Abstract
Enzymatic DNA synthesis (EDS) is a promising benchtop and user-friendly method of nucleic acid synthesis that, instead of solvents and phosphoramidites, uses mild aqueous conditions and enzymes. For applications such as protein engineering and spatial transcriptomics that require either oligo pools or arrays with high sequence diversity, the EDS method needs to be adapted and certain steps in the synthesis process spatially decoupled. Here, we have used a synthesis cycle comprising a first step of site-specific silicon microelectromechanical system inkjet dispensing of terminal deoxynucleotidyl transferase enzyme and 3' blocked nucleotide, and a second step of bulk slide washing to remove the 3' blocking group. By repeating the cycle on a substrate with an immobilized DNA primer, we show that microscale spatial control of nucleic acid sequence and length is possible, which, here, are assayed by hybridization and gel electrophoresis. This work is distinctive for enzymatically synthesizing DNA in a highly parallel manner with single base control.