Abstract

Understanding the interactions among biomolecules, such as proteins and nucleic acids, is fundamental to life’s processes and remains a central focus of biochemistry and molecular biology. To advance this understanding, researchers mix relevant molecules in controlled extracellular settings, a method crucial for detailed examination of molecular interactions and a key technique in field evolution. The theoretical bedrock of these experiments is chemical and enzyme kinetics, established over a century ago. Given its robustness and broad applicability, the theory has remained unaltered to this day. Yet, with the advent of modern techniques for molecular manipulation and observation, once beyond the theory’s foresight, there is now a significant opportunity to extend the theory, facilitating new experimental designs. Here, we revisit and expand the principles of chemical and enzyme kinetics to encompass a broader range of phenomena, proposing an experimental setup that more closely mirrors intracellular conditions. At first glance, the proposed experimental system, which deals with particles confined within nanospaces, might appear peculiar and unnatural; however, theoretical insights have demonstrated its closer alignment with ‘natural’ processes, defying initial assumptions. Illustratively, this approach has allowed for a fundamental derivation of Michaelis-Menten equation, a cornerstone formula in the field. We showcase its practical utility through the successful screening of molecular glues, a task previously considered lacking a rational methodology. Our theoretical framework does not merely open new experimental doors; it also provides a profound yet straightforward answer to the perennial question of why simple mixing of constituents in a test tube does not recreate life. Also, it introduces measures for previously ambiguous concepts such as ‘local concentration’ in molecular interactions and ‘artifacts’ in biomolecular modifications, offering a foundation for the quantitative analysis of these critical notions for the first time. The introduction of these measures promises to deepen our comprehension of molecular phenomena within cells.