Cross-Linkers Applications

Cross-Linkers Applications

One of the most commonly used intercalator cross-linkers is psoralen, which is used to probe nucleic acid secondary structure at specific points in both duplex and triplex DNA. Specifically, psoralen forms cross-links with thymidine. In duplex DNA, after intercalation, psoralen can form either monoadducts with one adjacent thymidine, or diadducts with two thymidines adjacent to it, depending on the particular UV wavelength it’s exposed to (1). These adducts can occur on the same or complementary strands. For triplex DNA, psoralen C6-modified homopyrimidine oligos are used to bind to a complementary homopurine-homopyrimidine duplex, thereby forming a triplex that can be cross-linked together at the triplex-duplex junction point (2). Demonstration of the existence of triple-helix-directed gene modification and the involvement of nucleotide excision repair mechanism in DNA interstrand cross-link repair are two examples of the use of psoralen-modified oligos as research tools (3-4). Halogenated bases are a second class of UV cross-linker used for probing biomolecular structure, particularly the structure of protein-DNA complexes. 5-Br-dC and 5-Br-dG have been incorporated into dG-dC oligos capable of easily changing into the Z-conformation. This property allowed such oligos to function as probes for detecting and studying Z-DNA binding proteins (5). Substituting 5-Br-dU at several thymine positions of oligos allowed them to be used to characterize the binding of Nuclear Factor BA1 with DNA (6). See the relevant tech sheets of the different halogenated bases offered by Gene Link for additional examples.

References

(1) Pieles, U., Englisch, U. Psoralen covalently linked to oligodeoxyribonucleotides: synthesis, sequence specific recognition of DNA and photo-cross-linking to pyrimidine residues of DNA. Nucleic Acids Res. (1989), 17: 285-299.
(2) Takasugi, M., Guendouz, A., Chassignol, M., Decout, J.L., Lhomme, J., Thuong, N.T., Helene, C. Sequence-specific photo-induced cross-linking of the two strands of double-helical DNA by a psoralen covalently linked to a triple-helix-forming oligonucleotide.Proc. Natl. Acad. Sci. USA (1991), 88: 5602-5606.
(3) Barre, F-X., Ait-Si-Ali, S., Giovannangeli, C., Luis, R., et al. Unambiguous demonstration of triple-helix-directed gene modification. Proc. Natl. Acad. Sci. USA (2000), 97: 3084-3088.
(4) Wang, X., Peterson, C.A., Zheng, H., Nairn, R.S., Legerski, R.J., Lei, L. Involvement of Nucleotide Excision Repair in a Recombination-Independent and Error-Prone Pathway of DNA Interstrand Cross-Link Repair.Mol. Cell. Biol. (2001), 21: 713-720.
(5) Herbert, A.G.; Rich, A. A method to identify and characterize Z-DNA binding proteins using a linear oligodeoxynucleotide. Nucleic Acids Res. (1993), 21: 2669-2672.
(6) Kardassis, D.; Zannis, V.I.; Cladaras, C. Purification and Characterization of the Nuclear Factor BA1. J. Biol. Chem.. (1990), 265: 21733-21740.
(7) Meisenheimer, K.M., Koch, T.H. Photocross-lining of nucleic acids to associated proteins. Crit. Rev. Biochem. Mol. Biol. (1997), 32: 101-140.
(8) Steen, H., Jensen, O.N. Analysis of protein-nucleic acid interactions by photochemical cross-linking and mass spectrometry. Mass Spectrometry Reviews (2002), 21: 163-182.
(9) Chodosh, L.A. UV Crosslinking of Proteins to Nucleic Acids. Curr. Prot. Mol. Bio. (2001), 12.5.1-12.5.8.
(10) Kurz, M., Gu, K., Lohse, P.A. Psoralen photo-crosslinked mRNA-puromycin conjugates: a novel template for the rapid and facile preparation of mRNA-protein fusions. Nucleic Acids Res. (2000), 28: E83.