PT  - JOURNAL ARTICLE
AU  - Hoff, K.
AU  - Halpain, M.
AU  - Garbagnati, G.
AU  - Edwards, J.
AU  - Zhou, W.
TI  - Rapid and dynamic nucleic acid hybridization enables enzymatic oligonucleotide synthesis by cyclic reversible termination: A novel mechanism for enzymatic DNA synthesis
AID  - 10.1101/561092
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 561092
4099  - http://biorxiv.org/content/early/2019/04/15/561092.short
4100  - http://biorxiv.org/content/early/2019/04/15/561092.full
AB  - Phosphoramidite chemistry for DNA synthesis remains the industry standard despite limitations on length and yield of the resulting oligonucleotides, time restrictions, and the production of hazardous waste. Herein, we demonstrate the synthesis of single-stranded oligos on a solid surface by DNA polymerases and reverse transcriptases. We report single base extension of the surface-bound oligonucleotide which transiently hybridizes to a neighboring strand with as few as the last two bases. Additionally, when multiple transient hybridization structures are possible, each templating a different base, a DNA polymerase or reverse transcriptase can extend the oligonucleotide with either of these two bases, and therefore the sequence of the newly synthesized fragment can be controlled by adding only the desired base (dNTP deoxyribonucleic acid triphosphate) to create custom oligonucleotides. We used this enzymatic approach to synthesize a 20 base oligonucleotide by incorporating reversible terminator dNTPs 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.