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Sequence-specific triple helix formation with genomic DNA.

Zhaoyang Ye1, Ramareddy V Guntaka, Ram I Mahato

  • 1Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.

Biochemistry
|September 12, 2007
PubMed
Summary
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This study confirms that psoralen-modified triplex-forming oligonucleotides (TFOs) bind effectively to alpha1(I) collagen DNA in hepatic stellate cells (HSCs). This binding correlates with significant inhibition of gene transcription, supporting therapeutic applications.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • Previous work demonstrated targeted delivery of alpha1(I) collagen triplex-forming oligonucleotides (TFOs) to hepatic stellate cells (HSCs).
  • The correlation between TFO-induced triplex formation and transcriptional inhibition remained to be established.

Purpose of the Study:

  • To investigate the correlation between triplex formation and transcription inhibition of the alpha1(I) collagen gene.
  • To quantify triplex formation of modified TFOs in various cellular contexts.

Main Methods:

  • Alpha1(I) collagen TFOs (APS165) were modified with psoralen for enhanced stability.
  • Triplex formation was quantified using a real-time PCR-based method with a purification step.
  • Experiments were conducted on naked genomic DNA, isolated nuclei, and whole HSC-T6 cells.

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Main Results:

  • Psoralen modification was essential for maintaining triplex structure under denaturing conditions.
  • Significant triplex formation was observed: 82.2% on naked DNA, 36.7% in isolated nuclei (90 min), and 50% in living cells (12 h).
  • Triplex formation was dose-dependent, and on a plasmid construct, 75.3% triplex formation correlated with 73.6% transcription inhibition.

Conclusions:

  • Psoralen-modified TFOs form stable triplex structures with the alpha1(I) collagen gene promoter in vitro and in vivo.
  • The extent of triplex formation directly correlates with the inhibition of gene transcription.
  • These findings support the therapeutic potential of TFOs for targeting fibrotic diseases.