Multivalent glycan arrays

Glycan microarrays have become a powerful technology to study biological processes, such as cell–cell interaction, inflammation, and infections. Yet, several challenges, especially in multivalent display, remain. In this introductory lecture we discuss the state-of-the-art glycan microarray technology, with emphasis on novel approaches to access collections of pure glycans and their immobilization on surfaces. Future directions to mimic the natural glycan presentation on an array format, as well as in situ generation of combinatorial glycan collections, are discussed

Quantitative prediction of multivalent ligand–receptor binding affinities for influenza, cholera, and anthrax inhibition

Multivalency achieves strong, yet reversible binding by the simultaneous formation of multiple weak bonds. It is a key interaction principle in biology and promising for the synthesis of high-affinity inhibitors of pathogens. We present a molecular model for the binding affinity of synthetic multivalent ligands onto multivalent receptors consisting of n receptor units arranged on a regular polygon. Ligands consist of a geometrically matching rigid polygonal core to which monovalent ligand units are attached via flexible linker polymers, closely mimicking existing experimental designs. The calculated binding affinities quantitatively agree with experimental studies for cholera toxin (n = 5) and anthrax receptor (n = 7) and allow to predict optimal core size and optimal linker length. Maximal binding affinity is achieved for a core that matches the receptor size and for linkers that have an equilibrium end-to-end distance that is slightly longer than the geometric separation between ligand core and receptor sites. Linkers that are longer than optimal are greatly preferable compared to shorter linkers. The angular steric restriction between ligand unit and linker polymer is shown to be a key parameter. We construct an enhancement diagram that quantifies the multivalent binding affinity compared to monovalent ligands. We conclude that multivalent ligands against influenza viral hemagglutinin (n = 3), cholera toxin (n = 5), and anthrax receptor (n = 7) can outperform monovalent ligands only for a monovalent ligand affinity that exceeds a core-size dependent threshold value. Thus, multivalent drug design needs to balance core size, linker length, as well as monovalent ligand unit affinity

Nanoparticles for live cell microscopy: A surface-enhanced Raman scattering perspective.

Surface enhanced Raman scattering (SERS) nanoparticles are an attractive alternative to fluorescent probes for biological labeling because of their photostability and multiplexing capabilities. However, nanoparticle size, shape, and surface properties are known to affect nanoparticle-cell interactions. Other issues such as the formation of a protein corona and antibody multivalency interfere with the labeling properties of nanoparticle-antibody conjugates. Hence, it is important to consider these aspects in order to validate such conjugates for live cell imaging applications. Using SERS nanoparticles that target HER2 and CD44 in breast cancer cells, we demonstrate labeling of fixed cells with high specificity that correlates well with fluorescent labels. However, when labeling live cells to monitor surface biomarker expression and dynamics, the nanoparticles are rapidly uptaken by the cells and become compartmentalized into different cellular regions. This behavior is in stark contrast to that of fluorescent antibody conjugates. This study highlights the impact of nanoparticle internalization and trafficking on the ability to use SERS nanoparticle-antibody conjugates to monitor cell dynamics

Discovery of Stable and Selective Antibody Mimetics from Combinatorial Libraries of Polyvalent, Loop-Functionalized Peptoid Nanosheets.

The ability of antibodies to bind a wide variety of analytes with high specificity and high affinity makes them ideal candidates for therapeutic and diagnostic applications. However, the poor stability and high production cost of antibodies have prompted exploration of a variety of synthetic materials capable of specific molecular recognition. Unfortunately, it remains a fundamental challenge to create a chemically diverse population of protein-like, folded synthetic nanostructures with defined molecular conformations in water. Here we report the synthesis and screening of combinatorial libraries of sequence-defined peptoid polymers engineered to fold into ordered, supramolecular nanosheets displaying a high spatial density of diverse, conformationally constrained peptoid loops on their surface. These polyvalent, loop-functionalized nanosheets were screened using a homogeneous Förster resonance energy transfer (FRET) assay for binding to a variety of protein targets. Peptoid sequences were identified that bound to the heptameric protein, anthrax protective antigen, with high avidity and selectivity. These nanosheets were shown to be resistant to proteolytic degradation, and the binding was shown to be dependent on the loop display density. This work demonstrates that key aspects of antibody structure and function-the creation of multivalent, combinatorial chemical diversity within a well-defined folded structure-can be realized with completely synthetic materials. This approach enables the rapid discovery of biomimetic affinity reagents that combine the durability of synthetic materials with the specificity of biomolecular materials

Synthesis of tripodal catecholates and their immobilization on zinc oxide nanoparticles

A common approach to generate tailored materials and nanoparticles (NPs) is the formation of molecular monolayers by chemisorption of bifunctional anchor molecules. This approach depends critically on the choice of a suitable anchor group. Recently, bifunctional catecholates, inspired by mussel-adhesive proteins (MAPs) and bacterial siderophores, have received considerable interest as anchor groups for biomedically relevant metal surfaces and nanoparticles. We report here the synthesis of new tripodal catecholates as multivalent anchor molecules for immobilization on metal surfaces and nanoparticles. The tripodal catecholates have been conjugated to various effector molecules such as PEG, a sulfobetaine and an adamantyl group. The potential of these conjugates has been demonstrated with the immobilization of tripodal catecholates on ZnO NPs. The results confirmed a high loading of tripodal PEG-catecholates on the particles and the formation of stable PEG layers in aqueous solution

Size Dependence of Steric Shielding and Multivalency Effects for Globular Binding Inhibitors

Competitive binding inhibitors based on multivalent nanoparticles have shown great potential for preventing virus infections. However, general design principles of highly efficient inhibitors are lacking as the quantitative impact of factors such as virus concentration, inhibitor size, steric shielding, or multivalency effects in the inhibition process is not known. Based on two complementary experimental inhibition assays we determined size- dependent steric shielding and multivalency effects. This allowed us to adapt the Cheng–Prusoff equation for its application to multivalent systems. Our results show that the particle and volume normalized IC50 value of an inhibitor at very low virus concentration predominantly depends on its multivalent association constant, which itself exponentially increases with the inhibitor/virus contact area and ligand density. Compared to multivalency effects, the contribution of steric shielding to the IC50 values is only minor, and its impact is only noticeable if the multivalent dissociation constant is far below the virus concentration, which means if all inhibitors are bound to the virus. The dependence of the predominant effect, either steric shielding or multivalency, on the virus concentration has significant implications on the in vitro testing of competitive binding inhibitors and determines optimal inhibitor diameters for the efficient inhibition of viruses

Temperature-sensitive protein–DNA dimerizers

Programmable DNA-binding polyamides coupled to short peptides have led to the creation of synthetic artificial transcription factors. A hairpin polyamide-YPWM tetrapeptide conjugate facilitates the binding of a natural transcription factor Exd to an adjacent DNA site. Such small molecules function as protein-DNA dimerizers that stabilize complexes at composite DNA binding sites. Here we investigate the role of the linker that connects the polyamide to the peptide. We find that a substantial degree of variability in the linker length is tolerated at lower temperatures. At physiological temperatures, the longest linker tested confers a "switch"-like property on the protein-DNA dimerizer, in that it abolishes the ability of the YPWM moiety to recruit the natural transcription factor to DNA. These observations provide design principles for future artificial transcription factors that can be externally regulated and can function in concert with the cellular regulatory circuitry