(402a) A Rapid and Quantitative Assay for DNA Binding

Protein-DNA interactions are typically assessed using the electrophoretic mobility shift assay (EMSA) or an adaptation of the EMSA. However, the EMSA and its adaptations are often not quantitative and suffer from low throughput. In this work, we describe a rapid and quantitative DNA binding assay that employs fluorescent DNA and radiolabeled protein.

We synthesize hexahistidine-tagged DNA-binding proteins using cell-free protein synthesis (CFPS). The open system of CFPS allows us to easily radiolabel the protein of interest. The hexahistidine tag allows us to capture the protein-DNA complex using metal chelation affinity media. A fluorescent signal from the DNA and a radioactivity signal from the protein are then measured. With this information, we can quantify the DNA-to-protein ratio and estimate the fraction of protein sample that is competent for DNA binding. This is system is amenable for high throughput evaluation of DNA binding activity, useful when exploring different expression conditions for optimizing production yields of difficult-to-produce DNA-binding proteins.