Locked nucleic acid oligomers as handles for single molecule manipulation.

Single-molecule manipulation (SMM) techniques use applied force, and measured elastic response, to reveal microscopic physical parameters of individual biomolecules and details of biomolecular interactions. A major hurdle in the application of these techniques is the labeling method needed to immobilize biomolecules on solid supports. A simple, minimally-perturbative labeling strategy would significantly broaden the possible applications of SMM experiments, perhaps even allowing the study of native biomolecular structures. To accomplish this, we investigate the use of functionalized locked nucleic acid (LNA) oligomers as biomolecular handles that permit sequence-specific binding and immobilization of DNA. We find these probes form bonds with DNA with high specificity but with varied stability in response to the direction of applied mechanical force: when loaded in a shear orientation, the bound LNA oligomers were measured to be two orders of magnitude more stable than when loaded in a peeling, or unzipping, orientation. Our results show that LNA provides a simple, stable means to functionalize dsDNA for manipulation. We provide design rules that will facilitate their use in future experiments

Counting the ions surrounding nucleic acids.

Nucleic acids are strongly negatively charged, and thus electrostatic interactions-screened by ions in solution-play an important role in governing their ability to fold and participate in biomolecular interactions. The negative charge creates a region, known as the ion atmosphere, in which cation and anion concentrations are perturbed from their bulk values. Ion counting experiments quantify the ion atmosphere by measuring the preferential ion interaction coefficient: the net total number of excess ions above, or below, the number expected due to the bulk concentration. The results of such studies provide important constraints on theories, which typically predict the full three-dimensional distribution of the screening cloud. This article reviews the state of nucleic acid ion counting measurements and critically analyzes their ability to test both analytical and simulation-based models

Spatial Organization of Phase-separated DNA Droplets

Many recent studies of liquid-liquid phase separation in biology focus on phase separation as a dynamic control mechanism for cellular function, but it can also result in complex mesoscopic structures. We primarily investigate a model system consisting of DNA nanostars: finite-valence, self-assembled particles that form micron-scale liquid droplets via a binodal phase transition. We demonstrate that, upon phase separation, nanostar droplets spontaneously form hyperuniform structures, a type of disordered material with `hidden order' that combines the long-range order of crystals with the short-range isotropy of liquids. We find that the hyperuniformity of the DNA droplets reflects near-equilibrium dynamics, where phase separation drives the organization of droplets that then relax toward equilibrium via droplet Brownian motion. We engineer a two-species system of immiscible DNA droplets and find two distinctly hyperuniform structures in the same sample, but with random cross-species droplet correlations, which rules out explanations that rely on droplet-droplet hydrodynamic interactions. In addition, we perform experiments on the electrostatic coacervation of peptides and nucleotides which exhibit hyperuniform structures indistinguishable from DNA nanostars, indicating the phenomenon generally applies to phase-separating systems that experience Brownian motion. Our work on near-equilibrium droplet assembly and structure provides a foundation to investigate droplet organizational mechanisms in driven/biological environments. This approach also provides a clear path to implement phase-separated droplet patterns as exotic optical or mechanical metamaterials, or as efficient biochemical reactors.Comment: Additional experiments and discussion of hyperuniformity in droplets formed by electrostatic coacervatio

Validated Stability-indicating HPTLC Determination of Baclofen in Bulk Drug, Pharmaceutical Formulations and Real Human Urine and Plasma.

A simple, highly selective and stability-indicating high-performance thin-layer chromatographic method was developed and validated for the analysis of baclofen in bulk powder, pharmaceutical formulations and human urine and in and real human plasma. The method employed TLC aluminum plates precoated with silica gel 60 F254 as the stationary phase. The solvent system consisted of butanol–acetic acid–water (3.0: 0.5: 0.5, v/v/v). This system was found to give compact spots for baclofen (Rf value of 0.54). Densitometric analysis was carried out in the absorbance mode at 238 nm. The linear regression analysis data for the calibration plot showed good linear relationship (r2 = 0.9983) in the concentration range 1.5-7.5 µg per spot. The analytical performance of the method was fully validated, and the results were satisfactory. The limits of detection and quantitation were 0.31 and 1.03 µg per spot, respectively. Baclofen was subjected to acid and alkali hydrolysis, oxidation and photodegradation. The degraded product was well separated from the pure drug. Results indicate that the drug is stable against light and basic conditions. However, additional peaks were observed at Rf value of 0.65 and at Rf value of 0.14 with hydrogen peroxide and hydrochloric acid respectively, indicating that the drug is susceptible to oxidation and acid degradation. The method was applied for the analysis of baclofen in commercial tablets and the results were similar to those obtained using the reference method. As the method could effectively separate the drug from its degradation product, it can be employed as a stability-indicating one. The high sensitivity of the proposed method allowed determination of baclofen in real human urine and plasma

Single-molecule stretching shows glycosylation sets tension in the hyaluronan-aggrecan bottlebrush

Large bottlebrush complexes formed from the polysaccharide hyaluronan (HA) and the proteoglycan aggrecan contribute to cartilage compression resistance and are necessary for healthy joint function. A variety of mechanical forces act on these complexes in the cartilage extracellular matrix, motivating the need for a quantitative description which links their structure and mechanical response. Studies using electron microscopy have imaged the HA-aggrecan brush but require adsorption to a surface, dramatically altering the complex from its native conformation. We use magnetic tweezers force spectroscopy to measure changes in extension and mechanical response of an HA chain as aggrecan monomers bind and form a bottlebrush. This technique directly measures changes undergone by a single complex with time and under varying solution conditions. Upon addition of aggrecan, we find a large swelling effect manifests when the HA chain is under very low external tension (i.e. stretching forces less than ~1 pN). We use models of force-extension behavior to show that repulsion between the aggrecans induces an internal tension in the HA chain. Through reference to theories of bottlebrush polymer behavior, we demonstrate that the experimental values of internal tension are consistent with a polydisperse aggrecan population, likely caused by varying degrees of glycosylation. By enzymatically deglycosylating aggrecan, we show that aggrecan glycosylation is the structural feature which causes HA stiffening. We then construct a simple stochastic binding model to show that variable glycosylation leads to a wide distribution of internal tensions in HA, causing variations in the mechanics at much longer length-scales. Our results provide a mechanistic picture of how flexibility and size of HA and aggrecan lead to the brush architecture and mechanical properties of this important component of cartilage

Bandpass Filtering of DNA Elastic Modes Using Confinement and Tension

During a variety of biological and technological processes, biopolymers are simultaneously subject to both confinement and external forces. Although significant efforts have gone into understanding the physics of polymers that are only confined, or only under tension, little work has been done to explore the effects of the interplay of force and confinement. Here, we study the combined effects of stretching and confinement on a polymer’s configurational freedom. We measure the elastic response of long double-stranded DNA molecules that are partially confined to thin, nanofabricated slits. We account for the data through a model in which the DNA’s short-wavelength transverse elastic modes are cut off by applied force and the DNA’s bending stiffness, whereas long-wavelength modes are cut off by confinement. Thus, we show that confinement and stretching combine to permit tunable bandpass filtering of the elastic modes of long polymers

Single-stranded nucleic acid elasticity arises from internal electrostatic tension

Charged, flexible polymers, such as single-stranded nucleic acids (ssNAs), are ubiquitous in biology and technology. Quantitative description of their solution conformation has remained elusive due to the competing effects of polymer configurational freedom and salt-screened electrostatic repulsion between monomers. We investigate this by measuring the elastic response of single ssNA molecules over a range of salt concentrations. The data are well described by a model, inspired by a mean-field approach, in which intrapolymer electrostatic repulsion creates a salt-dependent internal tension whose interplay with the external force determines the elasticity. The internal tension can be related to the polymer’s charge spacing; thus, our results show how mesoscopic polymer conformation emerges from microscopic structure