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Inhibition of transcription by platinated triplex-forming oligonucleotides

Mindy K Graham1, Paul S Miller

  • 1Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21212, USA.

Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry
|September 12, 2012
PubMed

Insights

Platinated triplex-forming oligonucleotides (TFOs) can block DNA transcription and gene expression. These TFOs show potential for therapeutic applications by inhibiting gene activity in mammalian cells.

Area of Science:

  • Medicinal Chemistry
  • Molecular Biology
  • Biochemistry

Background:

  • Triplex-forming oligonucleotides (TFOs) are short DNA strands that bind to specific DNA sequences.
  • Platination of TFOs introduces platinum-based cross-links to target DNA.
  • Understanding TFO-DNA interactions is crucial for developing gene-targeting therapies.

Purpose of the Study:

  • To investigate the cross-linking efficiency of platinated TFOs with target DNA.
  • To evaluate the impact of platinated TFOs on gene transcription and expression.
  • To assess the role of DNA repair mechanisms in response to platinated TFOs.

Main Methods:

  • Synthesis of platinated TFOs with platinum at various positions.
  • DNA cross-linking assays to determine binding and linkage efficiency.
  • Transfection of DNA reporter plasmids into mammalian cells (CHO).
  • Luciferase gene expression assays to measure transcriptional inhibition.
  • Comparison of gene expression in normal versus nucleotide excision repair (NER) deficient cells.

Main Results:

  • Platinated TFOs form mono- and interstrand cross-links with target DNA, with greatest efficiency at the 3' end.
  • Cross-linking is reduced by contiguous guanines in the TFO binding site or adjacent cytosines.
  • Platinated TFOs significantly inhibit transcription elongation, reducing luciferase expression by up to 95%.
  • NER-deficient cells showed higher inhibition (99%), suggesting NER repairs the lesion.
  • The 3'-G(Pt) moiety protects TFOs from serum exonuclease degradation.

Conclusions:

  • Platinated TFOs, particularly TFO-G(Pt), effectively cross-link target DNA and inhibit transcription.
  • These findings suggest platinated TFOs can be developed as gene expression inhibitors.
  • The 3'-G(Pt) modification enhances TFO stability and therapeutic potential.

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