Optical tweezers are microscopic tools with extraordinary precision in the determination of the position (±2 nm) of a colloid (diameter: ∼2.0 μm) in 3D-space and in the measurement of small forces in the range between 0.1 and 100 pN (pN=10−12 N). Experiments are reported in which single double-stranded (ds)-DNA chains of different length [2,000 base pairs (bp), 3,000, 4,000, and 6,000 bp] are spanned between two colloidal particles by use of appropriate molecular linkers. For the forces applied (≤40 pN) a fully reversible and well reproducible force–extension dependence is found. The data can be well described by both the worm-like chain model or by an approach developed by R. G. Winkler. For the resulting persistence length, a pronounced dependence on the ionic concentration in the surrounding medium is found.
Moenomycin A is an amphiphilic phosphoglycolipid antibiotic that interferes with the transglycosylation step in peptidoglycan biosynthesis. The antibiotic consists of a branched pentasaccharide moiety, connected to the moenocinol lipid via a glycerophosphate linker. We have previously described the selection of aptamers that require the lipid group and the disaccharide epitopes of the oligosaccharide moiety for moenomycin binding. Here we report that the enriched moenomycin-binding library contains sequences that evolved for specific recognition of the unpolar lipid group of the antibiotic. These results suggest that the evolution of hydrophobic binding pockets in RNA molecules may be much more common than previously assumed.
The kinetics of binding for the histone-like protein TmHU (from Thermotoga maritima) to DNA is analyzed on a single molecule level by use of optical tweezers. For the reaction rate a pronounced concentration-dependence is found with an "all or nothing"-limit which suggests the cooperative nature of the binding-reaction. By analyzing the statistics of mechanically induced dissociation-events of TmHU from DNA multiple reaction sites are observed to become more likely with increasing TmHU concentration. This is interpreted as a hint for a secondary organizational level of the TmHU/DNA complex. The reaction rate of TmHU binding to DNA is remarkably higher than that of the HU protein from Escherichia coli which will be discussed.
Optical tweezers (OT) are ideally suited to study the interaction of single receptor-ligand bonds. Here we introduce a newly developed assay using OT to investigate the interactions between Protein A from Staphylococcus aureus and Immunoglobulin G from rabbit serum (RIgG). We demonstrate that the rupture forces depend on the loading rate and on the sodium chloride concentration. The measured loading rate effect is well known in the literature and the data we obtained were found to be in good agreement with an already published theoretical model. The dependence of the rupture forces on the salt concentration demonstrates the influence of hydrophobic interactions on the bond strength. Our experimental setup can probe the interaction between a single receptor and its specific ligand under changing conditions and hence offers manifold applications in single molecule biotechnology.
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