EXTENSION OF HELIX-II OF AN HIV-1-DIRECTED HAMMERHEAD RIBOZYME WITH LONG ANTISENSE FLANKS DOES NOT ALTER KINETIC-PARAMETERS IN-VITRO BUT CAUSES LOSS OF THE INHIBITORY POTENTIAL IN LIVING CELLS

When designed to cleave a target RNA in trans, the hammerhead ribozyme contains two antisense flanks which form helix I and helix III by pai ring with the complementary target RNA. The sequences forming helix II are contained on the ribozyme strand and represent a major structural component of the hammerhead structure. In the case of an inhibitory 4 29 nucleotides long trans-ribozyme (2as-Rz12) which was directed again st the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1), helix II was not pre-formed in the single-stranded molecule. Thus, major structural changes are necessary before cleavage can occu r. To study whether pre-formation of helix II in the non-paired 2as-Rz 12 RNA could influence the observed cleavage rate in vitro and its inh ibitory activity on HIV-1 replication, we extended the 4 base pair hel ix II of 2as-Rz12 to 6, 16, 21, and 22 base pairs respectively. Limite d RNase cleavage reactions performed in vitro at 37 degrees C and at p hysiological ion strength indicated that a helix II of the hammerhead domain was pre-formed when its length was at least six base pairs. Thi s modification neither affected the association rate with target RNA n or the cleavage rate in vitro. In contrast to this, extension of helix II led to a significantly decreased inhibition of HIV-1 replication i n human cells. Together with the finding of others that shortening of helix II to less than two base pairs reduces the catalytic activity in vitro, this observation indicates that the length of helix II in the naturally occurring RNAs with a hammerhead domain is already close or identical to the optimal length for catalytic activity in vitro and in vivo.