Efficient sequence-directed psoralen targeting using pseudocomplementary Peptide nucleic acids.
A pair of decameric pseudocomplementary PNAs which bind to their mixed purine-pyrimidine sequence target in duplex DNA by double duplex invasion has been synthesized with a derivative of 8-methoxypsoralen conjugated to one of the PNAs. It is shown that this pair of psoralen-conjugated pseudocomplementary PNA oligomers, which target a site in the pBluescriptKS+ vector, upon irradiation with long-wavelength UV light (UVA) with high efficiency and specificity form photoadducts to an adjacent 5'-TA site, and more than 50% of these adducts are DNA interstrand cross-links. Transcription elongation by T7 or T3 RNA polymerase is specifically arrested at the psoralen cross-linking site, yielding more than 90% arrested product. These results emphasize the potential of pseudocomplementary PNA oligomers for highly specific gene targeting, in particular, with respect to sequence-directed psoralen photomodification of double-stranded DNA. Thus, such psoralen-PNA conjugates could be very useful in a range of biology and drug discovery applications.
Kim
Ki-Hyun
KH
Department of Cellular and Molecular Medicine, University of Copenhagen, The Panum Institute, Blegdamsvej 3c, 2200 Copenhagen N, Denmark.
Fan
Xue-Jun
XJ
Nielsen
Peter E
PE
D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D012321:DNA-Directed RNA Polymerases; D024202:Electrophoretic Mobility Shift Assay; D005363:Ficusin / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D018390:Gene Targeting; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D009692:Nucleic Acid Heteroduplexes; D020135:Peptide Nucleic Acids; D004247:DNA; C081591:bacteriophage T3 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D005363:Ficusin