title: The subcellular localization and length of hammerhead ribozymes determine efficacy in human cells.
abstract: The length requirements of the antisense portion of hammerhead ribozymes for efficacy in living cells was investigated. The HIV-1tat-directed asymmetric hammerhead ribozyme alphaYRz195 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 kass ranged between 0.3 and 1.8x104/M/s). The cleavage rate constants (kcleav) were independent of the length of the antisense flank and ranged between 0.8 and 1.2x10-4/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.
authors.affiliation: Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Hormes
authors.forename: M
authors.initials: M
authors.lastname: Homann
authors.forename: I
authors.initials: I
authors.lastname: Oelze
authors.forename: P
authors.initials: P
authors.lastname: Marschall
authors.forename: M
authors.initials: M
authors.lastname: Tabler
authors.forename: F
authors.initials: F
authors.lastname: Eckstein
authors.forename: G
authors.initials: G
authors.lastname: Sczakiel
mesh_terms: D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics; D003593:Cytoplasm / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000502:physiology; D006801:Humans; D008845:Microinjections; D008969:Molecular Sequence Data; D016372:RNA, Antisense / Q000378:metabolism; D016337:RNA, Catalytic / Q000008:administration & dosage / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics
chemical_list: D016372:RNA, Antisense; D016337:RNA, Catalytic