De novo DNA synthesis using polymerase-nucleotide conjugates

@article{Palluk2018DeND,
  title={De novo DNA synthesis using polymerase-nucleotide conjugates},
  author={Sebastian Palluk and Daniel H Arlow and Tristan de Rond and Sebastian Barthel and Justine S Kang and Rathin Bector and Hratch M. Baghdassarian and Alisa N Truong and Peter W Kim and Anup Kumar Singh and Nathan J. Hillson and Jay D. Keasling},
  journal={Nature Biotechnology},
  year={2018},
  volume={36},
  pages={645-650}
}
Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of ∼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the… 
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103 Citations

Enzymatic Synthesis of Designer DNA Using Cyclic Reversible Termination and a Universal Template.
TLDR
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.
Rapid and dynamic nucleic acid hybridization enables enzymatic oligonucleotide synthesis by cyclic reversible termination or A novel mechanism for enzymatic DNA synthesis
TLDR
It is demonstrated that single-stranded oligos on a solid surface can be extended by DNA polymerases and reverse transcriptases even when hybridization seems unlikely, and 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.
Sequence Preference and Initiator Promiscuity for De Novo DNA Synthesis by Terminal Deoxynucleotidyl Transferase
TLDR
Investigation of full permutational libraries of mono- to pentamers of d- DNA, l-DNA, and 2′O-methyl-RNA of differing directionality immobilized to glass surfaces, and generated via photolithographic in situ synthesis, shows that the efficiency of extension strongly depends on the nucleobase sequence.
Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis
TLDR
Two parallel efforts to improve the activity of TdT on hairpins are described, optimization of the concentrations of the divalent cation cofactors and engineering TdD for enhanced thermostability, enabling reactions at elevated temperatures.
Evolving a thermostable terminal deoxynucleotidyl transferase.
TLDR
An assay to identify thermostable TdT variants is developed, and after screening about 10,000 T dT mutants, a variant is identified that is 10 °C more thermostably than WT TdD, while preserving the catalytic properties of the WT enzyme.
Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids
TLDR
This work summarizes the different applications stemming from the incorporation of modified nucleotides by the TdT and discusses the structural, mechanistic, and biochemical properties of this polymerase.
Evaluation of 3′-phosphate as a transient protecting group for controlled enzymatic synthesis of DNA and XNA oligonucleotides
TLDR
The use of 3’-phosphate as a transient protecting group for the controlled enzymatic synthesis of DNA and XNA oligonucleotides is explored and a hitherto unknown phosphatase activity of various DNA polymerases is discovered.
Thermococcus sp. 9°N DNA polymerase exhibits 3′-esterase activity that can be harnessed for DNA sequencing
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It is reported that the 3′-esterase activity is intrinsic to the Thermococcus sp.
Towards the enzymatic formation of artificial metal base pairs with a carboxy-imidazole-modified nucleotide.
Rapid in vitro production of single-stranded DNA
TLDR
This work presents a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases and demonstrates that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.
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