Fig. 1: Overview of photon-directed multiplexed enzymatic DNA synthesis system.

a An array surface derivatized with single-stranded DNA initiator oligonucleotide is brought into contact with a master mix containing the appropriate buffers, Co²⁺ divalent cation cofactor, TdT enzyme, the desired nucleotide to be incorporated (dXTP), and photolabile DMNP-EDTA caging molecule provided in excess. All Co²⁺ ions are initially complexed with DMNP-EDTA, which causes TdT to remain in an inactive state until needed. Using photolithography, patterned UV light at 365 nm illuminates the array’s surface causing the complexed DMNP-EDTA to degrade releasing Co²⁺ ions and activates TdT in a spatially selective manner. The UV light is then turned off and the reaction is allowed to incubate for a short period of time. During this incubation, excess, non-complexed DMNP-EDTA spontaneously quenches the extension reaction by chelating free Co²⁺ causing active TdT to become inactive. The array surface is then washed and either the next synthesis cycle begins or material is retrieved from the surface for downstream applications. b Arrays are mounted into a simple flow cell with a reaction chamber inlet and outlet to waste or collection. Individually addressable patterning is a major advantage of our synthesis method, which is provided by the generation of reflective on-demand dynamic masks from the DMD through a ×10 objective from a collimated UV light source.