Quantification of synthetic errors during chemical synthesis of DNA and its suppression by non-canonical nucleosides

Abstract

Substitutions, insertions, and deletions derived from synthetic oligonucleotides are the hurdles for the synthesis of long DNA such as genomes. We quantified these synthetic errors by next-generation sequencing and revealed that the quality of the enzymatically amplified final combined product depends on the conditions of the preceding solid phase chemical synthesis, which generates the initial pre-amplified fragments. Among all possible substitutions, the G-to-A substitution was the most prominently observed substitution followed by G-to-T, C-to-T, T-to-C, and A-to-G substitutions. The observed error rate for G-to-A substitution was influenced by capping conditions, suggesting that the capping step played a major role in the generation of G-to-A substitution. Because substitutions observed in long DNA were derived from the generation of non-canonical nucleosides during chemical synthesis, non-canonical nucleosides resistant to side reactions could be used as error-proof nucleosides. As an example of such error-proof nucleosides, we evaluated 7-deaza-2´-deoxyguanosine and 8-aza-7-deaza-2´-deoxyguanosine and showed 50-fold decrease in the error rate of G-to-A substitution when phenoxyacetic anhydride was used as capping reagents. This result is the first example that improves the quality of synthesized sequences by using non-canonical nucleosides as error-proof nucleosides. Our results would contribute to the development of highly accurate template DNA synthesis technologies.

Figure 1

Phosphoramidite chemistry for DNA synthesis.

Figure 2

Synthetic error quantification of chemically synthesized oligonucleotides.

Figure 3

Reproducibility of observed error rates. Three oligonucleotides synthesized independently using the same synthetic conditions were compared. The synthetic conditions are 1H-tetrazole in anhydrous acetonitrile as an activator, acetic anhydride in THF as a capping reagent A, 10% 1-methylimidazole in 10% pyridine-THF as a capping reagent B, 0.02 M I₂ in THF/pyridine/H₂O (90:<1:10, v/v/v) as an oxidation reagent, and 3% trichloroacetic acid in dichloromethane (TCA) as a deblocking reagent. Q5 High-Fidelity DNA polymerase was used for assembling reaction.

Figure 4

Comparison of error rates derived from assembling reactions using different polymerases. The synthesized oligonucleotide in the same batch was split into three samples and prepared NGS library by using Q5 High-Fidelity DNA polymerase (Q5), Phusion High-Fidelity DNA polymerase (Phusion), or Takara Ex Taq (Ex). (a) Q5 vs Phusion. (b) Q5 vs Ex.

Figure 5

Error rates for substitutions, insertions, and deletions during standard oligonucleotide synthesis. The data were obtained from three independently synthesized oligonucleotides. The synthetic conditions are 1H-tetrazole in anhydrous acetonitrile as an activator, acetic anhydride in THF as a capping reagent A, 10% 1-methylimidazole in 10% pyridine-THF as a capping reagent B, 0.02 M I₂ in THF/pyridine/H₂O (90:<1:10, v/v/v) as an oxidation reagent, and 3% trichloroacetic acid in dichloromethane (TCA) as a deblocking reagent. Q5 High-Fidelity DNA polymerase was used for assembling reaction.

Figure 6

Synthetic condition dependency of error rates of G-to-A and T-to-C substitutions. The data were obtained from three independently synthesized oligonucleotides. (a) G-to-A substitutions. (b) T-to-C substitutions.

Figure 7

Mechanism of G-to-A substitutions. (a) Previously proposed mechanism of diaminopurine formation, which is slightly modified based on this study. (b) Optimized structure of the model of intermediates. (c) Positions of unnatural nucleosides. (d) Observed error rates related to deoxyguanosine. The synthetic conditions for Pac₂O (n = 2) are 5-benzylthio-1H-tetrazole in anhydrous acetonitrile as an activator, phenoxyacetic anhydride in THF as a capping reagent A, 10% 1-methylimidazole in 10% pyridine-THF as a capping reagent B, 0.02 M I₂ in THF/pyridine/H₂O (90:<1:10, v/v/v) as an oxidation reagent, and 3% trichloroacetic acid in dichloromethane (TCA) as a deblocking reagent. The synthetic conditions for Ac₂O (n = 1) are 1H-tetrazole in anhydrous acetonitrile as an activator, acetic anhydride in THF as a capping reagent A, 10% 1-methylimidazole in 10% pyridine-THF as a capping reagent B, 0.02 M I₂ in THF/pyridine/H₂O (90:<1:10, v/v/v) as an oxidation reagent, and 3% trichloroacetic acid in dichloromethane (TCA) as a deblocking reagent. Q5 High-Fidelity DNA polymerase was used for assembling reaction.