Using linkers to investigate the spatial separation of the conserved nucleotides A9 and G12 in the hammerhead ribozyme.

Two series of hammerhead-derived ribozymes, or 'minizymes', in which helix II has been replaced by linkers of non-nucleotidic moieties, have been synthesised by solid-phase methods. In the first series, the minizymes had linkers containing one, two, three, four or five repeated units of phosphopropanediol, so that the number of atoms in the chain connecting the 3'O of the conserved A9 to the 5'O of the conserved G12 varied from 7 to 31. In the second, more-limited series, the minizymes contained linkers of either tetra- or hexa-ethyleneglycol. The rates at which these minizymes cleaved their cognate 13-nucleotide substrate were determined at 30 degrees C, and compared with the rates of cleavage by an analogous series of minizymes containing from two to six repeated units of thymine deoxyribonucleotide in place of helix II. In all three series, the cleavage rates increased with increasing linker length, with a plateau being reached at the longer lengths tested. Relative cleavage rates within the phosphopropanediol and the thymidine series depended strongly on linker length, but maximal activity was achieved in both series with 25 atoms in the chain joining A9 and G12. The lengths of linkers required to achieve maximal activity of the minizymes are considerably greater than the linkers of 13 atoms which are sufficient to stabilise the ends of double-helices of DNA or RNA.