Design of experiments for the optimization of a copper-sensitive DNA enzymes

Copper is an essential metal ion in biological, and medical contexts, recently emerging as an important biomarker for cancer progression, and a key target for liquid biopsy. While mass spectrometry remains the gold standard for aqueous copper detection, its adoption is hindered by high costs, extensive labour requirements, and slow turnaround times. DNA-cleaving DNA enzymes are synthetically evolved DNA sequences that perform specific oxidative cleavage in the presence of copper ions. Compared to mass spectrometry, they offer higher sensitivity, robust storage stability, and molecular programmability. Despite the prevalence of copper-sensitive DNA enzymes in the literature, the reported buffer conditions vary widely, and we still lack a systematic investigation into how buffer components influence the enzyme kinetics.

In this talk, I will discuss a design of experiments approach to the optimisation of DNA enzymes buffer formulation. I will describe a novel buffer composition that enhances the activity of a previously described enzymes, that expands the range of detectable copper concentrations. I will also demonstrate how the initial choice of response variable fundamentally shapes the optimisation outcome. Finally, I will discuss how the optimised buffer is compatible with downstream strand-displacement modules and complex matrices such as cell lysates.