In vitro-synthesized mRNA containing nucleotide modifications has distinguished therapeutic potential to transiently express physiologically important proteins. One such protein is photolyase, which accelerates the removal of UV-induced DNA damages, including cyclobutane pyrimidine dimers (CPD) using the energy of visible light (photoreactivation). Photolyase however is absent in humans. In our previous work, mRNA encoding CPD-specific photolyase has been effectively transfected into HaCaT cells. The encoded protein was visualized in the nuclei. Using CPD-specific ELISA, we showed that CPD-photolyase removed 90% of CPDs in the mRNA transfected cells at one hour after photoreactivation.
Here, we found that the mechanism of DNA repair by CPD-photolyase shows a time-dependent behavior. To determine cell viability and apoptosis in photolyase mRNA transfected HaCaT keratinocytes flow cytometry was used. First, cells were exposed to UVB irradiation (20 mJ/cm2), and then photolyase was activated by photoreactivation at 6 h, 12 h and 24 h after UVB exposure. Photoreactivation performed at 6 h or 12 h after UVB irradiation the viability of cells was significantly higher compared to those kept in dark. However, no difference could be observed between the groups when the photoreactivation was delivered at 24 h after UVB irradiation, thus resulting significant keratinocytes death by apoptosis.
These findings contribute to better understanding of UVB-induced cellular processes and give an opportunity for developing potential therapeutic agents which is effective not only before but even after UVB irradiation.

Time-dependent investigation of UVB-induced cellular mechanisms in human keratinocytes using mRNA encoding cyclobutane pyrimidine dimer-specific photolyase