Development and Characterization of a Nucleoside Kinase That Accepts Unnatural Deoxyribonucleoside P, a Part of an Expanded DNA

One of the more ambitious long-term goals of synthetic biology seeks to construct living cells that accept and use biopolymers different from DNA, RNA, and proteins. For example, the Romesberg group at The Scripps Research Institute reported last year that a strain of Escherichia coli could maintain an unnatural nucleotide pair whose structure greatly deviates from the standard Watson-Crick geometry. Since the Scripps group could not obtain kinases to convert
fed nucleosides intracellularly to the triphosphates needed for DNA replication, they instead altered the E. coli to have transport systems that transported their nucleoside triphosphate analogs from the growth medium into the cell directly.
Our approach is different. We seek to construct a host cell that creates the triphosphates of components of an unnatural genetic system inside of the cell. This would allow the relatively inexpensive (and much more stable) nucleosides to be added to the growth medium, where we have now shown that natural nucleoside transporters bring them inside of the cells. This strategy requires (a) that the structure of the added nucleotides not differ too greatly from the structures of the standard nucleotides, so that (b) we can engineer standard nucleoside and nucleotide kinases to accept them in a synthetic metabolic pathway that makes the triphosphates intracellularly.
Over the past five years, we have developed a set of alternative nucleotides that create a
12 letter artificially expanded genetic information system (AEGIS) (Fig. 1). AEGIS pairs are joined by alternative patterns of hydrogen bond donor and acceptor groups. In my work, I have examined one of these AEGIS components, 2-amino-8-(1â??-?-D-2â??-deoxyribofuranosyl)- imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, trivially called P. DNA polymerases, RNA polymerases, and reverse transcriptases have all been developed to copy and transcribe DNA and RNA containing P and its pair, 6-amino-5-nitro-3-(1â??-?-D-2â??-deoxyribofuranosyl)-2(1H)-pyridone (trivially called Z).
A nucleoside kinase from Drosophila melanogaster (DmdNK) was engineered to accept unnatural nucleoside dP to produce unnatural nucleoside monophosphates dPMP. This is the first phosphorylation step to synthesize the corresponding nucleoside triphosphate dPTP, a substrate for DNA polymerases.
In this study, the activity of DmdNK Q81E mutant was measured in a three-step coupled- enzyme assay [1] and its kinetic parameters were calculated. These results showed that the Q81E
mutation allowed the engineered kinase to accept dP as a substrate. The Michaelis constant

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(KM ), the turnover rate (kcat ), and specificity constant (kcat /KM ) for dP were similar to those for standard nucleosides dA, dT, dC.
To formally complete the construction of an artificial metabolism in vitro, I am also developing nucleoside monophosphate kinases (NMPK) to phosphorylate dPMP to give dPDP. I will describe work adding these to nucleoside diphosphate kinase (NDPK), which I have already shown phosphorylate dPDP to give dPTP, the third phosphorylation step to synthesize triphosphate substrates for DNA polymerases. Once transferred into living cells, these enzymes should allow the formation of dPTP in vivo, and represent an example of a standard problem- solution in synthetic biology.

Figure 1. The expanded genetic alphabet is created by shuffling hydrogen bond donor and acceptor groups within the Watson-Crick pairing geometry. Standard base are in the box on the left. The Z:P pair is boxed in the center. The similarity between Z and P and natural nucleotides allows us to solve the metabolism problem that the Scripps group left unsolved. Also, once the Z:P pair is in place, the strategy allows further expansion to add three more base pairs.

Reference

[1] Gerth, M. L.; Lutz, S. (2007) Mutagenesis of non-conserved active site residues improves the activity and narrows the specificity of human thymidine kinase 2. Biochem. Biophys. Res. Commun., 354, 3: 802-807.

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