Ribozyme-mediated Cleavage Research Articles

Selective cleavage of closely-related mRNAs by synthetic ribozymes.

In Phaseolus vulgaris L. (French bean) glutamine synthetase (GS) is encoded by four closely-related genes termed gln-alpha, gln-beta, gln-gamma and gln-delta. We have constructed and characterised in vitro a number of hammerhead ribozymes designed to cleave individual RNAs encoded by these genes. The three ribozymes, termed J1, J2 and J3, were targeted to cleave RNA at the start of the gamma and beta, and the middle of the gamma, GS open reading frames respectively. All three ribozymes successfully discriminated between the four (alpha, beta, gamma and delta) highly homologous sequences, even though the targeted sites of cleavage shared up to 18 out of 22 identical bases with other gene family members. The ribozyme-mediated cleavage reactions were Mg2+ dependent and enhanced at higher temperatures, although the J1 ribozyme retained considerable activity at physiological temperatures. Both J1 and J2 demonstrated a time-dependent cleavage of their targeted GS RNAs, although these two ribozymes differed markedly in their ability to cleave multiple substrate molecules. The rate of cleavage by J1 was found to be reduced in the presence of related GS RNAs and by total leaf poly(A) RNAs. The implications of these results for ribozyme activity in vivo are discussed.

Hammerhead ribozyme-mediated cleavage of the long terminal repeat RNA of human immunodeficiency virus type 1.

Three ribozymes targeted against different sites of the long terminal repeat RNA (LTR RNA) of human immunodeficiency virus type 1 cleaved a LTR RNA transcript 1,000 x less efficiently than corresponding short synthetic oligoribonucleotide substrates. Varying the stem lengths of the ribozyme resulted in changes of the catalytic efficiency. Almost no cleavage was observed for a ribozyme forming only 10 base pairs instead of 14 with the LTR RNA. Increasing the base pairs to 16 or elongation of the stem formed within the ribozyme revealed only small changes in kcat/Km. The influence of chemical modifications within the ribozyme on the cleavage of the LTR RNA was also examined. 2'-Fluorocytidine substitutions as well as four terminal phosphorothioate internucleotidic linkages influenced the catalytic efficiency of ribozymes only negligibly. However, substitution of uridine by 2'-fluorouridine resulted in a 5-fold decrease of kcat/Km. A ribozyme containing all these modifications revealed only a 7-fold lower catalytic efficiency but a markedly increased stability in cell culture supernatant. These results demonstrate that it is possible to increase the stability of ribozymes toward nucleases without a serious loss in catalytic efficiency.

Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionein.

The c-fos gene product Fos has been implicated in many cellular processes, including signal transduction, DNA synthesis, and resistance to antineoplastic agents. A fos ribozyme (catalytic RNA) was designed to evaluate the effects of suppressing Fos protein synthesis on expression of enzymes involved in DNA synthesis, DNA repair, and drug resistance. DNA encoding the fos ribozyme (fosRb) was cloned into the pMAMneo expression plasmid, and the resultant vector was transfected into A2780DDP cells resistant to the chemotherapeutic agent cisplatin. The parental drug-sensitive A2780S cells were transfected with the pMMV vector containing the c-fos gene. Morphological alterations were accompanied by significant changes in pharmacological sensitivity in both c-fos- and fosRb-transfected cells. pMAMneo fosRb transfectants revealed decreased c-fos gene expression, concomitant with reduced thymidylate (dTMP) synthase, DNA polymerase beta, topoisomerase I, and metallothionein IIA mRNAs. In contrast, c-myc expression was elevated after fos ribozyme action. Insertion of a mutant ribozyme, mainly capable of antisense activity, into A2780DDP cells resulted in smaller reductions in c-fos gene expression and in cisplatin resistance than the active ribozyme. These studies establish a role for c-fos in drug resistance and in mediating DNA synthesis and repair processes by modulating expression of genes such as dTMP synthase, DNA polymerase beta, and topoisomerase I. These studies also suggest the utility of ribozymes in the analysis of cellular gene expression.

Ribozyme-Mediated Cleavage of an HIV-1gagRNA: The Effects of Nontargeted Sequences and Secondary Structure on Ribozyme Cleavage Activity

Catalytic antisense RNAs, or ribozymes, have great potential as inhibitors of gene expression and as antiviral therapeutic agents. The major advantage of ribozymes versus standard antisense RNAs is their catalytic capability, enabling these RNAs to cleave multiple substrates. We have been investigating the antiviral activity of ribozymes targeted to the HIV-1 genome. The successful use of these antisense agents in an intracellular milieu requires stabilization of the ribozymes by flanking, non-base-pairing sequences, or some modification of the sugar-phosphate backbone. We describe a systematic investigation of the effects of flanking, non-base-pairing sequences on the catalytic activity of an anti-HIV-1 gag ribozyme embedded in radically different transcripts. Amazingly, these complex ribozyme-containing transcripts maintain substantial catalytic activity. Finally, we describe a bacterial gene fusion system that has potential for the large scale production of catalytically active ribozymes.