THE SUBCELLULAR-LOCALIZATION AND LENGTH OF HAMMERHEAD RIBOZYMES DETERMINE EFFICACY IN HUMAN-CELLS

The length requirements of the antisense portion of hammerhead ribozym es for efficacy in living cells was investigated. The HIV-1 tot-direct ed asymmetric hammerhead ribozyme alpha YRz195 was used with a 195 nt 3'-antisense arm and a 3 nt 5'-antisense portion as well as a set of s uccessively 3'-shortened derivatives thereof. In the 3'-antisense arm a minimum length of 20 complementary nucleotides was required for effi cient association with a 645 nt target RNA transcript in vitro (for al l constructs k(ass) ranged between 0.3 and 1.8 x 10(4)/M/s), The cleav age rate constants (k(cleav)) were independent of the length of the an tisense flank and ranged between 0.8 and 1.2 x 10(-4)/s. However; the length of the antisense arms, as well as the mode of delivery and the subcellular location of the ribozymes, had a dramatic effect on effica cy in HIV-1-producing human cells. When proviral HIV-1 DNA and ribozym es were co-microinjected into the nucleus of human cells, a minimum le ngth of 51 nt in the antisense arm was necessary for antisense- and ri bozyme-mediated inhibition of HIV-1 replication. Ribozymes with shorte r antisense arms were almost ineffective, Conversely, short chain ribo zymes, including those with chemical modifications, were superior to l ong chain ribozymes when co-microinjected into the cytoplasm, When tra nsfected, all ribozymes showed an antisense effect as well as an addit ional ribozyme-mediated increase in inhibition. Consequences for the d esign and application of ribozymes are discussed.