Sequence-specific triple helix formation with genomic DNA

Zhaoyang Ye, Ramareddy Guntaka, Ram I. Mahato

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

We have previously demonstrated site-specific delivery of antiparallel phosphorothioate triplex forming oligonucleotide (TFO) specific to -165 to -141 promoter region of α1(I) collagen (abbreviated as APS165) to hepatic stellate cells (HSCs) of fibrotic rats after conjugation with mannose 6-phosphate-bovine serum albumin. However, we still need to determine whether there is correlation between transcription inhibition and triplex formation with genomic DNA. In this study, APS165 was modified with psoralen and the extent of triplex formation with α1(I) collagen DNA was determined in naked genomic DNA, isolated nuclei of HSC-T6 cells and whole cells by using a simple real-time PCR based method. In this method, a purification step was added to remove unbound APS165, which eliminated the possible artifacts during real-time PCR. Psoralen photoadduct formation was shown to be essential to retain triplex structure under denaturing conditions. On naked genomic DNA, 82.2% of DNA formed triplex and 36.7% of genomic DNA in isolated nuclei at 90 min contained triplex structure. As quantified by real-time PCR, 50% of genomic DNA in living cells at 12 h postincubation contained triplex structures. Furthermore, the triplex formation was dose-dependent with 26.5% and 50% of DNA having triplex structure at concentrations of 1 μM and 5 μM, respectively. Moreover, on a plasmid pCol-CAT220 containing rat α1(I) gene promoter (-225 to +113), 75.3% of triplex formation was observed, which was correlated with a 73.6% of transcription inhibition. These findings will further strengthen the therapeutic applications of APS165.

Original languageEnglish (US)
Pages (from-to)11240-11252
Number of pages13
JournalBiochemistry
Volume46
Issue number40
DOIs
StatePublished - Oct 9 2007

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DNA
Ficusin
Real-Time Polymerase Chain Reaction
Hepatic Stellate Cells
Collagen
Transcription
Rats
Bovine Serum Albumin
Genetic Promoter Regions
Oligonucleotides
Artifacts
Plasmids
Purification
Genes
Cells
triplex DNA
Therapeutics

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Sequence-specific triple helix formation with genomic DNA. / Ye, Zhaoyang; Guntaka, Ramareddy; Mahato, Ram I.

In: Biochemistry, Vol. 46, No. 40, 09.10.2007, p. 11240-11252.

Research output: Contribution to journalArticle

Ye, Zhaoyang ; Guntaka, Ramareddy ; Mahato, Ram I. / Sequence-specific triple helix formation with genomic DNA. In: Biochemistry. 2007 ; Vol. 46, No. 40. pp. 11240-11252.
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