Evaluation of Uranyl Photocleavage as a Probe to Monitor Ion Binding and Flexibility in RNAs Dolly Wittberger 1 , Christian Berens 1 , Christian Hammann 2 Eric Westhof 3 and Rene  e Schroeder 1 * 1 Institute of Microbiology and Genetics, Vienna Biocenter University of Vienna Dr. Bohrgasse 9 A-1030 Vienna, Austria 2 CRC Nucleic Acid Structure Research Group, Biochemistry Department, The University Dundee DD1 4HN, UK 3 Institut de Biologie Mole Âculaire et Cellulaire du CNRS, F-67084 Strasbourg France In order to evaluate uranyl photocleavage as a tool to identify and characterize structural and dynamic properties in RNA, we compared uranyl cleavage sites in ®ve RNA molecules with known X-ray struc- tures, namely the hammerhead and hepatitis delta virus ribozymes, the P4-P6 domain of the Tetrahymena group I intron, as well as tRNA Phe and tRNA Asp from yeast. Uranyl photocleavage was observed at speci®c pos- itions in all molecules investigated. In order to characterize the sites, photocleavage was performed in the absence and in increasing amounts of MgCl 2 . Uranyl photocleavage correlates well with sites of low calcu- lated accessibility, suggesting that uranyl ions bind in tight RNA pockets formed by close approach of phosphate groups. RNA foldings require ion binding, usually magnesium ions. Thus, upon the adoption of the native structure, uranyl ions can no longer bind well except in ¯exible and open to the solvent regions that can undergo induced-®t without dis- rupting the native fold. Uranyl photocleavage was compared to N-ethyl- N-nitrosourea and lead-induced cleavages in the context of the three- dimensional X-ray structures. Overall, the regions protected from ENU attack are sites of uranyl cleavage, indicating sites of low accessibility which can form ion binding sites. On the contrary, lead cleavages occur at ¯exible and accessible sites and correlate with the unspeci®c cleavages prevalent in dynamic and open regions. Applied in a magnesium-depen- dent manner, and only in combination with other backbone probing agents such as N-ethyl-N-nitrosourea, lead and Fenton cleavage, uranyl probing has the potential to reveal high-af®nity metal ion environments, as well as regions involved in conformational transitions. # 2000 Academic Press Keywords: RNA structure; chemical probing; metal ions; ribozyme; tRNA; hammerhead; HDV; Tetrahymena intron *Corresponding author Introduction The uranyl(VI) ion (UO 2 2 ) forms strong com- plexes with different inorganic and organic anions, including phosphate ions. Irradiation with light at wavelengths around 420 nm results in radical oxi- dation of ligands (Azenha et al., 1989; Burrows & Kemp, 1974; Cunningham & Srijaranai, 1990). These features can be exploited for cleavage of the phosphate backbone of nucleic acids to which the ion binds via electrostatic interactions. Upon irradiation, proximal riboses are oxidized. This leads to a break of the phosphodiester backbone, generating 3 0 or 5 0 -phosphate termini and free nucleobases (Jeppesen & Nielsen, 1989; Nielsen et al., 1988, 1990b). Thus, uranyl photocleavage can be used to probe the accessibility of phosphates in the DNA backbone (Jeppesen & Nielsen, 1989; Nielsen et al., 1988, 1990a) and to determine DNA local structure (Nielsen et al., 1990b). For DNA, the underlying principle for uranyl binding was shown to be the interaction with AT-rich sequences in the minor groove (Bailly et al., 1995; Mollegaard et al., 1997; Sonnichsen & Nielsen, 1996). However, when studied in a DNA four-way junction (Mollegaard et al., 1994), uranyl not only had the ability to recognize a speci®c architecture but also contributed to folding with an ef®ciency similar to that of magnesium and, thus, was suggested to E-mail address of the corresponding author: renee@gem.univie.ac.at Abbreviations used: ENU, N-ethyl-N-nitrosourea; HDV, hepatitis delta virus. doi:10.1006/jmbi.2000.3747 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 300, 339±352 0022-2836/00/020339±14 $35.00/0 # 2000 Academic Press