Polymer brush-functionalized nanomaterials offer interesting features for the design of gene delivery vectors as their physicochemical and structural properties can be designed independently of the chemistry, size and shape of the nanomaterial core. However, little is known of the parameters regulating the adsorption and infiltration of DNA molecules at the surface of positively charged polymer brushes, despite the importance of such processes for gene delivery. Here we investigate the role of the molecular environment (e.g., pH, type of buffer, concentration) on the interactions between plasmid DNA and positively charged poly(dimethylaminoethyl methacrylate) (PDMAEMA) brushes using a combination of light scattering, electrophoretic light scattering, in situ ellipsometry, and surface plasmon resonance. We show that the conformation of swollen PDMAEMA brushes is modulated by the surrounding buffer and that this impacts strongly on the ability of such brushes and nanomaterials based on these coatings to complex DNA molecules. In turn, the levels of transfection efficiency measured correlate with changes in brush conformation and DNA binding. Therefore, this work demonstrates the importance of molecular design of polymer brushes to control DNA complexation and release in order to optimize the performance of polymer brush-functionalized nanomaterials for gene delivery applications.