(489f) Exploring Sequence-Function Space of a Template-Independent DNA Polymerase for DNA Data Storage and Biorecording Applications

Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that is currently being explored for various applications, including enzymatic DNA synthesis, DNA data storage, and temporal recording of biosignals. TdTs have several unique features that make them a promising tool; they incorporate nucleotide bases probabilistically to single-stranded DNA (ssDNA) substrates, change base incorporation preference in response to their environment, and are catalytically active with several divalent cations. However, TdTs also have properties that can be limiting for various applications. For example, TdTs have relatively slow base incorporation rates compared to other DNA polymerases and cannot effectively incorporate nucleotides into ssDNA secondary structures such as hairpins. While we anticipate that such challenges can be addressed through TdT engineering, there is no adequate knowledge of the relationship between TdT domains and its catalytic properties nor is there a high-throughput screen for TdTs to gain in-depth understanding of sequence-function space. Therefore, we seek to explore the natural diversity of TdTs. To accomplish this, we have selected 10 TdTs that represent the maximum sequence diversity of TdT homologues; our goal is to screen these TdTs for useful and desirable characteristics for recording and data storage applications. These characteristics include favorable kinetics, large base preference biases for signals of interest, and the ability to extend single-stranded DNA primer for long lengths. Our study will correlate differences in TdT characteristics to differences in sequence space that should inform engineering of TdT for a range of applications.