Florian Seier

• Glycans play an important role in the intracellular interactions of pathogenic bacteria. Pathogenic bacteria possess binding proteins capable of recognizing certain sugar motifs on other cells, which are found in glycan structures. Artificial carbohydrate synthesis allows scientists to recreate those sugar motifs in a rational, precise, and pure form. However, due to the high specificity of sugar-binding proteins, known as lectins, to glycan structures, methods for identifying suitable binding agents need to be developed. To tackle this hurdle, the Fraunhofer Institute for Cell Therapy and Immunology (Fraunhofer IZI) and the Max-Planck Institute of Colloids and Interfaces (MPIKG) developed a binding assay for the high throughput testing of sugar motifs that are presented on modular scaffolds formed by the assembly of four DNA strands into simple, branched DNA nanostructures. The first generation of this assay was used in combination with bacteria that express a fluorescent protein as a proof-of-concept. Here, the assay was optimized to be used with bacteria not possessing a marker gene for a fluorescent protein by staining their genomic DNA with SYBR® Green. For the binding assay, DNA nanostructures were combined with artificially synthesized mannose polymers, typical targets for many lectins on the surface of bacteria, presenting them in a defined constellation to bind bacteria strongly due to multivalent cooperativity. The testing of multiple mannose polymers identified monomeric mannose with a 5’-carbon linker and 1,2-linked dimeric mannose with linker as the best binding candidates for E. coli, presumably due to binding with the FimH protein on the surface. Despite similarities between the FimH proteins of E. coli and K. pneumoniae, binding was only observed between E. coli and the different sugar molecules on DNA structures. Furthermore, the degree of free movement seemed to affect the binding of mannose polymers to targeted proteins, since when utilizing a more flexible DNA nanostructure, an increase in binding could be observed. An alternative to the simple DNA nanostructures described above is the use of larger, more complex DNA origami structures consisting of several hundred strands. DNA origami structures are capable of carrying dozens of modifications at the same time. The results for the DNA origami structure showed a successful functionalization with up to 71 1,2-linked dimeric mannose with linker molecules. These results point towards a solution for the high-throughput analysis of potential binding agents for pathogenic bacteria e.g. as an alternative treatment for antibiotic-resistant.