Nucleic Acids Research Advance Access originally published online on October 30, 2007
Nucleic Acids Research 2007 35(22):7604-7613; doi:10.1093/nar/gkm666
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Nucleic Acids Research, 2007, Vol. 35, No. 22 7604-7613
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids
1Departments of Therapeutic Radiology and Genetics, Yale University School of Medicine, P.O. Box 208040, New Haven, CT 06520-8040, USA and 2Department of Cellular and Molecular Medicine, University of Copenhagen, The Panum Institute, Blegdamsvej 3, Copenhagen, DK-2200, Denmark
*To whom correspondence should be addressed. Tel: 203 737 2788; Fax: 203 737 1467; Email: peter.glazer{at}yale.edu
Received April 11, 2007. Revised August 10, 2007. Accepted August 13, 2007.
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.
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