(427a) Engineering a Template-Independent DNA Polymerase for DNA Data Storage and Biosignal Recording Applications

Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that synthesizes single-stranded DNA (ssDNA) and is currently being explored for use in various applications, including DNA data storage, enzymatic DNA synthesis, and temporal recording of biosignals. Several unique features make TdT a compelling tool in these areas. TdT adds nucleotides to the 3’ termini of ssDNA probabilistically, with an inherent bias in its base incorporation. It exhibits base preference changes in response to changes in its environment, and can make use of several divalent cations for its enzymatic activity. However, TdT also has properties that can be limiting. For example, TdT cannot bind ssDNA that has formed secondary structures at the 3’ end, limiting the length of extension. Therefore, it would beneficial to modulate and tailor TdT properties for different applications. However, there are currently few examples of TdT engineering in the literature and limited high-throughput screening methods to pursue a directed evolution approach to TdT engineering. Therefore, we seek to adopt a compartmentalized beads labeling (CBL) method for evaluating large libraries of TdT variants. The screen’s workflow relies on encapsulating single E. coli cells expressing different TdT variants in aqueous droplets along with fluorescently labelled dUTPs and streptavidin beads bound with ssDNA and a plasmid capture sequence. Upon cell lysis, the TdT variant and plasmid (with the TdT gene) are released from the single cell into the droplet environment. Active TdT variants extend the bead-bound ssDNA using available fluorescent dUTPs and the plasmid is annealed to the plasmid capture sequence on the bead. Recovered beads are sorted based on their fluorescence and the active genotype is linked to the phenotype via the plasmid captured on the bead. The described screen will enable TdT engineering for a range of applications, with easy adaptation for an array of selective pressures. More broadly, this screen expands the tools available for TdT engineering.