Site-specific gene modification by PNAs conjugated to psoralen

Biochemistry. 2006 Jan 10;45(1):314-23. doi: 10.1021/bi051379a.


DNA-binding molecules, including triplex-forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs), can be utilized to introduce site-specific mutations or to promote recombination at selected genomic sites. To further evaluate the utility of PNAs for site-specific gene modification, we tested dimeric bis-PNAs conjugated to psoralen. These PNAs are designed to form a triplex-invasion complex within the supF reporter gene in an episomal shuttle vector and to direct site-specific photoadduct formation by the conjugated psoralen. The psoralen-bis-PNA conjugate was found to direct photoadduct formation to the intended 5'-TpA base step next to the PNA-binding site, and the photoadduct formation efficiency displayed both concentration and UVA irradiation dependence. The effect of PNA-targeted photoadducts in a mammalian system was tested by SV40-based shuttle vector assay. After in vitro binding, we found that photoadducts directed by PNAs conjugated to psoralen-induced mutations at frequencies in the range of 0.46%, 6.5-fold above the background. In a protocol for intracellular gene targeting in the episomal shuttle vector, the psoralen-PNA-induced mutation frequency was 0.13%, 3.5-fold higher than the background. Most of the induced mutations were deletions and single-base-pair substitutions at or adjacent to the targeted PNA-binding and photoadduct-formation sites. When the results are taken together, they demonstrate the ability of bis-PNAs conjugated with psoralen to mediate site-specific gene modification, and they further support the development of PNAs as tools for gene-targeting applications.

MeSH terms

  • Binding Sites
  • Carcinogens / pharmacology
  • Dose-Response Relationship, Radiation
  • Ficusin / metabolism*
  • Gene Targeting / methods*
  • Genes, Reporter
  • Genes, Suppressor
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism
  • Mutagenesis, Site-Directed*
  • Nucleic Acid Conformation
  • Oligonucleotides / metabolism*
  • Peptide Nucleic Acids / metabolism*
  • Photochemistry
  • RNA, Transfer / genetics
  • Recombination, Genetic / genetics
  • Simian virus 40 / genetics
  • Simian virus 40 / metabolism
  • Tetradecanoylphorbol Acetate / pharmacology
  • Ultraviolet Rays


  • Carcinogens
  • Oligonucleotides
  • Peptide Nucleic Acids
  • supF tRNA
  • RNA, Transfer
  • Ficusin
  • Tetradecanoylphorbol Acetate