[3] COPPER PHENANTHROLINE PROBING OF R N A 33 Acknowledgments B.S. is a member of the CNRS and was the recipient of a Marie Curie Research Training Fellowship from the European Union and a NATO Fellowship during the course of this investigation. This work was supported by a grant from the Wellcome Trust (040375) to C.W.J.S. [3] Structure and Distance Determination in RNA with Copper Phenanthroline Probing By THOMAS HERMANN and HERMANN HEUMANN Introduction Complexes of redox-active metals serve as chemical nucleases for prob- ing secondary and tertiary structure of RNA molecules. I-4 Among these metal complexes, 1,10-phenanthroline-copper (OP-Cu) is especially useful, as it cleaves RNA with high specificity in ordered single-stranded regions. 5-8 The cleavage reaction proceeds in an oxidative attack of OP-Cu on the ribose moiety of nucleotides followed by strand scission. 1-4'9'10 The character of the reactive species has not been finally established. 4 However, there is evidence that OP-Cu may generate diffusible hydroxyl radicals that attack riboses of nucleic acid by H-abstraction. 4'11-14 The structure specificity in OP-Cu-mediated cleavage of RNA originates from specific interactions of 1 D. S. Sigman and C. B. Chen, Annu. Rev. Biochem, 59, 207 (1990). 2 C. S. Chow and J. K. Barton, J. Am. Chem. Soc. 112, 2839 (1990). 3 D. M. Perrin, A. Mazumder, and D. S. Sigman, Progr. Nucleic Acid Res. Mol. Biol. 52, 23 (1996). 4 W. K. Pogozelski and T. D. Tullius, Chem. Rev. 98, 1089 (1998). 5 G. J. Murakawa, C. B. Chen, W. D. Kuwabara, D. P. Nierlich, and D. S. Sigman, Nucleic Acids Res. 17, 5361 (1989). 6 Y.-H. Wang, S. R. Sczekan, and E. C. Theil, Nucleic Acids. Res. 18, 4463 (1990). 7 A. Mazumder, C. B. Chen, R. Gaynor, and D. S. Sigman, Biochem. Biophys. Res. Commun. 187, 1503 (1992). 8 T. Hermann and H. Heumann, RNA L 1009 (1995). 9 T..E. Goyne and D. S. Sigman, J. Am. Chem. Soc. 109, 2846 (1987). 10 O. Zelenko, J. Gallagher, Y. Xu, and D. S. Sigman, Inorg. Chem. 37, 2198 (1998). 11 L. M. Pope, K. A. Reich, D. R. Graham, and D. S. Sigman, J. Biol. Chem. 257, 12121 (1982). 12H. R. Drew and A. A. Travers, Cell 37, 491 (1984), 13T. B. Thederahn, M. D. Kuwabara, T. A. Larsen, and D. S. Sigman, J. Am. Chem. Soc. 111, 4941 (1989). 14M. Dizdaroglu, O. I. Aruoma, and B. Haliwell, Biochemistry 29, 8447 (1990). Copyright© 2000by Academic Press All rightsof reproduction in any formreserved. METHODSIN ENZYMOLOGY, VOL.318 0076-6879/00 $30.00