A Tool for the Import of Natural and Unnatural Nucleoside Triphosphates into Bacteria.
A Tool for the Import of Natural and Unnatural Nucleoside Triphosphates into Bacteria.
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DOI：
10.1021/jacs.7b11404
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发表时间：
2018-01-31
影响因子：
15
通讯作者：
Romesberg FE
中科院分区：
文献类型：
--
作者：
Feldman AW;Fischer EC;Ledbetter MP;Liao JY;Chaput JC;Romesberg FE
核苷三磷酸在生物学中起着核心作用，但研究这些作用的努力已经被证明是困难的，因为三磷酸的水平在细胞中受到严格的调控，并且因为单个三磷酸可能难以标记或修饰。此外，许多合成生物学工作都集中在开发在细胞环境中执行特定功能的非天然核苷三磷酸上。通常，这两种努力都将通过将所需三磷酸盐直接引入细胞的一般手段来促进。以前，我们证明了重组表达的核苷三磷酸转运蛋白从三角褐指藻（PtNTT 2）在大肠杆菌中的功能，进口三磷酸被添加到介质中。在这里，探索这种方法的一般性和实用性，我们报告的PtNTT 2的结构活性关系的研究。使用传统的竞争性摄取抑制试验，我们的特点的影响，核碱基，糖，和三磷酸修饰，然后开发一个LC-MS/MS测定直接测量的影响，进口的修改。最后，我们使用转运蛋白导入放射性标记的或2′-氟修饰的三磷酸，并定量它们掺入DNA和RNA。结果表明PtNTT 2介导的天然或修饰的核苷三磷酸的进口用于不同的分子或合成生物学应用的一般效用。
Nucleoside triphosphates play a central role in biology, but efforts to study these roles have proven difficult because the levels of triphosphates are tightly regulated in a cell and because individual triphosphates can be difficult to label or modify. In addition, many synthetic biology efforts are focused on the development of unnatural nucleoside triphosphates that perform specific functions in the cellular environment. In general, both of these efforts would be facilitated by a general means to directly introduce desired triphosphates into cells. Previously, we demonstrated that recombinant expression of a nucleoside triphosphate transporter from Phaeodactylum tricornutum (PtNTT2) in Escherichia coli functions to import triphosphates that are added to the media. Here, to explore the generality and utility of this approach, we report a structure activity-relationship study of PtNTT2. Using a conventional competitive uptake inhibition assay, we characterize the effects of nucleobase, sugar, and triphosphate modification, and then develop an LC-MS/MS assay to directly measure the effects of the modifications on import. Lastly, we use the transporter to import radiolabeled or 2′-fluoro-modified triphosphates and quantify their incorporation into DNA and RNA. The results demonstrate the general utility of the PtNTT2-mediated import of natural or modified nucleoside triphosphates for different molecular or synthetic biology applications.
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影响因子：
15
作者：
Feldman AW;Dien VT;Romesberg FE
通讯作者：
Romesberg FE
影响因子：
15
作者：
Batra VK;Pedersen LC;Beard WA;Wilson SH;Kashemirov BA;Upton TG;Goodman MF;McKenna CE
通讯作者：
McKenna CE
影响因子：
3.2
作者：
MCKEOWN, M;KAHN, M;HANAWALT, P
通讯作者：
HANAWALT, P
DOI：
10.1073/pnas.0808862106
发表时间：
2009-03-03
影响因子：
11.1
作者：
Ast, Michelle;Gruber, Ansgar;Haferkamp, Ilka
通讯作者：
Haferkamp, Ilka
DOI：
10.1111/j.1432-1033.1987.tb13431.x
发表时间：
1987-10-15
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
HANSEN, SEW;JENSEN, N;MUNCHPETERSEN, A
通讯作者：
MUNCHPETERSEN, A