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The  in vitro  proliferation of prokaryotic and eukaryotic cells is remarkably hampered in the presence of heavy water (D 2 O). Impairment of gene expression at the transcription or translation level can be the base for this effect. However, insights into the underlying mechanisms are lacking. Here, we employ a cell-free expression system for the quantitative analysis of the effect of increasing percentages of D 2 O on the kinetics of  in-vitro  GFP expression. Experiments are designed to discriminate the rates of transcription, translation, and protein folding using pDNA and mRNA vectors, respectively. We find that D 2 O significantly stimulates GFP expression at the transcription level but acts as a suppressor at translation and maturation (folding) in a linear dose-dependent manner. At a D 2 O concentration of 60%, the GFP expression rate was reduced to 40% of an undisturbed sample. We observed a similar inhibition of GFP expression by D 2 O in a recombinant  Escherichia coli  strain, although the inhibitory effect is less pronounced. These results demonstrate the suitability of cell-free systems for quantifying the impact of heavy water on gene expression and establish a platform to further assess the potential therapeutic use of heavy water as antiproliferative agent.

Heavy Water Reduces GFP Expression in Prokaryotic Cell-Free Assays at the Translation Level While Stimulating Its Transcription