspension of saponite particles is measured. It is observed that the dynamics is fastened by the application of a moderate shear amplitude. Nevertheless, this acceleration does not affect the dynamics of the suspension when the shear is ceased. By applying a succession of shear of various amplitudes, we conclude that the dynamics of the suspension at a time t(w) after complete rejuvenation is independent of the shear history between times 0 and t(w) , as soon as the amplitude of the applied shear is smaller than the characteristic shear gamma(c) necessary to completely rejuvenate the suspension.

Flexibility of short ds-DNA intercalated by a dipyridophenazine ligand.

We use Förster Resonant Energy Transfer (FRET) in order to measure the increase of flexibility of short ds-DNA induced by the intercalation of dipyridophenazine (dppz) ligand in between DNA base pairs. By using a DNA double strand fluorescently labeled at its extremities, it is shown that the end-to-end length increase of DNA due to the intercalation of one dppz ligand is smaller than the DNA base pair interdistance. This may be explained either by a local bending of the DNA or by an increase of its flexibility. The persistence length of the formed DNA/ligand is evaluated. The described structure may have implications in the photophysical damages induced by the complexation of DNA by organometallic molecules.