Chemically Synthesized Ultra-long DNA as Building Blocks to Accelerate Complex Gene Construction in Synthetic Biology

Certain applications of synthetic biology rely on the construction of large and complex DNA sequences. Current DNA synthesis technologies are limited in their capacity to generate ultra-long oligonucleotide for complex gene construction with extensive repetitive motifs and uneven base distribution efficiently. Here, we report a novel platform named UCOS (short forUltralongComplexOligonucleotidesSynthesis) that enables the efficient synthesis of long, complex, and challenging DNA fragments. This platform employs nonporous silica microspheres as the solid support instead of traditional CPG (Controlled Pore Glass) solid support, full-length enrichment based on 5’ flank sequence hybridization and an error-removing enzyme for correct sequence selection, substantially enhancing the fidelity of intricate, ultralong oligonucleotides. Using this approach, we successfully synthesized challenging sequences up to 600nt in length, encompassing tandem repeats and uneven base distributions. Overall, this novel platform demonstrates exceptional efficiency and reliability in handling ultralong DNA fragments with highly repetitive and complex features. It provides a strong foundation for advancing synthetic biology and shows great potential as a powerful tool for constructing challenging genes and enabling the customized synthesis of functional genetic elements.