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.

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.

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.

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.

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.

Effects of non-Gaussian Brownian motion on direct force optical tweezers measurements of the electrostatic forces between pairs of colloidal particles.

Measurements of the electrostatic force with separation between a fixed and an optically trapped colloidal particle are examined with experiment, simulation and analytical calculation. Non-Gaussian Brownian motion is observed in the position of the optically trapped particle when particles are close and traps weak. As a consequence of this motion, a simple least squares parameterization of direct force measurements, in which force is inferred from the displacement of an optically trapped particle as separation is gradually decreased, contains forces generated by the rectification of thermal fluctuations in addition to those originating directly from the electrostatic interaction between the particles. Thus, when particles are close and traps weak, simply fitting the measured direct force measurement to DLVO theory extracts parameters with modified meanings when compared to the original formulation. In such cases, however, physically meaningful DLVO parameters can be recovered by comparing the measured non-Gaussian statistics to those predicted by solutions to Smoluchowski's equation for diffusion in a potential.

Developing a video tracking method to study interactions between close pairs of optically trapped particles in three dimensions.

We develop a video tracking method that utilizes an interpolation-based normalized cross-correlation approach to track the position of microscopic spherical particles in three dimensions. Subnanometer resolution is demonstrated. The method does not assume that the particle's image is radially symmetric, making it useful for determining the position when particles are close and their images overlap. This is demonstrated in a study of the electrostatic and hydrodynamic interactions between a pair of beads in dual laser tweezers traps.

DNA visualization in single molecule studies carried out with optical tweezers: Covalent versus non-covalent attachment of fluorophores.

In this study, we investigated the use of the covalent attachment of fluorescent dyes to double-stranded DNA (dsDNA) stretched between particles using optical tweezers (OT) and compared the mechanical properties of the covalently-functionalized chain to that of unmodified DNA and to DNA bound to a previously uncharacterized groove-binder, SYBR-gold. Modified DNA species were obtained by covalently linking azide-functionalized organic fluorophores onto the backbone of DNA chains via the alkyne moieties of modified bases that were incorporated during PCR. These DNA molecules were then constructed into dumbbells by attaching polystyrene particles to the respective chain ends via biotin or digoxigenin handles that had been pre-attached to the PCR primers which formed the ends of the synthesized molecule. Using the optical tweezers, the DNA was stretched by separating the two optically trapped polystyrene particles. Displacements of the particles were measured in 3D using an interpolation-based normalized cross-correlation method and force-extension curves were calculated and fitted to the worm-like chain model to parameterize the mechanical properties of the DNA. Results showed that both the contour and persistence length of the covalently-modified dsDNAs were indistinguishable from that of the unmodified dsDNA, whereas SYBR-gold binding perturbed the contour length of the chain in a force-dependent manner.

Two beam surface fluctuation specular reflection spectroscopy.

In surface fluctuation specular reflection spectroscopy (SFSRS) deflections of a specularly reflected laser beam are used to characterize thermally excited surface waves. Here we report on a new two beam version of SFSRS in which the deflections of two reflected laser beams from separate locations on a surface are correlated. We demonstrate that this new two beam SFSRS technique can be used to determine directly the power spectrum of height fluctuation of thermally excited surface waves over a large range of both frequencies and wavevectors. In addition, we show that the technique is well suited for materials ranging from simple liquids to complex liquids and soft solids, including turbid materials.

A rheo-optical study of shear rate and optical anisotropy in wormlike micelles solutions.

The flow behaviour of wormlike micelles solutions composed of the surfactant cetylpyridinium chloride (CPCl) and counter-ion sodium salicylate (NaSal) at a molar ratio [CPCl]/[NaSal] = 2 in brine [NaCl] = 0.5 M in a cylindrical Couette geometry was examined using homodyne PCS and ellipsometry. Homodyne PCS was used to profile local shear rate and ellipsometry to concurrently profile local optical anisotropy of the fluid. Shear thinning was observed and was correlated to an increase in turbidity and a breakdown in the stress-optic law. A stress plateau observed in mechanical measurements was correlated with the partitioning of the fluid into regions of low shear rate/low turbidity/high birefringence and high shear rate/high turbidity/low birefringence. The partitioning observed was inconsistent with a simple interpretation of the lever rule.