ja7b11404_si_001.pdf (1.19 MB)
A Tool for the Import of Natural and Unnatural Nucleoside Triphosphates into Bacteria
journal contribution
posted on 2018-01-16, 21:19 authored by Aaron
W. Feldman, Emil C. Fischer, Michael P. Ledbetter, Jen-Yu Liao, John C. Chaput, Floyd E. RomesbergNucleoside 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.