Dimer-specific photolyase; eGFP – enhanced Green Fluorescent Protein; ELISA – Enzyme Linked Immunosorbent Assay; IL-6 – Interleukin-6; SDHA – Succinate Dehydrogenase Complex, Subunit

24 UVB irradiation induces harmful photochemical reactions, including formation of 25 cyclobutane pyrimidine dimers (CPDs) in DNA. Accumulation of unrepaired CPD lesions 26 causes inflammation, premature ageing and skin cancer. Photolyases are DNA repair enzymes 27 that can rapidly restore DNA integrity in a light-dependent process called photoreactivation, 28 but these enzymes are absent in humans. Here, we present a novel mRNA-based gene therapy 29 method that directs synthesis of a marsupial, Potorous tridactylus, CPD-photolyase in 30 cultured human keratinocytes. Pseudouridine was incorporated during in vitro transcription to 31 make the mRNA non-immunogenic and highly translatable. Keratinocytes transfected with 32 lipofectamine-complexed mRNA expressed photolyase in the nuclei for at least 2 days. 33 Exposing photolyase mRNA-transfected cells to UVB irradiation resulted in significantly less 34 CPD in those cells that were also treated with photoreactivating light, which is required for 35 photolyase activity. The functional photolyase also diminished other UVB-mediated effects, 36 including induction of IL-6 and inhibition of cell proliferation. These results demonstrate that 37 pseudouridine-containing photolyase mRNA is a powerful tool to repair UVB-induced DNA 38 lesions. The pseudouridine-modified mRNA approach has a strong potential to discern 39 cellular effects of CPD in UV-related cell biological studies. The mRNA-based transient 40 expression of proteins offers a number of opportunities for future application in medicine. 41