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

Abstract

Enzymatic oligonucleotide synthesis (EOS) has been attempted in many iterations for more than forty years, but chemical synthesis remains the industry standard despite hazardous waste produced, time restrictions, and length limitations of approximately 200 bases. Herein, we demonstrate that single-stranded oligos on a solid surface can transiently hybridize to neighboring strands and these structures can be recognized and extended by DNA polymerases and reverse transcriptases through a mechanism we describe as “bend and extend.” Additionally, we show that 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 3’-reversibly blocked nucleotides followed by subsequent cleavage of the 3’-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 3’ end.