Barcoded DNA origami structures for multiplexed optimization and enrichment of DNA-based protein-binding cavities

• Published in: Nature chemistry 2020-09, Vol.12 (9), p.852-859
• Main Authors: Aghebat Rafat, Ali, Sagredo, Sandra, Thalhammer, Melissa, Simmel, Friedrich C.
• Format: Article
• Language: English
• Subjects: 631/57/2265; 639/925/926/1049; 639/925/926/1050; Analytical Chemistry; Aptamers; Aptamers, Nucleotide - chemistry; Aptamers, Nucleotide - metabolism; Bar codes; Binding sites; Biochemistry; Chemistry; Chemistry and Materials Science; Chemistry/Food Science; Deoxyribonucleic acid; Design parameters; DNA; DNA - chemistry; DNA - metabolism; Humans; Inorganic Chemistry; Mechanical properties; Microscopy, Atomic Force; Molecular interactions; Nanostructures - chemistry; Nucleic Acid Conformation; Optimization; Organic Chemistry; Physical Chemistry; Protein Binding; Proteins; Proteins - chemistry; Proteins - metabolism; Scaffolds; Streptavidin - chemistry; Streptavidin - metabolism; Thrombin - chemistry; Thrombin - metabolism
• ISSN: 1755-4330; 1755-4349
• DOI: 10.1038/s41557-020-0504-6

Simultaneous binding of molecules by multiple binding partners is known to strongly reduce the apparent dissociation constant of the corresponding molecular complexes, and can be used to achieve strong, non-covalent molecular interactions. Based on this principle, efficient binding of proteins to DNA nanostructures has been achieved previously by placing several aptamers in close proximity to each other onto DNA scaffolds. Here, we develop an approach for exploring design parameters, such as the geometric arrangement or the nanomechanical properties of the binding sites, that use two-dimensional DNA origami-based nanocavities that bear aptamers with known mechanical properties at defined distances and orientations. The origami structures are labelled with barcodes, which enables large numbers of binding cavities to be investigated in parallel and under identical conditions, and facilitates a direct and reliable quantitative comparison of their binding yields. We demonstrate that binding geometry and mechanical properties have a dramatic effect on origami-based multivalent binding sites, and that optimization of linker spacings and flexibilities can improve the effective binding strength of the sites substantially. Multivalent binding is a common strategy to enhance the interactions between weak binding partners. Now, following this principle, DNA origami scaffolds have been used to arrange DNA aptamers into specific geometries and to optimize linker spacings and flexibilities, which results in artificial binding sites with very high affinities for their corresponding ligands.