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Kim H.-J.,Foundation for Applied Molecular Evolution FfAME | Kim H.-J.,Firebird Biomolecular Sciences, LLC | Leal N.A.,Foundation for Applied Molecular Evolution FfAME | Leal N.A.,Firebird Biomolecular Sciences, LLC | And 4 more authors.
Journal of Organic Chemistry | Year: 2014

Rearranging hydrogen bonding groups adds nucleobases to an artificially expanded genetic information system (AEGIS), pairing orthogonally to standard nucleotides. We report here a large-scale synthesis of the AEGIS nucleotide carrying 2-amino-3-nitropyridin-6-one (trivially Z) via Heck coupling and a hydroboration/oxidation sequence. RiboZ is more stable against epimerization than its 2′-deoxyribo analogue. Further, T7 RNA polymerase incorporates ZTP opposite its Watson-Crick complement, imidazo[1,2-a]-1,3,5-triazin-4(8H)one (trivially P), laying grounds for using this "second-generation" AEGIS Z:P pair to add amino acids encoded by mRNA. © 2014 American Chemical Society. Source


Kim H.-J.,Foundation for Applied Molecular Evolution FfAME | Kim H.-J.,Westheimer Institute for Science and Technology TWIST | Chen F.,Foundation for Applied Molecular Evolution FfAME | Chen F.,Westheimer Institute for Science and Technology TWIST | And 3 more authors.
Journal of Organic Chemistry | Year: 2012

6-Aminopyridin-2-ones form Watson-Crick pairs with complementary purine analogues to add a third nucleobase pair to DNA and RNA, if an electron-withdrawing group at position 5 slows oxidation and epimerization. In previous work with a nucleoside analogue trivially named dZ, the electron withdrawing unit was a nitro group. Here, we describe an analogue of dZ (cyano-dZ) having a cyano group instead of a nitro group, including its synthesis, pK a, rates of acid-catalyzed epimerization, and enzymatic incorporation. © 2012 American Chemical Society. Source


Chen F.,Foundation for Applied Molecular Evolution FfAME | Chen F.,Westheimer Institute for Science and Technology TWIST | Yang Z.,Foundation for Applied Molecular Evolution FfAME | Yang Z.,Westheimer Institute for Science and Technology TWIST | And 6 more authors.
Nucleic Acids Research | Year: 2011

To explore the possibility of using restriction enzymes in a synthetic biology based on artificially expanded genetic information systems (AEGIS), 24 type-II restriction endonucleases (REases) were challenged to digest DNA duplexes containing recognition sites where individual Cs and Gs were replaced by the AEGIS nucleotides Z and P [respectively, 6-amino-5-nitro-3-(1′- β-d-2′-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1′- β-d-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one]. These AEGIS nucleotides implement complementary hydrogen bond donor-donor-acceptor and acceptor-acceptor-donor patterns. Results allowed us to classify type-II REases into five groups based on their performance, and to infer some specifics of their interactions with functional groups in the major and minor grooves of the target DNA. For three enzymes among these 24 where crystal structures are available (BcnI, EcoO109I and NotI), these interactions were modeled. Further, we applied a type-II REase to quantitate the fidelity polymerases challenged to maintain in a DNA duplex C:G, T:A and Z:P pairs through repetitive PCR cycles. This work thus adds tools that are able to manipulate this expanded genetic alphabet in vitro, provides some structural insights into the working of restriction enzymes, and offers some preliminary data needed to take the next step in synthetic biology to use an artificial genetic system inside of living bacterial cells. © 2011 The Author(s). Source


Yang Z.,Foundation for Applied Molecular Evolution FfAME | Yang Z.,Westheimer Institute for Science and Technology TWIST | Chen F.,Foundation for Applied Molecular Evolution FfAME | Chen F.,Westheimer Institute for Science and Technology TWIST | And 4 more authors.
Journal of the American Chemical Society | Year: 2011

The next goals in the development of a synthetic biology that uses artificial genetic systems will require chemistry-biology combinations that allow the amplification of DNA containing any number of sequential and nonsequential nonstandard nucleotides. This amplification must ensure that the nonstandard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technology is needed to sequence artificial genetic DNA molecules. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four standard nucleotides (G, A, C, and T) and two additional nonstandard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theoretical cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence "six-letter" GACTZP DNA. These are all not yet reported for any other synthetic genetic system. © 2011 American Chemical Society. Source

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