De novo DNA synthesis using polymerase-nucleotide conjugates

Nat Biotechnol. 2018 Aug;36(7):645-650. doi: 10.1038/nbt.4173. Epub 2018 Jun 18.

Abstract

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. This approach may form the basis of an enzymatic oligonucleotide synthesizer.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • DNA Nucleotidylexotransferase / chemistry
  • DNA Nucleotidylexotransferase / genetics
  • DNA Replication / genetics*
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics*
  • Nucleosides / chemistry
  • Nucleosides / genetics*
  • Oligonucleotides / biosynthesis
  • Oligonucleotides / chemistry
  • Oligonucleotides / genetics*
  • Organophosphorus Compounds / chemistry

Substances

  • Nucleosides
  • Oligonucleotides
  • Organophosphorus Compounds
  • phosphoramidite
  • DNA Nucleotidylexotransferase
  • DNA-Directed DNA Polymerase