Cross-Linkers Design and Protocols

Cross-Linkers Design / Protocol

Oligonucleotides containing photo-crosslinker modifications can be valuable research tools for probing the structure of DNA-protein complexes. In particular, it is those amino acids and bases in contact with each other that can be cross-linked, and thus identified as the specific units involved in binding DNA to protein. Cross-linking can be generated either by steady-state UV irradiation or pulsed lasers. For such studies, 5-halogenated uracils/cytosines are commonly used as cross-linker modifications in the DNA template. (7,8), and several excellent protocols are publicly available (9). Positioning of the halogenated bases within the DNA can be done systematically by progressive substitution along the DNA until cross-linking with an amino acid is achieved, or other structural information can be used to guide the choice of where to place the modified base(s).

When inter- or intra-strand cross-linking between duplex or triplex DNA at an thymidine position is desired, the cross-linking intercalator psoralen is typically chosen. The amount of cross-linking achieved can be tightly controlled by varying the dose of 360 nm UV light applied. Although the use of psoralen-modified oligos is primarily considered with respect to their ability to cross-link duplex or triplex DNA, cross-linking to mRNA is also possible. A 5'-psoralen-modified DNA oligo containing puromycin can be cross-linked to the 3'-end of a long mRNA template. The resulting photo-crosslinked product efficiently forms mRNA-protein fusion products (10).

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.