One horizon in synthetic biology seeks alternative forms of DNA that store, transcribe, and support the evolution of biological information. Here, hydrogen bond donor and acceptor groups are rearranged within a Watson-Crick geometry to get 12 nucleotides that form 6 independently replicating pairs. Such artificially expanded genetic information systems (AEGIS) support Darwinian evolution . To move AEGIS into living cells, metabolic pathways are next required to make AEGIS triphosphates economically from their nucleosides, eliminating the need to feed these expensive compounds in growth media. We report that "polyphosphate kinases" can be recruited for such pathways, working with natural diphosphate kinases and engineered nucleoside kinases. This pathway makes AEGIS triphosphates, including third-generation triphosphates having improved ability to survive in living bacterial cells. In α-P-labeled forms, produced here for the first time, they were used to study DNA polymerases, finding cases where third-generation AEGIS triphosphates perform better with natural enzymes than second-generation AEGIS triphosphates.
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Synthetic Biology Pathway to Nucleoside Triphosphates for Expanded Genetic Alphabets.
ACS Synth Biol
June 2023
Foundation for Applied Molecular Evolution, 13709 Progress Blvd., Alachua, Florida 32615, United States.
One horizon in synthetic biology seeks alternative forms of DNA that store, transcribe, and support the evolution of biological information. Here, hydrogen bond donor and acceptor groups are rearranged within a Watson-Crick geometry to get 12 nucleotides that form 6 independently replicating pairs. Such artificially expanded genetic information systems (AEGIS) support Darwinian evolution .
View Article and Find Full Text PDFA Single Deoxynucleoside Kinase Variant from Drosophila melanogaster Synthesizes Monophosphates of Nucleosides That Are Components of an Expanded Genetic System.
ACS Synth Biol
March 2017
The Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Blvd., Box 17, Alachua, Florida 32615, United States.
Deoxynucleoside kinase from D. melanogaster (DmdNK) has broad specificity; although it catalyzes the phosphorylation of natural pyrimidine more efficiently than natural purine nucleosides, it accepts all four 2'-deoxynucleosides and many analogues, using ATP as a phosphate donor to give the corresponding deoxynucleoside monophosphates. Here, we show that replacing a single amino acid (glutamine 81 by glutamate) in DmdNK creates a variant that also catalyzes the phosphorylation of nucleosides that form part of an artificially expanded genetic information system (AEGIS).
View Article and Find Full Text PDFAssays To Detect the Formation of Triphosphates of Unnatural Nucleotides: Application to Escherichia coli Nucleoside Diphosphate Kinase.
ACS Synth Biol
March 2016
Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
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.
View Article and Find Full Text PDFDNA polymerases engineered by directed evolution to incorporate non-standard nucleotides.
Front Microbiol
November 2014
Foundation for Applied Molecular Evolution Gainesville, FL, USA.
Article Synopsis
- DNA polymerases, which traditionally accept only natural nucleotides, have been refined over billions of years to ensure high accuracy in DNA synthesis.
- There is increasing interest in developing polymerases that can incorporate unnatural nucleotides for various biotechnological applications.
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Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern.
Nucleic Acids Res
January 2007
Foundation for Applied Molecular Evolution, 1115 NW 4th Street, Gainesville, FL 32601, USA.
To support efforts to develop a 'synthetic biology' based on an artificially expanded genetic information system (AEGIS), we have developed a route to two components of a non-standard nucleobase pair, the pyrimidine analog 6-amino-5-nitro-3-(1'-beta-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson-Crick complement, the purine analog 2-amino-8-(1'-beta-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP). These implement the pyDDA:puAAD hydrogen bonding pattern (where 'py' indicates a pyrimidine analog and 'pu' indicates a purine analog, while A and D indicate the hydrogen bonding patterns of acceptor and donor groups presented to the complementary nucleobases, from the major to the minor groove). Also described is the synthesis of the triphosphates and protected phosphoramidites of these two nucleosides.
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