Journal Article

Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids

Ki-Hyun Kim, Peter E. Nielsen and Peter M. Glazer

in Nucleic Acids Research

Volume 35, issue 22, pages 7604-7613
Published in print December 2007 | ISSN: 0305-1048
Published online October 2007 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/gkm666
Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids

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  • Chemistry
  • Biochemistry
  • Bioinformatics and Computational Biology
  • Genetics and Genomics
  • Molecular and Cell Biology

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Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences by Watson–Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules.

Journal Article.  5855 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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