Assays To Detect the Formation of Triphosphates of Unnatural Nucleotides: Application to Escherichia coli Nucleoside Diphosphate Kinase
| Autor: | Myong-Jung Kim, Shuichi Hoshika, Nilesh B. Karalkar, Steven A. Benner, Hyo-Joong Kim, Myong-Sang Kim, Jennifer D. Moses, Ryan W. Shaw, Mariko F. Matsuura |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
DNA polymerase Deoxyribonucleotides Biomedical Engineering Biochemistry Genetics and Molecular Biology (miscellaneous) 03 medical and health sciences Synthetic biology Genes Reporter Polyphosphates Escherichia coli Nucleotide Polymerase chemistry.chemical_classification 030102 biochemistry & molecular biology biology Nucleotides Kinase Oligonucleotide Escherichia coli Proteins General Medicine Nucleoside-diphosphate kinase 030104 developmental biology Biochemistry chemistry Nucleoside-Diphosphate Kinase biology.protein Chromatography Thin Layer Nucleoside |
| Zdroj: | ACS Synthetic Biology. 5:234-240 |
| ISSN: | 2161-5063 |
| DOI: | 10.1021/acssynbio.5b00172 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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