Nucleic Acids Research, Vol 25, Issue 11 2182-2188, Copyright © 1997 by Oxford University Press
J Joseph, JC Kandala, D Veerapanane, KT Weber and RV Guntaka
The rat alpha1(I) collagen promoter contains a unique polypurine-
polypyrimidine sequence between -141 and -200 upstream of the transcription
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 triplex formation with a
polypurine 30 nt parallel triplex-forming oligonucleotide (TFO)
corresponding to C1 and inhibition of transcriptional activity of the rat
alpha1(I) collagen promoter. In the present work we have tested
triplex-forming abilities of shorter (18 nt) purine and pyrimidine TFOs in
parallel and antiparallel orientation to the C1 purine sequence. Our
results show that purine antiparallel TFOs formed triplexes with the
highest binding affinities, while pyrimidine oligodeoxyribonucleotides
(ODNs) did not show appreciable binding. Phosphorothioate modification of
purine TFOs did not significantly reduce binding affinity. We also
demonstrate that preformed triplexes are quite stable when precipitated
with ethanol and resuspended in water. Further analysis was carried out
using two purine phosphorothioate antiparallel TFOs, 158 APS and 164 APS,
designed to bind to the promoter region from -141 to -158 and -147 to -164,
respectively, which were found to form triplexes even under physiological
conditions. DNase I footprinting experiments showed the ability of these
TFOs to protect target sequences in the promoter region; both purine
sequences (C1 and C2) were protected in the case of 158 APS. Transfection
experiments using preformed triplexes with a reporter plasmid containing
the collagen promoter sequence showed significant inhibition of
transcription when compared with a control phosphorothioate ODN. The effect
of 164 APS was greater than that of 158 APS. These results indicate that
this triplex strategy could be used in the down-regulation of collagen
synthesis in cultured cells and offer the potential to control fibrosis in
vivo.
ARTICLES
Antiparallel polypurine phosphorothioate oligonucleotides form stable triplexes with the rat alpha1(I) collagen gene promoter and inhibit transcription in cultured rat fibroblasts
Department of Medicine, Division of Cardiology, School of Medicine, University of Missouri-Columbia, Columbia, MO 65212, USA.
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