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Related Experiment Videos

Detection of covalent triplex within human cells

A L Guieysse1, D Praseuth, M Grigoriev

  • 1Laboratoire de Biophysique, INSERM U.201, CNRS URA 481, Paris, France. a-laure@laurel.mnhn.fr

Nucleic Acids Research
|November 1, 1996
PubMed
Summary

This study demonstrates that psoralen-linked triple helix-forming oligonucleotides can form covalent bonds with DNA inside living cells. This targeted DNA modification occurs after UV irradiation, offering a novel approach for gene regulation.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Triple helix-forming oligonucleotides (TFOs) conjugated with psoralen can cross-link DNA upon UV irradiation.
  • Previous work showed inhibition of gene transcription using psoralen-TFOs targeting the interleukin-2 receptor gene promoter.

Purpose of the Study:

  • To directly demonstrate covalent triple helix formation at the target DNA site within living cells.
  • To validate the efficacy of psoralen-TFOs for targeted DNA modification in a cellular context.

Main Methods:

  • Development of a primer extension assay using Taq polymerase to quantify DNA reaction.
  • Utilizing psoralen-oligonucleotide conjugates designed to form triple helices on specific DNA sequences.
  • Employing UV irradiation to induce photoaddition of psoralen to DNA.

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Main Results:

  • Direct evidence of covalent triple helix formation at the target DNA site inside cells was obtained.
  • The psoralen-TFO mediated DNA modification was confirmed using a primer extension assay.
  • Successful photoaddition was achieved whether the triplex was pre-electroporated or added post-plasmid electroporation.

Conclusions:

  • Psoralen-conjugated triple helix-forming oligonucleotides can be effectively used for targeted covalent DNA modification within living cells.
  • This method provides a direct means to alter specific DNA sequences in situ, with potential applications in gene regulation and therapy.