A concentration-independent paradigm rendering weak interactions inherently quantifiable

A vast class of weak, millimolar-affinity molecular interactions governs cellular function, yet their quantitative characterization has remained largely beyond conventional methods. For over a century, biochemistry has worked within a concentration-based framework where molarity scales with molecular number per volume (N/V), and experiments have usually, often implicitly, changed concentration by moving N while holding V fixed. The weak-interaction measurement bottleneck arises from this paradigm: reading weak binding through bulk concentration requires concentrations beyond practical limits, a framework constraint rather than one of instrumental sensitivity. Here we show that shifting experimental control from N to accessible volume V overcomes this bottleneck and opens previously intractable affinity ranges through nanoscale spatial confinement. Controlling V means controlling what biochemists have called "local concentration" and "proximity effects," recasting these long-ambiguous notions as quantitative variables grounded in first principles. Implemented in DNA nanocavities, the approach showed that geometric arrangement alone can override solution-phase binding hierarchies. The same spatial control quantified a protein-peptide interaction of order 10 mM from femtomoles per well, totalling under a picomole per titration. Even so, a standard plate reader gave a signal-to-noise ratio near 10^3, leaving headroom for still weaker interactions. The affinity-and-geometry readout also enabled rational screening for protein-protein-interaction modulators, identifying compounds that enhance weak associations by reweighting local encounters rather than binding tightly on their own or forming a stable ternary complex. Together, this volume-based paradigm and its implementation provide a general strategy for probing and modulating previously inaccessible biochemical phenomena.

Source: arXiv:2608.02865v1 - http://arxiv.org/abs/2608.02865v1 PDF: https://arxiv.org/pdf/2608.02865v1 Original Link: http://arxiv.org/abs/2608.02865v1