Biotinylation of reducing and non-reducing termini to create plug-and-play polysaccharides.

Single-molecule studies continue to grow in popularity. In cases where biopolymer samples of interest exhibit variations in fine-structure between individual chains such single-molecule studies uniquely offer the promise of revealing deep structure-function relationships. Polysaccharides are typically studied in bulk and, as such, their study could greatly benefit from the application of single-molecule techniques. However, while for example single-molecule optical tweezers (OT) studies have become commonplace for DNA, studies of polysaccharides have lagged behind somewhat, complicated by the difficulty of studying molecules that amongst other things have more complex end-group chemistry. Recently, divalent streptavidin linkers have been shown to be capable of concatenating two pieces of biotin-terminated DNA to produce robust composite strings that run intact through conventional gels, and can be used in single-molecule OT experiments (Mohandas, Kent, Raudsepp, Jameson, & Williams, 2022). By using two such streptavidin linkers, biotin-terminated polymers could be inserted between two sections of DNA in order to facilitate single-molecule experiments on biopolymers that are currently difficult to address by other means. Here, we describe a generic approach for placing the required biotin moieties at both ends of polysaccharide chains, producing plug-and-play polysaccharide inserts that can be incorporated into composite polymer strings using streptavidin linking hubs.

Modeling multiple duplex DNA attachments in a force-extension experiment.

Optical tweezers-based DNA stretching often relies on tethering a single end-activated DNA molecule between optically manipulated end-binding beads. Measurement success can depend on DNA concentration. At lower DNA concentrations tethering is less common, and many trials may be required to observe a single-molecule stretch. At higher DNA concentrations tethering is more common; however, the resulting force-extensions observed are more complex and may vary from measurement to measurement. Typically these more complex results are attributed to the formation of multiple tethers between the beads; however, to date there does not appear to have been a critical examination of this hypothesis or the potential usefulness of such data. Here we examine stretches at a higher DNA concentration and use analysis and simulation to show how the more complex force-extensions observed can be understood in terms of multiple DNA attachments.

Interfacial colloidal assembly guided by optical tweezers and tuned via surface charge.

HYPOTHESIS: The size, shape and dynamics of assemblies of colloidal particles optically-trapped at an air-water interface can be tuned by controlling the optical potential, particle concentration, surface charge density and wettability of the particles and the surface tension of the solution. EXPERIMENTS: The assembly dynamics of different colloidal particle types (silica, polystyrene and carboxyl coated polystyrene particles) at an air-water interface in an optical potential were systematically explored allowing the effect of surface charge on assembly dynamics to be investigated. Additionally, the pH of the solutions were varied in order to modulate surface charge in a controllable fashion. The effect of surface tension on these assemblies was also explored by reducing the surface tension of the supporting solution by mixing ethanol with water. FINDINGS: Silica, polystyrene and carboxyl coated polystyrene particles showed distinct assembly behaviours at the air-water interface that could be rationalised taking into account changes in surface charge (which in addition to being different between the particles could be modified systematically by changing the solution pH). Additionally, this is the first report showing that wettability of the colloidal particles and the surface tension of the solution are critical in determining the resulting assembly at the solution surface.

Estimating orientation of optically trapped, near vertical, microsphere dimers using central moments and off-focus imaging.

Near vertical optically trapped dimers, composed of pairs of microspheres, and constructed in situ, were imaged in bright-field in flow and at rest, and with displacement Δz from the transverse xy imaging plane of an inverted microscope. Image first central moments µ01 were measured, and their dependence on the imposed flow velocity of the surrounding fluid was calculated. This dependence was related to the at-rest restricted diffusion statistics. It was assumed that, for small perturbations, the torque T on the dimer was proportional to the velocity of flow v and resulting angular deflection Δθ so that T∝v∝Δθ. Displacements Δz at which vâˆΔµ01∝Δθ, which are typically off focus, were examined in more detail; in this range, Δθ=hΔµ01. The hydrodynamics of the dimer were modeled as that of a prolate ellipsoid, and the constant of proportionality h was determined by comparing the short-time mean-squared variation measured during diffusion to that predicted by the model calculation: h2⟨Δµ012(t)⟩=⟨Δθ2(t)⟩. With h determined, the optical trap stiffness kθ was determined from the long-time restricted diffusion of the dimer. The measured kθ and Δθ can then be used compute torque: T=kθΔθ, potentially enabling the near vertical optically trapped dimer to be used as a torque probe.

Progress toward Plug-and-Play Polymer Strings for Optical Tweezers Experiments: Concatenation of DNA Using Streptavidin Linkers.

Streptavidin is a tetrameric protein that is renowned for its strong binding to biotin. The robustness and strength of this noncovalent coupling has led to multitudinous applications of the pairing. Within the streptavidin tetramer, each protein monomer has the potential to specifically bind one biotin-bearing moiety. Herein, by separating various streptavidin species that have had differing numbers of their four potential binding sites blocked, several different types of "linking hub" were obtained, each with a different valency. The identification of these species and the study of the plugging process used to block sites during their preparation were carried out using capillary electrophoresis. Subsequently, a specific species, namely, a trans-divalent linker, in which the two open biotin-binding pockets are approximately opposite one another, was used to concatenate two ∼5 kb pieces of biotin-terminated double-stranded DNA. Following the incubation of this DNA with the prepared linker, a fraction of ∼10 kb strings was identified using gel electrophoresis. Finally, these concatenated DNA strings were stretched in an optical tweezer experiment, demonstrating the potential of the methodology for coupling and extending molecules for use in single-molecule biophysical experiments.

Changes in the properties of membrane tethers in response to HP1α depletion in MCF7 cells.

Plasma membrane tension is known to regulate many cell functions, such as motility and membrane trafficking. Membrane tether pulling is an effective method for measuring the apparent membrane tension of cells and exploring membrane-cytoskeleton interactions. In this article, the mechanical properties of HP1α-depleted MCF7 breast cancer cells are explored in comparison to controls, by pulling membrane tethers using optical tweezers. These studies were inspired by previous findings that a loss of HP1α correlates with an increase in the invasive potential of malignant cancer cells. Specifically, the membrane tension and force relaxation curves for tethers pulled from MCF7 breast cancer cells with HP1α knockdown and their matched controls were measured, and shown to be significantly different.

Optical microlever assisted DNA stretching.

Optical microrobotics is an emerging field that has the potential to improve upon current optical tweezer studies through avenues such as limiting the exposure of biological molecules of interest to laser radiation and overcoming the current limitations of low forces and unwanted interactions between nearby optical traps. However, optical microrobotics has been historically limited to rigid, single-body end-effectors rather than even simple machines, limiting the tasks that can be performed. Additionally, while multi-body machines such as microlevers exist in the literature, they have not yet been successfully demonstrated as tools for biological studies, such as molecule stretching. In this work we have taken a step towards moving the field forward by developing two types of microlever, produced using two-photon absorption polymerisation, to perform the first lever-assisted stretches of double-stranded DNA. The aim of the work is to provide a proof of concept for using optical micromachines for single molecule studies. Both styles of microlevers were successfully used to stretch single duplexes of DNA, and the results were analysed with the worm-like chain model to show that they were in good agreement.

Inside the ensemble: unlocking the potential of one-at-a-time experiments with lab-on-a-chip automation.

The advent of technologies that allow the interactions of individual microscopic particles to be probed "one-at-a-time" has paved the way for new experimental avenues of enquiry in colloidal systems. For example, investigating whether a particular pair of colloidal particles isolated from a macroscopic sample might adhere to each other when brought into close proximity is certainly possible. However, given the probabilistic nature of the process (different particles within the ensemble may have slightly different surface charge distributions and asperities, and interaction energies involved can be close to thermal values), it is important that many hundreds or thousands of pairs of particles are tested under each set of experimental conditions of interest. Currently it is still an arduous task to perform such an experiment a sufficient number of times in order to acquire a data-set that truly represents the ensemble. Herein an automated particle collider for measuring particle-particle interactions has been realized by combining elements of microfluidics, holographic optical tweezers and image processing. Each individual measurement consists of confining two particles within a predetermined chemical micro-environment, and observing whether their interactions lead to aggregation. To automate the measurements, computer software consisting of LabVIEW and Red Tweezers with a custom plugin was used. Preliminary experiments carried out using 1 µm diameter polystyrene particles demonstrated that many hundreds of pairwise-interaction measurements could be carried out autonomously within a matter of hours. Further exemplar real-world experiments, designed to examine the stickiness of emulsion drops as a function of bulk measurements of the ζ-potential (zeta potential) of the sample, were then performed. It is envisaged that such robust approaches to the automation of "one-at-a-time" experiments will find applications in a large number of areas, and enable previously unthinkable experiments to be carried out in a timely fashion, thus allowing the focus to shift away from tedious experimental frustrations to more profound scientific questions.

Complexes of ß-lactoglobulin and high methyl-esterified pectin as a one-shot delivery system for reinforcing oil/water interfaces.

Electrostatic complexation of negatively charged polysaccharides with ß-lactoglobulin (ß-lg) has been shown to bolster the protein films at oil/water interfaces thereby improving emulsion stability. However, recent sub-phase exchange experiments demonstrated that highly charged polysaccharides such as low methyl-esterified pectin are complementary only if sequentially introduced to a pre-formed interfacial ß-lg film. In this study, results of transient interfacial shear rheology show that, by using high-methylesterified pectins instead, complexes can be formed in pre-mixed solutions with ß-lg at pH 4 that can lead to reinforced protein films at dodecane/water interfaces. Using this one-shot adsorption of such complexes, pectins as well as short chain polysaccharides like homogalacturonan nearly doubled the steady state shear elastic moduli as compared to that of a pure ß-lg film. The lag times of film formation were established to be primarily decided by the charge density and pattern on the polysaccharide. Based on the results from mixed solutions of ß-lg monomers, it is proposed that the polysaccharide at pH 4 strengthens the resulting interfacial layer by concatenating adsorbed ß-lg molecules thereby establishing cross-links in the aqueous phase.

Depletion of HP1α alters the mechanical properties of MCF7 nuclei.

Within the nucleus of the eukaryotic cell, DNA is partitioned into domains of highly condensed, transcriptionally silent heterochromatin and less condensed, transcriptionally active euchromatin. Heterochromatin protein 1α (HP1α) is an architectural protein that establishes and maintains heterochromatin, ensuring genome fidelity and nuclear integrity. Although the mechanical effects of changes in the relative amount of euchromatin and heterochromatin brought about by inhibiting chromatin-modifying enzymes have been studied previously, here we measure how the material properties of the nuclei are modified after the knockdown of HP1α. These studies were inspired by the observation that poorly invasive MCF7 breast cancer cells become more invasive after knockdown of HP1α expression and that, indeed, in many solid tumors the loss of HP1α correlates with the onset of tumor cell invasion. Atomic force microscopy (AFM), optical tweezers (OT), and techniques based on micropipette aspiration (MA) were each used to characterize the mechanical properties of nuclei extracted from HP1α knockdown or matched control MCF7 cells. Using AFM or OT to locally indent nuclei, those extracted from MCF7 HP1α knockdown cells were found to have apparent Young's moduli that were significantly lower than nuclei from MCF7 control cells, consistent with previous studies that assert heterochromatin plays a major role in governing the mechanical response in such experiments. In contrast, results from pipette-based techniques in the spirit of MA, in which the whole nuclei were deformed and aspirated into a conical pipette, showed considerably less variation between HP1α knockdown and control, consistent with previous studies reporting that it is predominantly the lamins in the nuclear envelope that determine the mechanical response to large whole-cell deformations. The differences in chromatin organization observed by various microscopy techniques between the MCF7 control and HP1α knockdown nuclei correlate well with the results of our measured mechanical responses and our hypotheses regarding their origin.

Optical Micromachines for Biological Studies.

Optical tweezers have been used for biological studies since shortly after their inception. However, over the years research has suggested that the intense laser light used to create optical traps may damage the specimens being studied. This review aims to provide a brief overview of optical tweezers and the possible mechanisms for damage, and more importantly examines the role of optical micromachines as tools for biological studies. This review covers the achievements to date in the field of optical micromachines: improvements in the ability to produce micromachines, including multi-body microrobots; and design considerations for both optical microrobots and the optical trapping set-up used for controlling them are all discussed. The review focuses especially on the role of micromachines in biological research, and explores some of the potential that the technology has in this area.

Polysaccharide Structures in the Outer Mucilage of Arabidopsis Seeds Visualized by AFM.

Evidence is presented that the polysaccharide rhamnogalacturonan I (RGI) can be biosynthesized in remarkably organized branched configurations and surprisingly long versions and can self-assemble into a plethora of structures. AFM imaging has been applied to study the outer mucilage obtained from wild-type (WT) and mutant (bxl1-3 and cesa5-1) Arabidopsis thaliana seeds. For WT mucilage, ordered, multichain structures of the polysaccharide RGI were observed, with a helical twist visible in favorable circumstances. Molecular dynamics (MD) simulations demonstrated the stability of several possible multichain complexes and the possibility of twisted fibril formation. For bxl1-3 seeds, the imaged polymers clearly showed the presence of side chains. These were surprisingly regular and well organized with an average length of ∼100 nm and a spacing of ∼50 nm. The heights of the side chains imaged were suggestive of single polysaccharide chains, while the backbone was on average 4 times this height and showed regular height variations along its length consistent with models of multichain fibrils examined in MD. Finally, in mucilage extracts from cesa5-1 seeds, a minor population of chains in excess of 30 µm long was observed.

Aggregation and coalescence of partially crystalline emulsion drops investigated using optical tweezers.

The solid content of viscoelastic emulsion drops is known to affect their propensity for aggregation and their subsequent coalescence behaviour, where the balance between the drive to reduce surface tension and the straining of an internal viscoelastic network is able to create a plethora of stable partially-coalesced states. The latter has previously been elegantly demonstrated in synthetic systems, generated using oil containing different phase volumes of added solids, with micro-pipette experiments carried out on emulsion drops of several tens of microns in size. Herein we carry out experiments in the same spirit but aided by optical tweezers (OT) and using smaller micron-sized emulsion drops generated from milk fat. Given the size dependence of Brownian fluctuations and Laplace pressure the experimental investigation of these smaller drops is not necessarily a trivial extension of the previous work. The solid content of initially separated drops is controlled using a temperature-cycling regime in the sample preparation protocol, and subsequently the propensity for drops to remain joined or not after being brought into contact was examined. Aggregated pairs of drops were then subjected to an increase in temperature, either locally using a high-powered laser, or more globally using a custom-made Peltier temperature-controller. By heating to different degrees, the amount of fat crystals in the drops was able to be controlled, with progressively more compact partially-coalesced states, and eventually complete coalescence generated as the solid content was reduced. While in contrast to previous studies, the emulsion studied here was quite different in size and nature, and the solid content was controlled using temperature, the same underlying physics was nevertheless observed.

Lubrication by biomacromolecules: mechanisms and biomimetic strategies.

Biomacromolecules play a key role in protecting human biointerfaces from friction and wear, and thus enable painless motion. Biomacromolecules give rise to remarkable tribological properties that researchers have been eager to emulate. In this review, we examine how molecules such as mucins, lubricin, hyaluronic acid and other components of biotribological interfaces provide a unique set of rheological and surface properties that leads to low friction and wear. We then highlight how researchers have used some of the features of biotribological contacts to create biomimetic systems. While the brush architecture of the glycosylated molecules present at biotribological interfaces has inspired some promising polymer brush systems, it is the recent advance in the understanding of synergistic interaction between biomacromolecules that is showing the most potential in producing surfaces with a high lubricating ability. Research currently suggests that no single biomacromolecule or artificial polymer successfully reproduces the tribological properties of biological contacts. However, by combining molecules, one can enhance their anchoring and lubricating capacity, thus enabling the design of surfaces for use in biomedical applications requiring low friction and wear.

Effects of Pressure and pH on the Physical Stability of an I-Motif DNA Structure.

The thermodynamic stability of a cytosine(C)-rich i-motif tract of DNA, which features pH-sensitive [C..H..C]+ moieties, has been studied as function of both pressure (0.1-200 MPa) and pH (3.7-6.2). Careful attention was paid to correcting citrate buffer pH for known variations that stem from changes in pressure. Once pH-corrected, (i) at pH >4.6 the i-motif becomes less stable as pressure is increased (KD decreases), giving a small negative volume change for dissociation (ΔD V°) of the i-motif - a conclusion opposite to that which would be drawn if the buffer pH was not corrected for the effects of pressure; (ii) the i-motif's melting temperature increases by more than 30 K between pH 6.5 and 4.5, the consequence of an enthalpy for dissociation (ΔD H°) of 77(3) and 90(3) kJ (mol H+ )-1 at 0.1 and 200 MPa, respectively; (iii) below pH 4.6 at 0.1 MPa (pH 4.3 at 200 MPa) the melting temperature decreases as a result of double protonation of cytosine pairs, and ΔD H° and ΔD V° change signs; and (iv) the combination of ΔD H° and ΔD V° lead to the melting temperature at pH 4.3 being 3 K higher at 200 MPa than at 0.1 MPa.

Resolving solution conformations of the model semi-flexible polyelectrolyte homogalacturonan using molecular dynamics simulations and small-angle x-ray scattering.

The conformation of polyelectrolytes in the solution state has long been of interest in polymer science. Herein we utilize all atom molecular dynamics simulations (MD) and small-angle x-ray scattering experiments (SAXS) to elucidate the molecular structure of the model polyelectrolyte homogalacturonan. Several degrees of polymerization were studied and in addition partial methylesterification of the otherwise charge-carrying carboxyl groups was used in order to generate samples with varying intra-chain charge distributions. It is shown that at length scales above around 1nm the conformation of isolated chains has surprisingly little dependence on the charge distribution or the concentration of attendant monovalent salts, reflective of the intrinsic stiffness of the saccharide rings and the dynamical constraints of the glycosidic linkage. Indeed the conformation of isolated chains over all accessible length scales is well described by the atomic coordinates available from fibre diffraction studies. Furthermore, in more concentrated systems it is shown that, after careful analysis of the SAXS data, the form of the inter-particle effects heralded by the emergence of a so-called polyelectrolyte peak, can be extracted, and that this phenomena can be reproduced by multiple chain MD simulations.

On the electrophoretic mobilities of partially charged oligosaccharides as a function of charge patterning and degree of polymerization.

Fully or partially charged oligosaccharide molecules play a key role in many areas of biology, where their fine structures are crucial in determining their functionality. However, the separation of specific charged oligosaccharides from similar moieties that typically coexist in extracted samples, even for those that are unbranched, and in cases where each saccharide moiety can only carry a single charge or not, is far from trivial. Typically such molecules are characterized by a degree of polymerization n and a number m (and distribution) of charged residues, and must be separated from a plethora of similar species possessing different combinations of n and m. Furthermore, the separation of the possible n!/m!(n-m)! isomers of each species of fixed n and m is a formidable challenge to analytical chemists. Herein, we report the results of molecular dynamics simulations that have been performed in order to calculate the free solution electrophoretic mobilities of galacturonides and charged oligosaccharides derived from digests of the important plant cell-wall polysaccharide pectin. The simulations are compared with an experiment and are found to correctly predict the loss of resolution of fully charged species above a critical degree of polymerization n and the ionic strength dependence of the electrophoretic mobilities of different partially charged oligosaccharides. It is expected that having a predictive tool for the calculation of the electrophoretic mobilities of differently charged oligosaccharide species in hand will allow experimental conditions that optimize the resolution of particular species to be ascertained and understood.

Overstretching partially alkyne functionalized dsDNA using near infrared optical tweezers.

The force-extension behaviour of synthesized double-stranded DNAs (dsDNAs) designed to have 2.1% or 6.6% of the thymine bases alkyne functionalized was studied using near infrared (NIR) optical tweezers. Measurements were carried out on substrates with and without flurophores covalently attached to the alkyne moiety over an extended force range (F=0-70 pN) and results were compared to those obtained from an unmodified control. In accordance with earlier work [1] (measured over a force range F=0-5 pN), the force-extension of the dsDNA containing 2.1% modified-bases agreed well with that of the control. By contrast, the force-extension of the dsDNA containing 6.6% modified-bases showed an increasing deviation from that of the control as the dsDNA extension approached the molecule's contour length. These results indicate that incorporating alkyne functionalized bases can modify the mechanical properties of the dsDNA and that degree of functionalization should be carefully considered if a fluorescent mechanical analogue is required. A discrepancy between 1) the control dsDNA force-extension measured in Ref. [1] and that measured here and 2) dsDNA extensions carried out on the same duplex at different laser powers was noted; this was attributed to beam heating by the NIR trapping laser which was estimated to raise the local temperature at the optical traps by ΔT≈10-15°C.

Structural Analysis of Polysaccharide Networks by Transmission Electron Microscopy: Comparison with Small-Angle X-ray Scattering.

Polysaccharide gels assembled from the anionic biopolymers pectin and carrageenan have been studied using transmission electron microscopy (TEM). Gels were formed in several different ways: for pectin, hydrogen bonding was used to form junction zones between strands, whereas for carrageenan systems, several different ion types were used to form ionotropic networks. Using this approach, several distinct network architectures were realized. In addition to preparing gelled samples for electron microscopy, a set of samples was taken without performing the additional treatment necessitated by the TEM measurements, and these were studied directly by small-angle X-ray scattering (SAXS). Taking careful consideration of the relative merits of different image sizes and available processing techniques, the real-space images acquired by TEM were used via radial integration of the Fourier transform to produce simulated scattering patterns. These intensity-versus-wavevector plots were compared with the results of SAXS experiments carried out on the unadulterated gels using synchrotron radiation. Although information regarding chain thicknesses and flexibilities was found to be modified by labeling and changes in the dielectric constant and mechanical properties of the surroundings in the TEM, the studies carried out here show that careful protocols can produce data sets where information acquired above ∼20 nm is broadly consistent with that obtained by SAXS studies carried out on unadulterated samples. The fact that at larger length scale the structure of these water-rich networks seems largely preserved in the TEM samples suggests that three-dimensional (3D) TEM tomography experiments carried out with careful sample preparation will be valuable tools for measuring network architecture and connectivity; information that is lost in SAXS owing to the intrinsic averaging nature of the technique.

Effects of Pressure and pH on the Hydrolysis of Cytosine: Implications for Nucleotide Stability around Deep-Sea Black Smokers.

The relatively low chemical stability of cytosine compared with other nucleobases is a key concern in origin-of-life scenarios, but the effect of pressure on the rate of hydrolysis of cytosine to uracil remains unknown. Through in situ NMR spectroscopy measurements, it has been determined that the half-life of cytosine at 373.15 K decreases from (18.0±0.7) days at ambient pressure (0.1 MPa) to (8.64±0.18) days at high pressure (200 MPa). This yields an activation volume for hydrolysis of (-11.8±0.5) cm3 mol-1 ; a decrease that is similar to the molar volume of water (18.0 cm3 mol-1 ) and consistent with a tetrahedral 3,3-hydroxyamine transition-state/intermediate species. Similar behaviour was also observed for cytidine. At both ambient and high pressures, the half-life of cytosine decreases significantly as the pH decreases from 7.0 to 6.0. These results provide scant support for the notion that RNA-based life forms originated in high-temperature, high-pressure, acidic environments.