Sequence Preference and Initiator Promiscuity for <i>De Novo</i> DNA Synthesis by Terminal Deoxynucleotidyl Transferase

Schaudy, Erika; Lietard, Jory; Somoza, Mark M.

doi:10.1021/acssynbio.1c00142

Cited by 24 publications

(18 citation statements)

References 61 publications

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“…This nonzero level of background fluorescence is consistent with loss of the DMT protecting group and extension of the single-base layer by TdT. Fluorescent patterning can still be observed despite this loss of specificity, as the (dT) 5 homopolymer is extended more quickly than the single dTTP residue …”

Section: Resultssupporting

confidence: 54%

Spatially Selective Electrochemical Cleavage of a Polymerase-Nucleotide Conjugate

et al. 2023

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Novel enzymatic methods are poised to become the dominant processes for de novo synthesis of DNA, promising functional, economic, and environmental advantages over the longstanding approach of phosphoramidite synthesis. Before this can occur, however, enzymatic synthesis methods must be parallelized to enable production of multiple DNA sequences simultaneously. As a means to this parallelization, we report a polymerase-nucleotide conjugate that is cleaved using electrochemical oxidation on a microelectrode array. The developed conjugate maintains polymerase activity toward surfacebound substrates with single-base control and detaches from the surface at mild oxidative voltages, leaving an extendable oligonucleotide behind. Our approach readies the way for enzymatic DNA synthesis on the scale necessary for DNA-intensive applications such as DNA data storage or gene synthesis.

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Section: Resultssupporting

confidence: 54%

Spatially Selective Electrochemical Cleavage of a Polymerase-Nucleotide Conjugate

et al. 2023

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“…Notably, microarrays have proven instrumental for optimizing the parallel synthesis of oligonucleotides on TiOES platforms, including the impact of initiating strands and chemically modified NTPs on enzymatic DNA synthesis. Microarrays have also prompted early considerations for DNA nanofabrication, synthesis multiplexing and compatibility of enzymes with alternative polymerization methods 169 – 172 .…”

Section: Emerging Commercialized Technologiesmentioning

confidence: 99%

DNA synthesis technologies to close the gene writing gap

Vellacott²,

et al. 2023

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Synthetic DNA is of increasing demand across many sectors of research and commercial activities. Engineering biology, therapy, data storage and nanotechnology are set for rapid developments if DNA can be provided at scale and low cost. Stimulated by successes in next generation sequencing and gene editing technologies, DNA synthesis is already a burgeoning industry. However, the synthesis of >200 bp sequences remains unaffordable. To overcome these limitations and start writing DNA as effectively as it is read, alternative technologies have been developed including molecular assembly and cloning methods, template-independent enzymatic synthesis, microarray and rolling circle amplification techniques. Here, we review the progress in developing and commercializing these technologies, which are exemplified by innovations from leading companies. We discuss pros and cons of each technology, the need for oversight and regulatory policies for DNA synthesis as a whole and give an overview of DNA synthesis business models.

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“…This is in contrast with 3′- O -benzoyl and 3′- O -pivaloyl-protected LNA-TTPs which displayed a marked preference for Co 2+ over Mn 2+ ( Flamme et al, 2022b ). The rather low yields observed with nucleotides 1 , 2 , 3 , and 5 might also be partially ascribed to the sequence bias of the TdT polymerase since 3′-terminal cytosine nucleotides on initiators are known to negatively impact the processivity of this enzyme ( Schaudy et al, 2021 ).…”

Section: Resultsmentioning

confidence: 99%

Towards the controlled enzymatic synthesis of LNA containing oligonucleotides

et al. 2023

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Enzymatic, de novo XNA synthesis represents an alternative method for the production of long oligonucleotides containing chemical modifications at distinct locations. While such an approach is currently developed for DNA, controlled enzymatic synthesis of XNA remains at a relative state of infancy. In order to protect the masking groups of 3′-O-modified LNA and DNA nucleotides against removal caused by phosphatase and esterase activities of polymerases, we report the synthesis and biochemical characterization of nucleotides equipped with ether and robust ester moieties. While the resulting ester-modified nucleotides appear to be poor substrates for polymerases, ether-blocked LNA and DNA nucleotides are readily incorporated into DNA. However, removal of the protecting groups and modest incorporation yields represent obstacles for LNA synthesis via this route. On the other hand, we have also shown that the template-independent RNA polymerase PUP represents a valid alternative to the TdT and we have also explored the possibility of using engineered DNA polymerases to increase substrate tolerance for such heavily modified nucleotide analogs.

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Sequence Preference and Initiator Promiscuity for De Novo DNA Synthesis by Terminal Deoxynucleotidyl Transferase

Cited by 24 publications

References 61 publications

Spatially Selective Electrochemical Cleavage of a Polymerase-Nucleotide Conjugate

Spatially Selective Electrochemical Cleavage of a Polymerase-Nucleotide Conjugate

DNA synthesis technologies to close the gene writing gap

Towards the controlled enzymatic synthesis of LNA containing oligonucleotides

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