title: Assays To Detect the Formation of Triphosphates of Unnatural Nucleotides: Application to Escherichia coli Nucleoside Diphosphate Kinase.
abstract: One frontier in synthetic biology seeks to move artificially expanded genetic information systems (AEGIS) into natural living cells and to arrange the metabolism of those cells to allow them to replicate plasmids built from these unnatural genetic systems. In addition to requiring polymerases that replicate AEGIS oligonucleotides, such cells require metabolic pathways that biosynthesize the triphosphates of AEGIS nucleosides, the substrates for those polymerases. Such pathways generally require nucleoside and nucleotide kinases to phosphorylate AEGIS nucleosides and nucleotides on the path to these triphosphates. Thus, constructing such pathways focuses on engineering natural nucleoside and nucleotide kinases, which often do not accept the unnatural AEGIS biosynthetic intermediates. This, in turn, requires assays that allow the enzyme engineer to follow the kinase reaction, assays that are easily confused by ATPase and other spurious activities that might arise through "site-directed damage" of the natural kinases being engineered. This article introduces three assays that can detect the formation of both natural and unnatural deoxyribonucleoside triphosphates, assessing their value as polymerase substrates at the same time as monitoring the progress of kinase engineering. Here, we focus on two complementary AEGIS nucleoside diphosphates, 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 assays provide new ways to detect the formation of unnatural deoxyribonucleoside triphosphates in vitro and to confirm their incorporation into DNA. Thus, these assays can be used with other unnatural nucleotides.
authors.affiliation: Department of Chemistry, University of Florida , Gainesville, Florida 32611, United States.|Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Mariko F
authors.initials: MF
authors.lastname: Matsuura
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences LLC , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Ryan W
authors.initials: RW
authors.lastname: Shaw
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences LLC , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Jennifer D
authors.initials: JD
authors.lastname: Moses
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences LLC , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Hyo-Joong
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences LLC , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Myong-Jung
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences LLC , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Myong-Sang
authors.initials: MS
authors.lastname: Kim
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences LLC , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Hoshika
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences LLC , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Nilesh
authors.initials: N
authors.lastname: Karalkar
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences LLC , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Benner
mesh_terms: D002855:Chromatography, Thin Layer; D003854:Deoxyribonucleotides / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry*; D011122:Polyphosphates / Q000032:analysis / Q000378:metabolism*
chemical_list: D003854:Deoxyribonucleotides; D029968:Escherichia coli Proteins; D009711:Nucleotides; D011122:Polyphosphates; D009701:Nucleoside-Diphosphate Kinase; C005692:triphosphoric acid
keywords: DNA polymerase; artificially expanded genetic information systems (AEGIS); assays; kinase; synthetic biology; triphosphates