Antiparallel polypurine phosphorothioate oligonucleotides form stable triplexes with the rat α1(I) collagen gene promoter and inhibit transcription in cultured rat fibroblasts

Jacob Joseph, Jagan C. Kandala, Dange Veerapanane, Karl Weber, Ramareddy Guntaka

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

The rat α1(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 α1(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.

Original languageEnglish (US)
Pages (from-to)2182-2188
Number of pages7
JournalNucleic acids research
Volume25
Issue number11
DOIs
StatePublished - Aug 2 1997

Fingerprint

Phosphorothioate Oligonucleotides
Collagen
Fibroblasts
Genes
Oligodeoxyribonucleotides
Genetic Promoter Regions
Transcription Initiation Site
Deoxyribonuclease I
Oligonucleotides
Transfection
purine
Cultured Cells
Plasmids
Fibrosis
Ethanol
Down-Regulation
Water

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Antiparallel polypurine phosphorothioate oligonucleotides form stable triplexes with the rat α1(I) collagen gene promoter and inhibit transcription in cultured rat fibroblasts. / Joseph, Jacob; Kandala, Jagan C.; Veerapanane, Dange; Weber, Karl; Guntaka, Ramareddy.

In: Nucleic acids research, Vol. 25, No. 11, 02.08.1997, p. 2182-2188.

Research output: Contribution to journalArticle

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abstract = "The rat α1(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 α1(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.",
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