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+) concentrations 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.