(389bo) Molecular Dynamics and Free Energy Simulations of ?-Hydroxybutyric Acid Binding to Nucleotides for Bioprocessing Applications

β-hydroxybutyric acid (BHB) is a biologically active metabolite and a precursor for the synthesis of polyhydroxybutyrate (PHB), a biodegradable biopolymer of growing interest. While BHB has been studied computationally in pharmacological contexts, it has not been for bioprocessing applications. Extracting BHB from fermentation broths remains a challenge in bioprocessing, and aptamer-based separation systems offer a promising route for selective recovery. However, molecular-level understanding of BHB’s interactions with potential aptamer components is lacking, limiting rational design of aptamer technologies.

This study employs all-atom molecular dynamics (MD) simulations and free energy calculations to investigate the binding behavior of BHB with small nucleotide bases, such as uracil and cytosine, as a precursor to computational aptamer design. Atomistic force fields were applied using CHARMM36 for solutes and TIP3P for water, while BHB’s electrostatic point charges were computed via the CHELPG method at the CBS-Q3 level using Gaussian. Binding free energies were calculated through umbrella sampling and the Weighted Histogram Analysis Method (WHAM).

These simulations provide insight to the stability and strength of BHB-nucleotide interactions in explicit solvent. This bottom-up simulation approach lays the groundwork for future in silico design of aptamers tailored for BHB extraction, bridging molecular modeling with bioprocess development and advancing strategies for sustainable biopolymer production.