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Author Notes:

Email: chenfei@big.ac.cn (FC); sbenner@ffame.org (SAB)

Conceptualization: FC SL SAB.

Funding acquisition: FC SAB.

Investigation: FC YZ ABD ZY RS MXD.

Software: YZ.

Supervision: FC SL SAB.

Writing – original draft: FC.

Writing – review & editing: FC.

Subjects:

Research Funding:

This research was funded by the National Natural Science Foundation of China (No. 21472182, 31270846) and the ‘100-Talent Program’ of the Chinese Academy of Sciences (No. Y3CAS81554).

This research was also based on work supported by the NSF under Grant No. 1412869, and the Defense Advanced Research Projects Agency Microsystems Technology (DSO) under contract CLIO: N66001-12C-4019, ARPA Order No. 8657\00.

Biological phosphorylation of an Unnatural Base Pair (UBP) using a Drosophila melanogaster deoxynucleoside kinase (DmdNK) mutant

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Journal Title:

PLoS ONE

Volume:

Volume 12, Number 3

Publisher:

, Pages e0174163-e0174163

Type of Work:

Article | Final Publisher PDF

Abstract:

One research goal for unnatural base pair (UBP) is to replicate, transcribe and translate them in vivo. Accordingly, the corresponding unnatural nucleoside triphosphates must be available at sufficient concentrations within the cell. To achieve this goal, the unnatural nucleoside analogues must be phosphorylated to the corresponding nucleoside triphosphates by a cascade of three kinases. The first step is the monophosphorylation of unnatural deoxynucleoside catalyzed by deoxynucleoside kinases (dNK), which is generally considered the rate limiting step because of the high specificity of dNKs. Here, we applied a Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) to the phosphorylation of an UBP (a pyrimidine analogue (6-amino-5-nitro-3-(1’-b-d-2’-deoxyribofuranosyl)-2(1H)-pyridone, Z) and its complementary purine analogue (2-amino-8-(1’-b-d-2’-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, P). The results showed that DmdNK could efficiently phosphorylate only the dP nucleoside. To improve the catalytic efficiency, a DmdNK-Q81E mutant was created based on rational design and structural analyses. This mutant could efficiently phosphorylate both dZ and dP nucleoside. Structural modeling indicated that the increased efficiency of dZ phosphorylation by the DmdNK-Q81E mutant might be related to the three additional hydrogen bonds formed between E81 and the dZ base. Overall, this study provides a groundwork for the biological phosphorylation and synthesis of unnatural base pair in vivo.

Copyright information:

© 2017 Chen et al

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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