ANTIPARALLEL POLYPURINE PHOSPHOROTHIOATE OLIGONUCLEOTIDES FORM STABLETRIPLEXES WITH THE RAT ALPHA-1(I) COLLAGEN GENE PROMOTER AND INHIBIT TRANSCRIPTION IN CULTURED RAT FIBROBLASTS

The rat alpha 1(I) collagen promoter contains a unique polypurine-poly pyrimidine sequence between -141 and -200 upstream of the transcriptio n start site. The polypurine sequence from -171 to -200 (C2) is on the coding strand and the adjacent polypurine sequence from -141 to -170 (C1) is on the non-coding strand. Earlier we demonstrated tripler form ation with a polypurine 30 nt parallel tripler-forming oligonucleotide (TFO) corresponding to C1 and inhibition of transcriptional activity of the rat alpha 1(I) collagen promoter. In the present work we have t ested tripler-forming abilities of shorter (18 nt) purine and pyrimidi ne TFOs in parallel and antiparallel orientation to the C1 purine sequ ence. Our results show that purine antiparallel TFOs formed triplexes with the highest binding affinities, while pyrimidine oligodeoxyribonu cleotides (ODNs) did not show appreciable binding. Phosphorothioate mo dification of purine TFOs did not significantly reduce binding affinit y. We also demonstrate that preformed triplexes are quite stable when precipitated with ethanol and resuspended in water. Further analysis w as carried out using two purine phosphorothioate antiparallel TFOs, 15 8 APS and 164 APS, designed to bind to the promoter region from -141 t o -158 and -147 to -164, respectively, which were found to form triple xes even under physiological conditions. DNase I footprinting experime nts showed the ability of these TFOs to protect target sequences in th e promoter region; both purine sequences (C1 and C2) were protected in the case of 158 APS. Transfection experiments using preformed triplex es with a reporter plasmid containing the collagen promoter sequence s howed significant inhibition of transcription when compared with a con trol phosphorothioate ODN. The effect of 164 APS was greater than that of 158 APS. These results indicate that this tripler strategy could b e used in the down-regulation of collagen synthesis in cultured cells and offer the potential to control fibrosis in vivo.