Figure 2.

Schematic overview of the kinase library screening assay with fluorescent NAs. Upon incubation of bacteria with the target fNA, cells that carry a functional kinase variant for the specific substrate will accumulate the fNA monophosphate. Hosts cells with the highest fluorescence intensity can be identified and isolated by fluorescence-activated cell sorting (FACS) for subsequent characterization of the library member or, if desirable, another round of directed evolution. For concept validation, E. coli TOP10 expressing exogenous DmdNK with thymidine kinase activity (+kinase) was compared to host expressing thymidine kinase-deficient human dCK (−kinase). The histogram shows the 20- to 30-fold increase in cellular fluorescence upon incubation with fT in the presence of the exogenous thymidine kinase activity.