Single-Molecule\nMultivalent Interactions Revealed\nby Plasmon-Enhanced Fluorescence

Multivalency as an interaction principle is widely utilized\nin\nnature. It enables specific and strong binding by multiple weak interactions\nthrough enhanced avidity and is a core process in immune recognition\nand cellular signaling, which is also a current concept in drug design.\nHere, we use the high signals from plasmon-enhanced fluorescence of\nnanoparticles to extract binding kinetics and dynamics of multivalent\ninteractions on the single-molecule level and in real time. We study\nmono-, bi-, and trivalent binding interactions using a DNA Holliday\nJunction as a model construct with programmable valency and introduce\na step-binding model for binding kinetics relevant for structured\nmacromolecules by including an experimentally extractable binding\nrestriction term ω to quantify the effects from conformation,\nsteric effects, and rigidity. We used this approach to explore how\nlength and flexibility of the DNA ligands affect binding restriction\nand binding strength, where the overall binding strength decreased\nwith spacer length. For trivalent systems, increasing spacer length\nadditionally activated binding in the trivalent state, giving insight\ninto the design of multivalent drug or targeting moieties. By systematically\nchanging the receptor density, we explored the binding super selectivity\nof the multivalent HJ at the single-molecule level. We find a polynomial\nbehavior of the trivalent binding strength that clearly shows receptor-density-dependent\nselective binding. Interestingly, we could exploit the rapidly decaying\nnear fields of the plasmon that induce a strong dependence of the\nsignal on the position of the dye to observe binding dynamics during\nsingle multivalent binding events.