Rapid and dynamic nucleic acid hybridization enables enzymatic oligonucleotide synthesis by cyclic reversible termination

Phosphoramidite chemistry for DNA synthesis remains the industry standard despite limitations on length and yield, time restrictions, and the production of hazardous waste. Herein, we demonstrate that single-stranded oligos on a solid surface can be extended by DNA polymerases and reverse transcriptases even when hybridization seems unlikely. We report extension using one polymerase and one transcriptase despite the fact that only the last two bases of the surface-bound oligonucleotide can hybridize to a neighboring strand. Additionally, when two hybridization structures are possible, each templating a different base, the oligonucleotide can be extended with either of these two bases, and the sequence of the newly synthesized fragment can be controlled to create custom oligonucleotides. We used this enzymatic approach to synthesize 20 bases on a solid surface through a two-step cyclic reversible termination process with stepwise efficiency over 98%. In our approach, a nascent DNA strand that serves as both primer and template is extended through polymerase-controlled sequential addition of 39-reversibly blocked nucleotides followed by subsequent cleavage of the 39-capping group. This process enables oligonucleotide synthesis in an environment not permitted by traditional phosphoramidite methods, eliminates the need for hazardous chemicals, has the potential to provide faster and higher yield results, and synthesizes DNA on a solid support with a free 39 end.