De novo DNA synthesis using polymerase-nucleotide conjugates

Palluk, Sebastian; Arlow, Daniel H.; de Rond, Tristan; Barthel, Sebastian; Kang, Justine S.; Bector, Rathin; Baghdassarian, Hratch M.; Truong, Alisa N.; Kim, Peter W.; Singh, Anup K.; Hillson, Nathan J.; Keasling, Jay D.

doi:10.1038/nbt.4173

Title: De novo DNA synthesis using polymerase-nucleotide conjugates

Journal Article · Mon Jun 18 00:00:00 EDT 2018 · Nature Biotechnology

DOI:https://doi.org/10.1038/nbt.4173· OSTI ID:1461176

Palluk, Sebastian ^[1]; Arlow, Daniel H. ^[2]; de Rond, Tristan ^[2]; Barthel, Sebastian ^[1]; Kang, Justine S. ^[2]; Bector, Rathin ^[2]; Baghdassarian, Hratch M. ^[2]; Truong, Alisa N. ^[3]; Kim, Peter W. ^[4]; Singh, Anup K. ^[4];

^[5];

^[6]

Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Technische Univ. Darmstadt, Darmstadt (Germany)
Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States); Technical Univ. of Denmark, Horsholm (Denmark)

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 tethered dNTP, the 3' end of the primer remains covalently bound to TdT and is inaccessible to other TdT-dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT-dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10-20 s, and that the scheme can be iterated to write a defined sequence. Furthermore, this approach may form the basis of an enzymatic oligonucleotide synthesizer.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1461176

Journal Information:: Nature Biotechnology, Vol. 36, Issue 7; ISSN 1087-0156

Publisher:: Springer NatureCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 112 works

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