(a) Agarose gel electrophoresis characterization of the binding of DegP6A633SA (labelled at genetically introduced cysteine residues with A633) to diverse 6p120 constructs, differing in the number (NcA1; from 1 to 18) and spatial arrangement of the ligands within the cavity (constructs I to XI, b). The results indicate successful binding for all constructs, with maximal efficiency for a radial distribution of ligands (constructs X and XI). Lane M contained 1 kbp DNA ladder (Roth). The DNA origami structures migrate between the 1.5 and 2.0 kbp bands of the ladder. Gel running conditions: 0.75% agarose in 1 × TBEMg buffer, 4 °C, 3 h at 80 V. Gel imaging was performed with a Typhoon FL900 upon illumination at selected wavelengths to allow detection of the protein (Alexa633), peptide ligand (TAMRA) and DNA (upon ethidium bromide staining). (c) Single-molecule fluorescence characterization of the 6p construct, bearing 18 convergent PAs hybridized with TAMRA-tagged peptide ligands and loaded with a DegP6A647SA protein. Molecules were immobilized on a coverslip surface and were measured using TIRF microscopy. TAMRA (red spots in d) and Alexa647 (blue spots in e) detection channels have been overlapped, indicating clear co-localization of the two species (violet spots showing energy transfer from the donor to the acceptor fluorophore in f, which shows a zoom-in view of the highlighted region in d,e).