A Modular Protein-DNA Multimer Model to Explore How Valence and Monomer Affinity Shape Multivalent DNA Binding

Multivalent interactions enhance binding strength through the cooperative effects of multiple weak interactions. While multivalency is known to improve sensitivity, increased valency can also enhance nonspecific interactions, potentially compromising selectivity. Insights into the relationship between valency, affinity, and detection performance are needed to optimize both sensitivity and selectivity in multivalent systems. Here, we developed modular protein-DNA multimers (valency 1-4) with uniform spatial arrangement, enabling an investigation of valency-dependent interactions between DNA strands across a broad range of monomeric affinities (subnanomolar to millimolar). Using surface plasmon resonance (SPR) and enzyme-linked immunosorbent assays (ELISA), we quantitatively analyzed how multimerization affects detection sensitivity and selectivity. We found that the optimal range of DNA monomer affinities for effective detection shifted from the nanomolar to the micromolar scale with increasing valency. For example, monomers with K_D values between a few micromolar and ∼50 μM showed the greatest signal amplification upon tetramerization under our conditions. We also discovered that monomers with K_D ≥ ∼50 μM remained largely undetectable even with tetramerization, indicating the presence of an affinity threshold for valency-driven signal enhancement. We further identified a steep transition zone in ELISA signals, where small changes in K_D led to large differences in detection, highlighting a narrow affinity window with maximal selectivity. This K_D window consistently shifted toward weaker affinities as valency increased. These findings show how a modular protein-DNA multimer model can derive quantitative trends for multivalent design, while laying a foundation for future studies in more complex protein systems.