Figure 1.

Translational inhibition by unmodified in vitro transcribed mRNA. (A) In vitro transcribed mRNAs encoding Renilla luciferase (Ren) and firefly luciferase (Luc) were synthesized with and without Ψ modifications then mixed (1 : 1 mass ratio) as indicated. The mixed mRNA was complexed with lipofectin and added to HEK293T cells seeded in 96-well plates (0.25 µg RNA/well). Cells were lysed 4 h after transfection and dual luciferase measurements were performed in aliquots (1/20th) of the lysates. Values presented are normalized to cells transfected with Ren and Luc mRNAs when both contained Ψ modifications. Error bars indicate the standard error of n = 3 samples. (B) Unmodified or pseudouridine-containing RNA was delivered to HEK293T cells by lipofection. Cells were subsequently incubated with ³⁵S-methionine/cysteine supplemented medium, lysed, and proteins were TCA precipitated. Data are presented as percentage of counts obtained from mock transfected cells. Data shown are mean values from three independent experiments ± SEM.