USING LINKERS TO INVESTIGATE THE SPATIAL SEPARATION OF THE CONSERVED NUCLEOTIDES A(9) AND G(12) IN THE HAMMERHEAD RIBOZYME

Two series of hammerhead-derived ribozymes, or 'minizymes', in which h elix II has been replaced by linkers of non-nucleotidic moieties, have been synthesised by solid-phase methods. In the first series, the min izymes had linkers containing one, two, three, four or five repeated u nits of phosphopropanediol, so that the number of atoms in the chain c onnecting the 3'O of the conserved A(9) to the 5'O of the conserved G( 12) varied from 7 to 31. In the second, more-limited series, the miniz ymes contained linkers of either tetra- or hexa-ethyleneglycol. The ra tes at which these minizymes cleaved their cognate 13-nucleotide subst rate were determined at 30 degrees C, and compared with the rates of c leavage by an analogous series of minizymes containing from two to six repeated units of thymine deoxyribonucleotide in place of helix II. I n all three series, the cleavage rates increased with increasing linke r length, with a plateau being reached at the longer lengths tested. R elative cleavage rates within the phosphopropanediol and the thymidine series depended strongly on linker length, but maximal activity was a chieved in both series with 25 atoms in the chain joining A(9) and G(1 2). 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.