Figure 5

Translation and stability (against skin surface ribonucleases) of cis-modified mRNA. Ribonuclease activity assay and translation of mRNA. In vitro transcription of capped eGFP mRNA was performed with non-modified NTP (norm), or with 2'-fluoro-2'-deoxy UTP (Fluoro U), 2'-amino-2'-deoxy UTP (Amino U) or 2'-azido-2'-deoxyuridine (Azido U). See A) for the position (R2) of the substituted residue. B) Transcripts were incubated with ribonucleases from Homo sapiens hand surface (final dilution 4×) or Mus musculus ear surface (20×) at 37°C for increasing time (indicated by the wedge): 15 min or 60 min, respectively. Samples without ribonucleases are indicated by a dash and were incubated for 60 min at 37°C. Negative images. C) Transcripts (10 μg) were also used to electroporate BHK21 cells. Expression of eGFP was monitored in the FL1 channel by FACS analysis. For each transcript (open histograms) the expression is shown relative to non-transfected cells (filled histograms). Numbers indicate the mean of fluorescence intensity of the cells transfected with the different transcripts (for non-transfected cells the mean is 6, 1).