Fluidic Interface\nfor Surface-based DNA Origami Studies

Traditionally, the use of DNA origami nanostructures\n(DONs) to\nstudy early cell signaling processes has been conducted using standard\nlaboratory equipment with DONs typically utilized in solution. Surface-based\ntechnologies simplify the microscopic analysis of cells treated with\nDON agents by anchoring them to solid substrates, thus avoiding the\ncomplications of receptor-mediated endocytosis. A robust microfluidic\nplatform for real-time monitoring and precise functionalization of\nsurfaces with DONs was developed here. The combination of controlled\nflow conditions with an upright total internal reflection fluorescence\nmicroscope enabled the kinetic analysis of the immobilization of DONs\non DNA-functionalized surfaces. The results revealed that DON morphology\nand binding tags influence the binding kinetics and that DON hybridization\non surfaces is more effective in microfluidic devices with larger-than-standard\ndimensions, addressing the low diffusivity challenge of DONs. The\nplatform enabled the decoration of DONs with protein-binding ligands\nand in situ investigation of ligand occupancy on DONs to produce high-quality\nbioactive surfaces. These surfaces were used to recruit and activate\nthe epidermal growth factor receptor (EGFR) through clustering in\nthe membranes of living cancer cells (MCF-7) using an antagonistic\nantibody (Panitumumab). The activation was quantified depending on\nthe interligand distances of the EGFR-targeting antibody.