Site-specific gene modification by PNAs conjugated to psoralen.
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.
Kim
Ki-Hyun
KH
Department of Therapeutic Radiology, Yale University School of Medicine, Post Office Box 208040, New Haven, Connecticut 06520-8040, USA.
Nielsen
Peter E
PE
Glazer
Peter M
PM
D001665:Binding Sites; D002273:Carcinogens / Q000494:pharmacology; D004307:Dose-Response Relationship, Radiation; D005363:Ficusin / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D016153:Genes, Suppressor; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism*; D020135:Peptide Nucleic Acids / Q000378:metabolism*; D010777:Photochemistry; D012343:RNA, Transfer / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014466:Ultraviolet Rays
D002273:Carcinogens; D009841:Oligonucleotides; D020135:Peptide Nucleic Acids; C096275:supF tRNA; D012343:RNA, Transfer; D005363:Ficusin; D013755:Tetradecanoylphorbol Acetate