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....Continue Reading
References
Dec 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·M A InnisM A Brow
Feb 1, 1993·Proceedings of the National Academy of Sciences of the United States of America·J V SkellyS Neidle
Aug 26, 1998·Chemistry & Biology·S O Kelley, J K Barton
Jun 3, 1999·Nucleic Acids Research·A F Gardner, W E Jack
Jun 26, 1999·Science·A Eschenmoser
Jan 15, 2002·Nucleic Acids Research·Andrew F Gardner, William E Jack
Jul 12, 2002·Nature·Gerald F Joyce
Aug 2, 2003·Journal of the American Chemical Society·John C Chaput, Jack W Szostak
Oct 25, 2003·Nucleic Acids Research·Veerle KempeneersPiet Herdewijn
Mar 3, 2005·Journal of the American Chemical Society·Justin K IchidaJack W Szostak
May 19, 2005·Journal of the American Chemical Society·Allen HorhotaJohn C Chaput
Sep 15, 2005·Nucleic Acids Research·Justin K IchidaJack W Szostak
Dec 6, 2008·Nucleic Acids Research·Bahram Arezi, Holly Hogrefe
Aug 25, 2010·Chembiochem : a European Journal of Chemical Biology·Nadine Staiger, Andreas Marx
Feb 23, 2012·Nature Chemistry·Hanyang YuJohn C Chaput
Apr 21, 2012·Science·Vitor B PinheiroPhilipp Holliger
Nov 28, 2012·Chemistry & Biology·John C ChaputSu Zhang
Jan 29, 2013·Bioorganic & Medicinal Chemistry Letters·Su Zhang, John C Chaput
Feb 26, 2013·Journal of the American Chemical Society·Hanyang YuJohn C Chaput
Citations
Feb 11, 2016·ACS Chemical Biology·Matthew R DunnJohn C Chaput
Sep 22, 2015·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Stella Diafa, Marcel Hollenstein
Feb 20, 2016·The Journal of Organic Chemistry·Sujay P SauJohn C Chaput
Feb 6, 2017·Chembiochem : a European Journal of Chemical Biology·Sydney L RosenblumAaron M Leconte
Mar 26, 2016·Methods : a Companion to Methods in Enzymology·Kirsten N MeekJennifer M Heemstra
Jan 17, 2017·Bioorganic & Medicinal Chemistry Letters·Pascal RöthlisbergerMarcel Hollenstein
Dec 18, 2015·Physical Chemistry Chemical Physics : PCCP·Yevgen P YurenkoRadek Marek
Apr 6, 2016·Nature Communications·Andrew C LarsenJohn C Chaput
May 10, 2019·Frontiers in Molecular Biosciences·Andrew F GardnerWilliam E Jack
May 28, 2019·Chemical Communications : Chem Comm·Takenori ShimoSatoshi Obika
Aug 8, 2018·Nucleic Acids Research·Alexandra E RangelJennifer M Heemstra
Jan 3, 2020·ACS Synthetic Biology·Kendall HoffWei Zhou