DNA polymerase-mediated synthesis of unbiased Threose Nucleic Acid (TNA) polymers requires 7-deazaguanine to suppress G

G mispairing during TNA transcription

Matthew R. Dunn, Andrew C. Larsen, Walter J. Zahurancik, Nour Eddine Fahmi, Madeline Meyers, Zucai Suo, John C. Chaput

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Threose nucleic acid (TNA) is an unnatural genetic polymer capable of undergoing Darwinian evolution to generate folded molecules with ligand-binding activity. This property, coupled with a nuclease-resistant backbone, makes TNA an attractive candidate for future applications in biotechnology. Previously, we have shown that an engineered form of the Archaean replicative DNA polymerase 9 N, known commercially as Therminator DNA polymerase, can copy a three-letter genetic alphabet (A,T,C) from DNA into TNA. However, our ability to transcribe four-nucleotide libraries has been limited by chain termination events that prevent the synthesis of full-length TNA products. Here, we show that chain termination is caused by tG:dG mispairing in the enzyme active site. We demonstrate that the unnatural base analogue 7-deazaguanine (7dG) will suppress tGTP misincorporation by inhibiting the formation of Hoogsteen tG:dG base pairs. DNA templates that contain 7dG in place of natural dG residues replicate with high efficiency and >99% overall fidelity. Pre-steady-state kinetic measurements indicate that the rate of tCTP incorporation is 5-fold higher opposite 7dG than dG and only slightly lower than dCTP incorporation opposite either 7dG or dG. These results provide a chemical solution to the problem of how to synthesize large, unbiased pools of TNA molecules by polymerase-mediated synthesis.

Original languageEnglish (US)
Pages (from-to)4014-4017
Number of pages4
JournalJournal of the American Chemical Society
Volume137
Issue number12
DOIs
StatePublished - Apr 1 2015

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Nucleic acids
DNA-Directed DNA Polymerase
Transcription
Nucleic Acids
Polymers
DNA
Molecules
Biotechnology
Nucleotides
Base Pairing
Catalytic Domain
Enzymes
Ligands
7-deazaguanine
erythrose
Kinetics

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

DNA polymerase-mediated synthesis of unbiased Threose Nucleic Acid (TNA) polymers requires 7-deazaguanine to suppress G : G mispairing during TNA transcription. / Dunn, Matthew R.; Larsen, Andrew C.; Zahurancik, Walter J.; Fahmi, Nour Eddine; Meyers, Madeline; Suo, Zucai; Chaput, John C.

In: Journal of the American Chemical Society, Vol. 137, No. 12, 01.04.2015, p. 4014-4017.

Research output: Contribution to journalArticle

Dunn, Matthew R. ; Larsen, Andrew C. ; Zahurancik, Walter J. ; Fahmi, Nour Eddine ; Meyers, Madeline ; Suo, Zucai ; Chaput, John C. / DNA polymerase-mediated synthesis of unbiased Threose Nucleic Acid (TNA) polymers requires 7-deazaguanine to suppress G : G mispairing during TNA transcription. In: Journal of the American Chemical Society. 2015 ; Vol. 137, No. 12. pp. 4014-4017.
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