Chemically Synthesized High-Fidelity Oligos up to 600 nt as Building Blocks to Accelerate Complex Gene Construction in Synthetic Biology

Abstract Synthetic biology and advanced genetic engineering applications rely heavily on the efficient construction of large and complex DNA sequences. Current DNA synthesis technologies have limited capacity to efficiently generate ultralong oligonucleotides for complex gene construction, particularly those with extensive repetitive motifs and uneven base distribution. Here, we report a novel platform named, UCOS (Ultralong Complex Oligonucleotides Synthesis), 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 600 nt 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. This novel platform provides a strong foundation for advancing synthetic biology and metabolic engineering, showing great potential as a powerful tool for constructing challenging genes and enabling the customized synthesis of functional genetic elements for complex genetic programs and synthetic genomics.