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Nanoscale organisation of receptor ligands has become an important approach to study the clustering behaviour of cell-surface receptors. Biomimetic substrates fabricated via different nanopatterning strategies have so far been applied to investigate specific integrins and cell types, but without multivalent control. Here we use DNA origami to surpass the limits of current approaches and fabricate nanoarrays to study different cell adhesion processes, with nanoscale spatial resolution and single-molecule control. Notably, DNA nanostructures enable the display of receptor ligands in a highly customisable manner, with modifiable parameters including ligand number, ligand spacing and most importantly, multivalency. To test the adaptability and robustness of the system we combined it with focused ion beam and electron-beam lithography nanopatterning to additionally control the distance between the origami structures (i.e. receptor clusters). Moreover, we demonstrate how the platform can be used to interrogate two different biological questions: (1) the cooperative effect of integrin and growth factor receptor in cancer cell spreading, and (2) the role of integrin clustering in cardiomyocyte adhesion and maturation. Thereby we find previously unknown clustering behaviour of different integrins, further outlining the importance for such customisable platforms for future investigations of specific receptor organisation at the nanoscale.

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Probing the nanoscale organisation and multivalency of cell surface receptors: DNA origami nanoarrays for cellular studies with single-molecule control