Arnold Hampel

A unique mechanism for RNA catalysis: the role of metal cofactors in hairpin ribozyme cleavage

BACKGROUND Ribozymes are biological catalysts that promote the hydrolysis and transesterification of phosphate diesters of RNA. They typically require divalent magnesium ions for activation, although it has proven difficult to differentiate structural from catalytic roles for the magnesium ions and to identify the molecular mechanism of catalysis. Direct inner-sphere coordination is usually invoked in the catalytic step, although there is no evidence to support the generality of such a pathway for all ribozymes. RESULTS We studied the catalytic pathway for the hairpin class of ribozyme. The substitutionally inert transition metal complex cobalt hexaammine [Co(NH3)6(3+)] was shown to be as active as Mg2+(aq) in promoting hairpin ribozyme activity, demonstrating that inner-sphere pathways are not used by this class of ribozyme. These results were confirmed by studies with Rp- and Sp-phosphorothioate substrate analogs which show a similar reactivity to that of the native substrate towards the magnesium-activated ribozyme. Monovalent cations enhance the activity of Co(NH3)6(3+)-promoted reactions, but inhibit Mg(2+)-activated catalysis, demonstrating a requirement for hydrated cations at several key sites in the ribozyme. CONCLUSIONS These results provide clear support for a model of RNA catalysis that does not involve direct coordination of magnesium to the phosphate ester, nor activation of a bound water molecule. A mechanism in which catalysis is carried out by functional groups on the RNA ribozyme itself is possible; such functional groups are likely to have pKa values that are appropriate for carrying out this catalysis. The metal cofactor would then serve to define the architecture of the catalytic pocket and contribute to the stabilization of transient species, as has been described earlier. Hydrolytic pathways in nucleic acid reactions are apparently more diverse than was previously thought, and the hairpin ribozyme falls into a mechanistically distinct class from the Tetrahymena and the hammerhead ribozymes.

AN EFFICIENT METHOD FOR THE ISOLATION AND PURIFICATION OF OLIGORIBONUCLEOTIDES

Abstract Problems associated with the use of tetrabutylammonium fluoride like incomplete desilylation and removal of the tetrabutylammonium salts during large scale syntheses of oligoribonucleotides (RNA) have been eliminated by the use of triethylamine trihydrofluoride and precipitation of the RNA with 1-butanol. An efficient anion-exchange HPLC method has been developed for the purification of chemically synthesized RNA and the resulting product precipitated directly by the addition of 1-propanol. A new activator, 5-ethylthio-1H-tetrazole significantly enhances the synthesis quality and yield of oligoribonucleotides. RNA synthesized using these improvements has been shown to be biologically active by a comparative ribozyme-substrate assay.

In vitro and in vivo characterization of a second functional hairpin ribozyme against HIV-1

We have constructed a hairpin ribozyme targeted to cleave a conserved sequence in the HIV-1 pol gene. The ribozyme was modified to include a structure-stabilizing tetraloop. In vitro studies revealed a cleavage efficiency unprecedented for hairpin ribozymes (Kcat/Km = 75 min-1 microM-1). Stable retroviral vector transduction of this ribozyme gene in T-cell lines resulted in long-term ribozyme expression. As compared to control vector transduced T-cells, the pol ribozyme-transduced cells exhibited significant inhibition of different strains of HIV-1 virus production; this protection was greater when ribozyme expression was driven from an internal pol III promoter (adenovirus VA1) than when driven by a pol II promoter (the MMLV LTR). These results further demonstrate the potential of hairpin ribozymes as anti-HIV gene therapy agents and suggest possibilities for employing combinations of independently targeted hairpin ribozymes.

The Hairpin Ribozyme: Discovery, Two-Dimensional Model, and Development for Gene Therapy

This review chronicles the discovery of the hairpin ribozyme, its characterization, and determination of the two-dimensional structure, culminating with its use for human gene therapy as an AIDS therapeutic. The minimal sequence constituting the hairpin ribozyme catalytic domain was identified from a small plant viral satellite RNA. Biochemical characterization showed it to be among the most efficient of all known ribozymes. Mutagenesis determined that the two-dimensional structure had four helices, consisting of 17 Watson-Crick base pairs and one A:G pair for a total of 18 bp. The helices were interspersed with five single-stranded loops. Helices 1 and 2 were located between the ribozyme and substrate, allowing the ribozyme to recognize the substrate. The substrate had a sequence preference of BN*GUC where * is the site of cleavage and N*GUC the substrate loop between these two helices. By using sequences of this type, it was possible to design the ribozyme to base pair with the substrate and cleave heterologous RNA substrates-leading to design of the hairpin ribozyme for gene therapy. The HIV-1 sequence was searched for suitable target sites, and ribozymes were designed, optimized, catalytically characterized, and tested in vivo against HIV-1 targets. Two ribozymes had excellent in vitro catalytic parameters and inhibited in vivo expression of viral proteins by 3-4 logs in tissue culture cells. Viral replication was inhibited as well. They have been developed as human AIDS therapeutics, and will likely be the first ribozymes to be tested as human drugs in clinical trials.

Altered leucyl-transfer RNA synthetase from a mammalian cell culture mutant

Altered leucyl-tRNA synthetase from a mammalian cell culture temperature-sensitive mutant, tsHl, was compared with enzyme from normal wild type Chinese hamster ovary cells. The mutant enzyme had a Km for leucine four times larger than that of wild type and enzyme levels 3-10% that of wild type. The presence of tRNA was necessary during in vitro heating of the mutant enzyme to allow expression of thermolability while the presence of tRNA protected wild type enzyme against thermal inactivation. The tsHl enzyme was stable when heated alone or in the presence of tRNA, leucine, and ATP simultaneously. The mutants enzymes aminoacylated tRNALeu, tRNAVal, and tRNAIle with fidelity in vitro as determined by cochromatography of the amino-acyl-tRNA isoacceptors on RPC-5 reversed phase chromatography. The mutant failed to show any defect other than the direct formation of leucyl tRNALeu by leucyl-tRNA synthetase.

Characterization of postribosomal aminoacyl-tRNA synthetases in cultured Chinese hamster ovary cells.

Abstract Postribosomal aminoacyl-tRNA synthetases from cultured Chinese hamster ovary cells are found in forms of defined size. Sedimentation distribution profiles on calibrated 10–30% (w/v) exponential sucrose gradients in two different buffer systems show that the number and nature of aminoacyl-tRNA synthetase complexes vary in a defined manner. Synthetases sediment in three general size classes in buffer B (10 mM KCl, 10 mM Tris-HCl (pH 7.4 at 25°C), 1.0 mM MgCl 2 and 0.1 mM dithiothreitol) sucrose gradients. Alanyl-, asparaginyl-, seryl-, tryptophanyl- and tyrosyl-tRNA synthetases have a single activity peak that sediments at 6–10 S; cysteinyl-, histidyl-, phenylalanyl- and valyl-tRNA synthetases have a 15–21 S form plus an 8–10 S form; and aspartyl-, arginyl-, glutaminyl-, glutamyl-, isoleucyl-, leucyl-, lysyl-, methionyl-, prolyl- and threonyl-tRNA synthetases have a 30 S form plus, in some cases, one or more slower sedimenting forms. In buffer A (same as buffer B except containing 100 mM KCl and 1.5 mM MgCl 2 ), alanyl-, asparaginyl-, and tryptophanyl-tRNA synthetases have very similar distribution profiles as those in buffer B sucrose gradients. Arginyl-, cysteinyl-, glutaminyl-, glutamyl-, isoleucyl-, leucyl-, lysyl-, methionyl-, prolyl-, seryl-, threonyl-, tyrosyl-, and valyl-tRNA synthetases have similarly shaped distribution profiles in both buffer systems, but appear to shift to a slightly slower sedimentation rate from buffer B to buffer A. Aspartyl-, histidyl-, and phenylalanyl-tRNA synthetase profiles differ quite markedly in the two buffer systems providing evidence all three are in separate complexes. The synthetase distributions are compared with the distribution of total RNA and total protein in the postribosomal particles. Observations are discussed in terms of the number and nature of the aminoacyl-tRNA synthetase complexes that exist.

Large Scale Synthesis of Oligoribonucleotides on High-Loaded Polystyrene (HLP) Support

Abstract Large quantities of oligoribonucleotides (up to 200 μmole) were synthesized on the high-loaded polystyrene (HLP) support with phosphoramidite nucleosides and 5-ethylthio-1H-tetrazole as activator. The HLP support significantly reduces solvent and reagent consumption. RNA synthesized on HLP support at large scale was shown to have full biological activity by a comparative ribozyme-substrate assay.

Reformation of Leucyl-tRNA Synthetase Complexes in Revertants from CHO Mutant tsH1

A direct correlation was found to exist between increased thermolability of leucyl-tRNA synthetase and loss of the high-molecular-weight enzyme complexes in the CHO cell mutant tsH1 and its revenants. This was shown to occur apart from a differential thermostability between the complexes themselves and is supported by Michaelis constant determinations.

Permeability of Xenopus laevis embryos: Specific incorporation of precursors into eukaryotic proteins and nucleic acids

Abstract All amino acids and several nucleic acid precursors are taken up by Xenopus laevis embryos. The embryos are completely intact and not modified in any way. These precursors are directly incorporated into the macromolecules of Xenopus embryos and not prokaryotic contaminants as has been previously claimed. Radioactive leucine is incorporated into Xenopus laevis ribosomal proteins as characterized by sucrose gradient centrifugation. The uptake of the amino acids is cycloheximide sensitive and unaffected by chloramphenicol. Radioactive adenosine and orotic acid are taken up and incorporated into tRNA and rRNA at high levels as characterized by sucrose gradients and electrophoresis. These characterizations of labeled macromolecules unequivocally show that normal Xenopus laevis embryos will take up and incorporate labeled precursors to levels which are sufficient to study cellular biochemical events at such early stages of development.

Amino acid and thyroid hormone transport systems in Xenopus laevis

Abstract The nature of amino acid uptake by Xenopus laevis embryos was examined. Xenopus laevis embryos (stage 23–25) were incubated with 14 C-labeled amino acids or [ 125 I]triiodo- l -thyronine (T 3 ) under various conditions. The amino acids studied can be placed into two categories: those whose transport is Na + dependent and those whose transport is Na + independent. Structurally related amino acids or amino acid analogs were mutually inhibitory to transport, while amino acids with dissimilar side chains were mutually permissive regarding uptake. Glycine uptake was ouabain sensitive, while leucine uptake was ouabain insensitive, as was T 3 uptake. T 3 transport appeared to be a Na + -dependent process in 24- to 30-hr-old embryos, but became Na + independent by 48 hr after fertilization. T 3 transport was inhibited when Na + -independent substrates were present in the incubation media. The delineation of natural in vivo transport systems in Xenopus laevis makes it an inherently more useful model developmental system.