Stimulation of melanogenesis by DNA oligonucleotides: effect of size, sequence and 5' phosphorylation

It has been shown that the small DNA fragment thymidine dinucleotide, (pTpT) induces photoprotective responses in cultured cells and intact skin. These responses include increased melanogenesis, enhanced DNA repair, and induction of TNF-alpha, and are accomplished, at least in part, through the induction and activation of the p53 tumor suppressor and transcription factor. Here it is reported that other, but not all, larger oligonucleotides induce the pigmentation response even more efficiently than pTpT. A 9 base oligonucleotide (p9mer) stimulated pigmentation in Cloudman S91 murine melanoma cells to 6-times the level of control cells while a 5 base oligonucleotide (p5mer#1) was inactive. In addition, the p9mer increased p21 mRNA levels and inhibited cell proliferation to a greater degree than did pTpT, consistent with the presumptive mechanism of action involving p53. Smaller, truncated versions of the p9mer also stimulated pigmentation, although to a lesser extent than did the p9mer. The ability of these oligonucleotides to stimulate pigmentation was highly dependent on the presence of a 5' phosphate group on the molecule, which was shown by confocal microscopy and fluorescent activated cell sorter (FACS) analysis to greatly facilitate the uptake of these oligonucleotides into the cells. Although the melanogenic activity of the oligonucleotides was directly related to increased length and 5' phosphorylation, nucleotide sequence is also critical because a p20mer was efficiently internalized yet was a poor inducer of pigmentation.