Abstract
The length requirements of the antisense portion of hammerhead ribozymes for efficacy in living cells was investigated. The HIV-I fat-directed asymmetric hammerhead ribozyme αYRz195 was used with a 195 nt 3'-antisense arm and a 3 nt 5'-antisense portion as well as a set of successively 3'-shortened derivatives thereof. In the 3'-antisense arm a minimum length of 20 complementary nucleotides was required for efficient association with a 645 nt target RNA transcript in vitro (for all constructs k(ass) ranged between 0.3 and 1.8 x 10 4/M/s). The cleavage rate constants (k(cleav) were independent of the length of the antisense 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 efficacy in HIV-1-producing human cells. When proviral HIV-1 DNA and ribozymes were co-microinjected into the nucleus of human cells, a minimum length of 51 nt in the antisense arm was necessary for antisense- and ribozyme-mediated inhibition of HIV-1 replication. Ribozymes with shorter antisense arms were almost ineffective. Conversely, short chain ribozymes, including those with chemical modifications, were superior to long chain ribozymes when co-microinjected into the cytoplasm. When transfected, all ribozymes showed an antisense effect as well as an additional ribozyme-mediated increase in inhibition. Consequences for the design and application of ribozymes are discussed.
| Original language | English |
|---|---|
| Journal | Nucleic Acids Research |
| Volume | 25 |
| Issue number | 4 |
| Pages (from-to) | 769-775 |
| Number of pages | 7 |
| ISSN | 0305-1048 |
| DOIs | |
| Publication status | Published - 1997 |
Funding
We thank H.zur Hausen for continuous support and W.Nedbal for critical comments on this work. We acknowledge financial support by the Deutsche Forschungsgemeinschaft (SC14/2-1), by the Bundesministerium fr Bildung Forschung und Wissenschaft (01KV9517) and by HCM contract ERBCHRXCT930162 of the European Union.
