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.

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.

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.

Structure and Properties of a Non-processive, Salt-requiring, and Acidophilic Pectin Methylesterase from Aspergillus niger Provide Insights into the Key Determinants of Processivity Control.

Many pectin methylesterases (PMEs) are expressed in plants to modify plant cell-wall pectins for various physiological roles. These pectins are also attacked by PMEs from phytopathogens and phytophagous insects. The de-methylesterification by PMEs of the O6-methyl ester groups of the homogalacturonan component of pectin, exposing galacturonic acids, can occur processively or non-processively, respectively, describing sequential versus single de-methylesterification events occurring before enzyme-substrate dissociation. The high resolution x-ray structures of a PME from Aspergillus niger in deglycosylated and Asn-linked N-acetylglucosamine-stub forms reveal a 10⅔-turn parallel ß-helix (similar to but with less extensive loops than bacterial, plant, and insect PMEs). Capillary electrophoresis shows that this PME is non-processive, halophilic, and acidophilic. Molecular dynamics simulations and electrostatic potential calculations reveal very different behavior and properties compared with processive PMEs. Specifically, uncorrelated rotations are observed about the glycosidic bonds of a partially de-methyl-esterified decasaccharide model substrate, in sharp contrast to the correlated rotations of processive PMEs, and the substrate-binding groove is negatively not positively charged.

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.