Spatial Screening of Hemagglutinin on Influenza A Virus Particles: Sialyl-LacNAc Displays on DNA and PEG Scaffolds Reveal the Requirements for Bivalency Enhanced Interactions with Weak Monovalent Binders

Abstract

Attachment of the Influenza A virus onto host cells involves multivalent interactions between virus surface hemagglutinin (HA) and sialoside-containing glyco ligands. Despite the development of extremely powerful multivalent binders of the Influenza virus and other viruses, comparably little is known about the optimal spacing of HA ligands, which ought to bridge binding sites within or across the trimeric HA molecules. To explore the criteria for ligand economical high affinity binding, we systematically probed distance-affinity relationships by means of two differently behaving scaffold types based on (i) flexible polyethylene glycol (PEG) conjugates and (ii) rigid self-assembled DNA·PNA complexes. The bivalent scaffolds presented two sialyl-LacNAc ligands in 23-101 Å distance. A combined analysis of binding by means of microscale thermophoresis measurements and statistical mechanics models exposed the inherent limitations of PEG-based spacers. Given the distance requirements of HA, the flexibility of PEG scaffolds is too high to raise the effective concentration of glyco ligands above a value that allows interactions with the low affinity binding site. By contrast, spatial screening with less flexible, self-assembled peptide nucleic acid (PNA)·DNA complexes uncovered a well-defined and, surprisingly, bimodal distance-affinity relationship for interactions of the Influenza A virus HA with bivalent displays of the natural sialyl-LacNAc ligand. Optimal constructs conferred 10-fold binding enhancements with only two ligands. We discuss the existence of secondary binding sites and shine light on the preference for intramolecular rather than intermolecular recognition of HA trimers on the virus surface.

Sialyl‐LacNAc‐PNA⋅DNA Concatamers by Rolling‐Circle Amplification as Multivalent Inhibitors of Influenza A Virus Particles

“…The inhibitory constants of virusinduced hemagglutination ( ) improved 10 4 ‐fold upon trivalent presentation of sialyl‐lactose ligands. We used DNA‐programmed spatial screening to identify the optimal arrangement of sialyl‐LacNAc displays and showed that 1.360‐fold enhancements over monovalent binding can be achieved with only two sugar ligands, provided that the sugars are arranged at the 52–59 Å distance necessary to bind two of the three binding sites within a HA trimer simultaneously (Figure B) . Note, we also obtained the first evidence for target specificity: an optimized intratrimeric, bivalent HA binder inhibited hemagglutination induced by an H3N2 influenza A virus (IAV) ten times better than that by an H1N1 IAV.…”

“…The sequence design of the shorter tandem DNA (39 nt) was chosen so as to allow the adjacent annealing of three peptide nucleic acid (PNA) 13‐mers, that is, two sugar‐modified α2,6‐sialyl‐LacNAc‐PNA conjugates (PNAs 1 and 2 ) and the unmodified spacer PNA oligomer (PNA 3 ; Figure B). The synthesis of the conjugates was previously reported . Multiple linear hybridization enabled concatenation of sialyl‐LacNAc‐PNA conjugates.…”

“…Both scaffold types allowed the multivalent display of a bivalent arrangement that presents the sialyl‐LacNAc ligands in multiples of 17 nt. In our previous investigations, this distance proved optimal for the simultaneous binding of two binding sites on one HA …”

Affinity Enhancement of Protein Ligands by Reversible Covalent Modification of Neighboring Lysine Residues

“…[27] Similar procedures could be implemented for the discovery of reversible covalent binders by incorporating 2-hydroxybenzaldehyde or similar moieties during library construction. [28] It will be interesting to investigate whether more reactive lysine modifiers (or modifiers for other amino acids) can be used for pharmaceutical applications.From athermodynamic viewpoint, practical applications would be limited by the molar concentration of amino acid residues in serum and in tissues.H owever,s ince Schiff-base formation is ar elatively slow reaction, it may be conceivable that a" dock-and-lock" procedure may allow the selective in vivo modification of target proteins,i fk inetic parameters are judiciously chosen. [11,19] Moreover,DNA structures have been used to probe the magnitude of affinity gain by polydentate engagement with multivalent protein target.…”

Templated chemistry for bioorganic synthesis and chemical biology

“…It remains unknown which and how many of the several glyco ligands engage on binding and, therefore, these scaffolds do not provide information about the optimal spacing of HA ligands. In order to identify the criteria for enhanced binding at high ligand economy and learn about the arrangement of binding sites on the IAV particle, we used DNA‐programmed bivalent screening . We figured that a range of far reaching scaffolds would be required to assess the potential for enhanced interactions upon bridging of two sugar binding sites within an HA trimer and across HA trimers on the IAV surface (Figure B).…”