A Tool for the Import of Natural and Unnatural Nucleoside Triphosphates into Bacteria

Abstract

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.

Modified nucleic acids: replication, evolution, and next-generation therapeutics

“…Remarkably, Romesberg and colleagues have been able to engineer E. coli strains with the capacity to replicate and maintain their unnatural NaM-TPT3 base pair in both plasmid [130] and genomic [131] contexts. To achieve this, they engineered a nucleoside triphosphate transporter [132], invoked Cas9 to degrade sequences having lost the UBP, and made several tweaks to the E. coli DNA synthesis and repair machinery. They further demonstrated the compatibility of the UBP with translation machinery to site-specifically encode unnatural amino acids [133,134].…”

Nucleotide Transport and Metabolism in Diatoms

“…Such semi-synthetic expression systems have great potential in biotechnology and diatom NTTs continue to be part of such systems. Meanwhile, import efficiency is also considered as a factor in artificial nucleobase design [88].…”

Expansion of the genetic code via expansion of the genetic alphabet

“…To overcome the crucial obstacle of making unnatural triphosphates available for replication within the SSO, we turned to the plastids of Phaeodactylum tricornutum that use nucleoside triphosphate transporters (NTTs) to import triphosphates from the cytosol. Expression of the transporter Pt NTT2 enables uptake of a wide variety of natural and modified deoxy- and ribonucleotide triphosphates into the SSO [••37,38], including d NaM TP, d 5SICS TP, and d TPT3 TP. Thus, the first SSO (Fig.…”