Highly Ordered DNA Framework Interface Enables Efficient Enzymatic Oligonucleotide Synthesis

De novo DNA synthesis plays crucial roles in life science. Enzymatic oligonucleotide synthesis (EOS) has attracted interest due to longer synthesized chains, simple procedure, cost-effectiveness, and environmental friendliness. However, unlike chemical synthesis dominated by small molecule, the EOS relies on enzyme reacting with primers. It remains challenging due to restricted accessibility caused by the anisotropy of initiator primers and the spatial hindrance of Enzymes. Herein, this study developes a nanoscopic interface functioned with 3D DNA framework to achieve efficient EOS. The highly ordered DNA framework - tetrahedral DNA nanostructures (TDN) provide an ordered upright orientation and reasonable spacing for primers to enhance enzyme accessibility. Compared to single-stranded structures, the TDN scaffold significantly enhances the enzyme's substrate affinity and catalytic reaction kinetics. As for the synthesis of five given patterned sequences, TDN scaffold effectively reduces the occurrence of deletion errors with increasing yield. Finally, efficient TDN-based EOS is employed for DNA information storage by synthesizing a 60-nucletide DNA fragment with a stepwise yield of 96.82%, allowing the accurate retrieval of 15 bytes of text information. The TDN-based EOS paves the way for developing more efficient and accurate DNA synthesis methods, laying a robust foundation for future applications in DNA storage and genetic research.

DNA information storage; DNA synthesis efficiency; synthetic biology; tetrahedral DNA nanostructure.