Bioconjugate Chem. 2007, 18, 567−572
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Efficient Sequence-Directed Psoralen Targeting Using Pseudocomplementary Peptide Nucleic Acids Ki-Hyun Kim,† Xue-Jun Fan, and Peter E. Nielsen* Department of Cellular and Molecular Medicine, University of Copenhagen, The Panum Institute, Blegdamsvej 3c, 2200 Copenhagen N, Denmark. Received October 14, 2006; Revised Manuscript Received November 21, 2006
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 sequencedirected psoralen photomodification of double-stranded DNA. Thus, such psoralen-PNA conjugates could be very useful in a range of biology and drug discovery applications.
INTRODUCTION Design and discovery of chemical agents capable of sequencespecific DNA modification is a challenge with a wide potential for the development of gene knockdown and gene repair technologies in molecular biology and medicine. Many examples of such agents based on DNA minor groove binders (1), triple helix forming oligonucleotides (2), or zinc-finger DNA binding proteins (3) in combination with DNA alkylating reagents (4, 5), photoreactive psoralens (6-9), or nucleases (10, 11) have been described. Such reagents may guide the discovery and development of highly specific antigene anticancer drugs and may lead to new techniques for highly specific site-directed mutagenesis in vivo. In particular, sequence-specific repair of targeted genes in somatic cells could eventually have a very significant impact on modalities for the treatment of genetically based diseases. State of the art techniques for the latter typically result in a low percentage (90% complex). However, a significant amount of DNA cross-linking took place upon irradiation (Figure 5c, lane 7) in the presence of only PNA 2300. It was noted that the band in the gel was more diffuse, and the cross-linking is ascribed to random cross-linking in the DNA fragment of TA sites by the psoralen-PNA conjugate, which due to the positive charge (+4) has unspecific DNA affinity. This conclusion was supported by the results of a similar experiment performed in 140 mM K+ (Figure 5d) showing that the cross-linking by PNA 2300 alone is not seen under these conditions (lane 7), while the cross-linking in the presence of both PNAs (2222 and 2300) persists (lane 4), and in addition, the cross-linked band is sharper under these conditions, consistent with target-specific cross-linking. Quantification of the results presented in Figure 5d, lanes 2 and 4) shows that, at 80% binding of the PNA to the target, 47% cross-linking is achieved, giving a cross-linking efficiency of 58%. It is well-established that helix invasion by PNA oligomers is significantly inhibited by increasing ionic strength (23, 24) and accelerated by DNA negative supercoiling (25). The present system exhibits very similar behavior, as binding is negatively affected by 40 mM K+. However, binding to a supercoiled DNA target is 20-fold more efficient than to a relaxed target (Figure 6b). Therefore, in analogy to dsDNA targeting via triplex invasion complexes (25), in vivo dsDNA targeting based on double duplex invasion may also be facilitated by the topological and dynamic state of DNA in vivo.
CONCLUSIONS The present results clearly 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. It is noteworthy that (at least in the present system) the yield of photomodification is almost stoichiometric and that transcription elongation is effectively arrested by the adducts (while not by
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