title: DNA polymerase-mediated synthesis of unbiased threose nucleic acid (TNA) polymers requires 7-deazaguanine to suppress G:G mispairing during TNA transcription.
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.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Dunn
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Larsen
authors.affiliation: ∥Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United States.
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Zahurancik
authors.forename: Nour Eddine
authors.initials: NE
authors.lastname: Fahmi
authors.forename: Madeline
authors.initials: M
authors.lastname: Meyers
authors.affiliation: ∥Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United States.
authors.forename: Zucai
authors.initials: Z
authors.lastname: Suo
authors.forename: John C
authors.initials: JC
authors.lastname: Chaput
mesh_terms: D001105:Archaea / Q000201:enzymology*; D020029:Base Pairing; D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D009696:Nucleic Acids / Q000737:chemistry* / Q000378:metabolism; D013780:Tetroses / Q000737:chemistry* / Q000378:metabolism
chemical_list: D009696:Nucleic Acids; D013780:Tetroses; D006147:Guanine; D004259:DNA-Directed DNA Polymerase; C066856:7-deazaguanine; C073321:erythrose