Kinetic Modeling of Solid-Phase Oligonucleotide Synthesis: Mechanistic Insights and Reaction Dynamics

Abstract

Solid-phase oligonucleotide synthesis is a cornerstone of modern biotechnology, enabling the production of custom DNA and RNA sequences for therapeutic, diagnostic, and research applications. Despite its widespread use, the kinetics of this process remain incompletely understood, limiting efforts to enhance efficiency and yield and reduce environmental impact. This study presents a comprehensive kinetic model of solid-phase oligonucleotide synthesis, integrating mechanistic insights into the stepwise coupling, capping, oxidation, and detritylation reactions. Using a combination of computational simulations and experimental data, we identify rate-limiting steps and quantify the influence of reaction conditions─such as concentrations, step duration, and stoichiometry─on synthesis performance. The model is a first step to predicting strategies for process optimization, including adjusted cycle times and excess ratios. Validation against experimental synthesis runs demonstrates that the proposed model can be used for predictive purposes. These findings offer a quantitative framework for improving solid-phase oligonucleotide synthesis with implications for scalable production and cost-effective design of nucleic acid–based technologies.