PROJECT SUMMARY Chemical modifications to synthetic DNA are of central importance to both: 1) understanding biology and disease and 2) enabling a wide range of biomedical applications and research tools. However, despite their importance, most modification patterns are not possible to make and those that are are exorbitantly expensive to synthesize (by at least 10x), in contrast to the rapidly dropping costs for unmodified DNA synthesis. The central challenge is that the most advanced methods to create modified DNA oligos continue to rely on phosphoramidite synthesis, an organic chemistry process that uses harsh reaction conditions that require expensive chemically protected monomers and associated deprotection steps, or that simply prohibits certain types of modified DNAs from being synthesized at all. To address this challenge, we have developed a robust platform technology capable of pro- ducing chemically modified DNA. It is technologically differentiated from the current state of the art through its use of sustainable enzymatic synthesis in mild and low temperature conditions, compatibility with a diverse range of modification types, and low cost and recycleable templates and reagents. The proposed work seeks to ad- vance the commercial potential of this technology by: Aim I – creating modified DNA products that cannot be ordered from any existing vendor; Aim II – developing an innovation on our technology that will eliminate the most costly reagent in our synthesis process, the biotinylated template; and Aim III – establishing and mapping the breadth of modifications that our platform technology can incorporate into DNA oligos and their relative syn- thesis efficiencies.