LNA nucleotides improve cleavage efficiency of singular and binary hammerhead ribozymes Janne K. Christiansen, a Sune Lobedanz, a,  Khalil Arar, b Jesper Wengel c and Birte Vester a, * a Department of Biochemistry and Molecular Biology, The Nucleic Acid Center, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark b Sigma-Proligo, Genopole Campus 1, 5, rue Henri Desbrueres, 91030 Evry Cedex, France c Departments of Physics and Chemistry, The Nucleic Acid Center, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark Received 15 March 2007; accepted 13 June 2007 Available online 28 June 2007 Abstract—Variants of trans-acting hammerhead ribozymes were modified with Locked Nucleic Acid (LNA) nucleotides to reduce their size, to improve access to their RNA target and to explore combinational properties of binary constructs. Using low Mg 2+ concentrations and low substrate and ribozyme concentrations, it was found that insertion of LNA monomers into the substrate binding arms allowed these to be shortened and results in a very active enzyme under both single and multiple turnover conditions. Incorporation of a mix of LNA and DNA residues further increased the multiple turnover cleavage activity. At high Mg 2+ concen- trations or high substrate and ribozyme concentrations, the enhancing effect of LNA incorporation was even more prominent. Using LNA in the stem of Helix II diminished cleavage activity, but allowed deletion of the tetra-loop and thus separating the ribozyme into two molecules with each half binding to the substrate. Efficient, binary hammerhead ribozymes were pursued in a combinatorial approach using a 6-times 5 library, which was analysed concerning the best combinations, buffer conditions and fragment ratios. Ó 2007 Elsevier Ltd. All rights reserved. 1. Introduction Hammerhead ribozymes are relatively small RNA struc- tures of around 30 nucleotides that can cleave RNA tar- gets in cis or trans in a sequence-specific way. The natural hammerhead ribozymes are cis acting RNA structures coded on viroid RNA. 1 The cleavage is highly dependent on the presence of divalent metal ions with magnesium as the natural choice. The potential for clin- ical use of ribozymes as RNA cleaving pharmaceuticals is being exploited but their actual application has so far been limited. There are several explanations for this. The catalytic power of the known ribozymes is relatively poor compared to efficient proteins. In addition, the sta- bility of ribozymes is limited due to their susceptibility hydrolysis mediated by RNases. Also, the binding prop- erties of ribozymes to their RNA target by Watson– Crick base pairing set some inherent limitations influenced by the accessibility to the target sequence. Thus some improvements of ribozymes are needed to make them more applicable. Hammerhead ribozymes have been the subject of numerous studies (recently reviewed in Refs. 2–6). The idea of introducing nucleotide modifications in hammer- head ribozymes has been explored both to elucidate the RNA cleaving mechanism and to increase the nucleolyt- ic stability of the ribozymes. Specific nucleotides have been replaced by, for example, fluorobenzene-C-nucleo- sides, 2 0 -aminothymidine, 2 0 -deoxyuridine, 2 0 -O-methyl- RNA nucleosides or 3-phosphorothioate derivatives (Refs. 7–9 and references therein). A major break- through in understanding the structural arrangement underlying the ribozyme-mediated phosphodiester cleav- age came with the recent X-ray determination of a full-length Schistosoma mansoni hammerhead ribozyme. 10 The structure solved a long-standing puzzle to fit all biochemical data with previous Y-shaped hammerhead structures. Locked Nucleic Acids (LNA) are modified nucleosides with a conformationally locked ribose moiety. 11 They are capable of targeting complementary RNA and 0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2007.06.045 Keywords: Hammerhead ribozymes; LNA; Modified nucleotides; RNA cleavage. * Corresponding author. Tel.: +45 6550 2377; fax: +45 6550 2467; e-mail: b.vester@bmb.sdu.dk   Present address: Novozymes A/S, Krogshojvej 36, 2880 Bagsvaerd, Denmark. Bioorganic & Medicinal Chemistry 15 (2007) 6135–6143