Downregulation of collagen gene transcription by triplex forming oligonucleotides

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

Research output: Contribution to journalArticle

Abstract

The rat α1(I) collagen promoter contains a unique polypurine sequence between -141 and -170 upstream of the transcription start site(C1).Earlier, we demonstrated triplex formation with C1 using a polypurine 30 nucleotide (nt) parallel triplex forming oligonucleotide (TFO) and inhibition of transcriptional activity of the collagen promoter.(Kovacs et al(1996)J.Biol.Chem.271, 1805-1812).In the present work, we have tested triplex forming abilities of shorter (18 nt) purine and pyrimidine TFOs in parallel and antiparallel orientation to C1.Our results showed that purine antiparallel TFOs formed triplexes with the highest binding affinities, while pyrimidine oligonucleotides did not show appreciable binding. Phosphorothioate modification of purine TFOs did not significantly reduce binding affinities. Further analysis was carried out using two purine phosphorothioate antiparallel TFOs 158APS and 164APS. designed to bind to the promoter region from -141 to -158 and -147 to 164 respectively. These TFOs were chosen since they showed the highest triplex forming abilities. The results indicate that both these TFOs formed triplexes with target sequences in the promoter even under physiological conditions. Transfection experiments using preformed triplexes with a reporter plasmid containing the collagen promoter sequence showed dose dependent inhibition of transcription compared to a control phosphorothioate oligonucleotide, and that 164APS appears to be better than 158APS. These results indicate that this triplex strategy could be used in the downregulation of collagen synthesis in cultured cells and offer a potential to control fibrosis in vivo.

Original languageEnglish (US)
JournalFASEB Journal
Volume11
Issue number3
StatePublished - Dec 1 1997
Externally publishedYes

Fingerprint

Transcription
Oligonucleotides
Collagen
Down-Regulation
Genes
Phosphorothioate Oligonucleotides
Pyrimidine Nucleotides
Purine Nucleotides
Transcription Initiation Site
Genetic Promoter Regions
Transfection
Rats
Cultured Cells
Plasmids
Fibrosis
Nucleotides
Cells
purine
Experiments

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Downregulation of collagen gene transcription by triplex forming oligonucleotides. / Joseph, Jacob; Kandala, Jagan C.; Weber, Karl; Guntaka, Ramareddy.

In: FASEB Journal, Vol. 11, No. 3, 01.12.1997.

Research output: Contribution to journalArticle

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