Manufacture thousands of very long oligonucleotides with high fidelity.

Engineering Biology

Manufacture thousands of very long oligonucleotides with high fidelity.

Current State-of-the-Art

Existing synthesis chemistries manufacture oligonucleotides up to 200 nucleotides long with cycle efficiencies of 99.5% and yields of 35%. Parallel synthesis of oligonucleotides is carried out on solid supports, producing up to 300,000 oligonucleotides with defined sequences.¹

Breakthrough Capabilities

Highly efficient oligonucleotide synthesis to increase the number, length, and fidelity of oligonucleotides.

Robustly synthesize one million 200-mer oligonucleotides with a per-nucleotide error rate of fewer than one in 500 nucleotides.

Robustly synthesize 1,000-mer oligonucleotides with a per-nucleotide error rate of fewer than one in 1,000 nucleotides.

Bottleneck/Challenge: Current phosphoramidite-based chemistries have peaked at 99.5% per-nucleotide efficiencies, resulting in only 0.66% yields when producing 1,000-mers; efficiencies must be 99.9% to achieve more than 35% yields and cycle times must also be reduced for commercial scalability.

Potential Solution: Enzyme-based, non-templated synthesis (e.g., via terminal deoxynucleotidyl transferases) has the potential to achieve greater than 99.9% per-nucleotide efficiencies and synthesis rates exceeding one nucleotide-per-second.

Reduce per-nucleotide error rates for 1,000-mer oligonucleotide synthesis to fewer than one in 5,000 nucleotides.

Bottleneck/Challenge: Non-templated DNA synthesis is currently slow with lower fidelity than templated synthesis and improvements in enzyme substrate selectivity or substrate availability are needed to control sequence-specific synthesis.

Potential Solution: Significant bioprospecting, rational design, and directed evolution of enzymes responsible for non-templated DNA synthesis can improve selectivity and increase catalytic efficiencies.

Synthesize 10,000-mer oligonucleotides at 99.99% cycle efficiency within one minute with a per-nucleotide error rate of fewer than one in 30,000 nucleotides.

Bottleneck/Challenge: Multiple synergistic improvements are needed, including improved non-templated DNA polymerases, fast substrate switching at the nanoliter scale, multi-nucleotide same-cycle addition, and electronic control of substrate selection.

Potential Solution: Inspiration from natural DNA polymerases, ligases, recombinases, and helicases, working together in a dynamic molecular machine, potentially using non-natural nucleotides for greater specificity.

Footnotes

Kosuri, S., & Church, G. M. (2014). Large-scale de novo DNA synthesis: technologies and applications. Nature Methods, 11(5), 499–507. View publication.; Hughes, R. A., & Ellington, A. D. (2017). Synthetic DNA synthesis and assembly: putting the synthetic in synthetic biology. Cold Spring Harbor Perspectives in Biology, 9(1). View publication.