Development of a fluorescence-based assay for RecBCD activity using functional data analysis and design of experiments.

Abstract

Biochemical assays are essential tools in biological research and drug discovery, but optimisation of these assays is often a challenging and lengthy process due to the wide range of input variables and the complex effects of these variables on one another. Traditional 'one-factor-at-a-time' optimisation is both time-consuming and fails to explore the full range of input combinations. In contrast, the modern 'design of experiments' (DoE) approach enables simultaneous investigation of multiple input variables and their interactions, leading to more information-rich and efficient experimentation. We therefore sought to apply DoE to the optimisation of a new fluorescence-based assay for the enzyme RecBCD, a helicase-nuclease-ATPase complex involved in bacterial stress responses. A novel 'functional data analysis' (FDA) approach was used to predict the shape of RecBCD reaction curves in response to different combinations of input variables, which successfully identified assay conditions suitable for drug screening. Collectively, this work delivers a new assay for the antibiotic target RecBCD and demonstrates the potential of DoE and FDA to accelerate biochemical assay development.