Hairpin ribozyme cleavage catalyzed by aminoglycoside antibiotics and the polyamine spermine in the absence of metal ions.

The hairpin ribozyme is a small catalytic RNA that achieves an active configuration by docking of its two helical domains in an antiparallel fashion. Both docking and subsequent cleavage are dependent on the presence of divalent metal ions, such as magnesium, but there is no evidence to date for direct participation of such ions in the chemical cleavage step. We show that aminoglycoside antibiotics inhibit cleavage of the hairpin ribozyme in the presence of metal ions with the most effective being 5-epi-sisomicin and neomycin B. In contrast, in the absence of metal ions, a number of aminoglycoside antibiotics at 10 mM concentration promote hairpin cleavage with rates only 13-20-fold lower than the magnesium-dependent reaction. We show that neomycin B competes with metal ions by ion replacement with the postively charged amino groups of the antibiotic. In addition, we show that the polyamine spermine at 10 mM promotes efficient hairpin cleavage with rates similar to the magnesium-dependent reaction. Low concentrations of either spermine or the shorter polyamine spermidine synergize with 5 mM magnesium ions to boost cleavage rates considerably. In contrast, at 500 microM magnesium ions, 4 mM spermine, but not spermidine, boosts the cleavage rate. The results have significance both in understanding the role of ions in hairpin ribozyme cleavage and in potential therapeutic applications in mammalian cells.     

The harpin ribozyme: the roles of ions in RNA cleavage.

The cleavage reaction of the hairpin ribozyme is facilitated by divalent metal ions, such as Mg2+, or by non-metallic polycations, such as the polyamine spermine. We show substantial enhancement of cleavage with combinations of metallic and non-metallic ions. In addition we elucidate the locations of some ion binding sites by Fenton chemistry.     

Novel spermine-amino acid conjugates and basic tripeptides enhance cleavage of the hairpin ribozyme at low magnesium ion concentration.

Combinations of the polyamine spermine and magnesium ions synergize to dramatically enhance cleavage of the hairpin ribozyme. Certain synthetic basic tripeptides stimulate hairpin cleavage significantly at limiting magnesium ion concentration, notably the tripeptide of L-diaminobutyric acid (Dab). Of a range of novel synthetic spermine-amino acid conjugates, L-Dab-spermine (but not D-Dab nor other amino acid conjugates) was more effective than spermine itself.     

Base and sugar requirements for RNA cleavage of essential nucleoside residues in internal loop B of the hairpin ribozyme: implications for secondary structure.

The hairpin ribozyme is a small self-cleaving RNA that can be engineered for RNA cleavage in trans and has potential as a therapeutic agent. We have used a chemical synthesis approach to study the requirements of hairpin RNA cleavage for sugar and base moieties in residues of internal loop B, an essential region in one of the two ribozyme domains. Individual nucleosides were substituted by either a 2'-deoxy-nucleoside, an abasic residue, or a C3-spacer (propyl linker) and the abilities of the modified ribozymes to cleave an RNA substrate were studied in comparison with the wild-type ribozyme. From these results, together with previous studies, we propose a new model for the potential secondary structure of internal loop B of the hairpin ribozyme.     

Ionic and structural specificity effects of natural and synthetic polyamines on the aggregation and resolubilization of single-, double-, and triple-stranded DNA

DNA condensation, precipitation, and aggregation are related phenomena involving DNA-DNA interactions in the presence of multivalent cations, and studied for their potential implications in DNA packaging in the cell. Recent studies have shown that the condensation/aggregation is a prerequisite for the cellular uptake of DNA for gene therapy applications. To elucidate the ionic and structural factors involved in DNA aggregation, we studied the precipitation and resolubilization of high molecular weight and sonicated calf thymus DNA, two therapeutic oligonucleotides, and poly(dA).2Poly(dT) triplex DNA in the presence of the tetravalent polyamine spermine using a centrifugation assay, Tm measurements, and CD spectroscopy. The ability of spermine to provoke DNA precipitation was in the following order: triplex DNA > duplex DNA > single-stranded DNA. In contrast, their resolubilization at high polyamine concentrations followed a reverse order. The effective concentration of spermine to precipitate DNA increased with Na+ in the medium. Tm data indicated the DNA stabilizing effect of spermine even in the resolubilized state. CD spectroscopy revealed a series of sequential conformational alterations of duplex and triplex DNA, with the duplex form regaining the B-DNA conformation at high concentrations (approximately 200 mM) of spermine. The triplex DNA, however, remained in a Psi-DNA conformation in the resolubilized state. Chemical structural specificity effects were exerted by spermidine and spermine analogues in precipitating and resolubilizing sonicated calf thymus DNA, with N4-methyl substitution of spermidine and a heptamethylene separation of the imino groups of spermine having the maximal difference in the precipitating ability of the analogues compared to spermidine and spermine, respectively. Therapeutically important bis(ethyl) substitution reduced the precipitating ability of the analogues compared to spermine. The effect of the cationicity of polyamines was evident with the pentamines being much more efficacious than the tetramines and triamines. These results provide new insights into the mechanism of DNA precipitation by polyamines, and suggest the importance of polyamine structure in developing gene delivery vehicles for therapeutic applications.     

RNA double cleavage by a hairpin-derived twin ribozyme

The hairpin ribozyme is a small catalytic RNA that catalyses reversible sequence-specific RNA hydrolysis in trans. It consists of two domains, which interact with each other by docking in an antiparallel fashion. There is a region between the two domains acting as a flexible hinge for interdomain interactions to occur. Hairpin ribozymes with reverse-joined domains have been constructed by dissecting the domains at the hinge and rejoining them in reverse order. We have used both the conventional and reverse-joined hairpin ribozymes for the design of a hairpin-derived twin ribozyme. We show that this twin ribozyme cleaves a suitable RNA substrate at two specific sites while maintaining the target specificity of the individual monoribozymes. For characterisation of the studied ribozymes we have evaluated a quantitative assay of sequence-specific ribozyme activity using fluorescently labelled RNA substrates in conjunction with an automated DNA sequencer. This assay was found to be applicable with hairpin and hairpin-derived ribozymes. The results demonstrate the potential of hairpin ribozymes for multi-target strategies of RNA cleavage and suggest the possibility for employing hairpin-derived twin ribozymes as powerful tools for RNA manipulation in vitro and in vivo.     

Hairpin ribozyme specificity in vivo: a case of promiscuous cleavage

We have used differential display to address the question of ribozyme specificity in vivo. Stably transfected PC12 cells bearing either a hairpin ribozyme expression plasmid targeted to betaAPP mRNA or the vector alone were analyzed using nine different primer pairs. One of the few differentially expressed genes obtained from this screen corresponded to rat ribosomal protein L19. Steady-state levels of L19 mRNA were lower in ribozyme-transfected cells compared to either vector-transfected cells or native PC12 cells, and a sequence within the L19 message was cleaved by the betaAPP hairpin ribozyme in vitro. These data imply that sequence-specific unintended cleavage of non-target mRNAs may present a formidable problem to the use of hairpin ribozyme therapeutic agents.     

Comparative kinetic analysis of structural variants of the hairpin ribozyme reveals further potential to optimize its catalytic performance.

The hairpin ribozyme derived from the minus strand of the satellite RNA associated with the tobacco ringspot virus is one of the small catalytic RNAs that has been shown to catalyze trans-cleavage reactions. There is much interest in designing hairpin ribozymes with improved catalytic activity for the development of new therapeutic agents. Extensive mutagenesis studies as well as in vitro selection experiments have been performed to define the structure and optimize its catalytic activity. This communication describes a comparative kinetic analysis of four structural variants, introduced, either alone, or in combination, into the hairpin ribozyme. We have shown that extension of the helix 2 from 4 to 6 bp resulted in a significant decrease in K(M). Furthermore, the combination of this extension with the simultaneous stabilization of helix 4, led to a more than two-fold increase in the catalytic efficiency. This variant showed a 15-fold reduction in the K(M) value in respect to the wild-type ribozyme. This could be of great interest for the in vivo application of this catalytic motif. The 9-bp enlargement of helix 4 implied about a three-fold improvement in the catalytic activity. Similarly, the U39C substitution brought up the efficiency of the ribozyme slightly. However, introduction of nucleotides at the hinge region between A and B domains reduced the catalytic activity. This reduction was gradually increased with the number of nucleotides. Results obtained with variants carrying more than one modification always agreed with the ones obtained from each single variant.     

The role of essential pyrimidines in the hairpin ribozyme-catalysed reaction.

The hairpin ribozyme is an example of a small catalytic RNA that catalyses the endonucleolytic transesterification of RNA in a highly sequence-specific manner. We have utilised chemical synthesis of RNA to create mutants of the hairpin ribozyme in which a nucleoside analogue replaces one of the essential pyrimidines in the ribozyme. Individual pyrimidine nucleosides were substituted by 4-thiouridine, O4-methyluridine, O2-methyluridine or 2-pyrimidinone-1-beta-d-riboside. To facilitate the synthesis of oligoribonucleotides containing 4-thiouridine, we have devised a new synthetic route to the key intermediate 5'-O-(4, 4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-S-cyanoethyl-4-thiou ridine. The ability of the modified ribozymes to support catalysis was studied and the steady-state kinetic parameters were determined for each mutant. The range of analogues used in this study allows the important functional groups of the essential pyrimidines to be identified. The results demonstrate that each pyrimidine (U41, U42 and C25) plays an important role in hairpin ribozyme catalysis. The findings are discussed in terms of the various models that have been proposed for loop B of the hairpin ribozyme.     

A cis and trans adenine-dependent hairpin ribozyme against Tpl-2 target

Ribozymes are catalytic RNAs that possess the property of cutting an RNA target via site-specific cleavage after sequence-specific recognition. Ribozymes can moreover cleave multiple substrate molecules. An increasing number of studies show that ribozymes are particularly well adapted tools against cancer, silencing or down-regulating gene expression at the RNA level. We have constructed an adenine-dependent hairpin ribozyme that cleaves the sequence at nucleotides A(225)(downward arrow)G(226) relative to the start codon of translation of the Tpl-2 kinase mRNA; this serine/threonine kinase activates the mitogen-activated protein kinase pathway implicated in cell proliferation in breast cancer. An adenine-dependent hairpin ribozyme 1 (ADHR1) was previously isolated using the Systematic Evolution of Ligands by EXponential enrichment procedure. Switch on/switch off ribozymes are particularly useful since high amounts of stable ribozyme can be produced in the absence of adenine and the ribozyme specifically cleaves its target in the presence of adenine. The ADHR1 target sequence was replaced by a sequence derived from the Tpl-2 kinase mRNA. The resulting Tpl-2 ribozyme is active in cis cleavage: kinetic studies have been performed as a function of Mg2+ concentration, adenine concentration, as well as at different pH and with various cofactors. Finally, the Tpl-2 ribozyme was shown to cleave its target in trans successfully. These findings demonstrate that a potential therapeutic ribozyme can be produced by simple sequence modification.     

Screening for modulators of spermine tolerance identifies Sky1, the SR protein kinase of Saccharomyces cerevisiae, as a regulator of polyamine transport and ion homeostasis

Although most cells are capable of transporting polyamines, the mechanism that regulates polyamine transport in eukaryotes is still largely unknown. Using a genetic screen for clones capable of restoring spermine sensitivity to spermine-tolerant mutants of Saccharomyces cerevisiae, we have demonstrated that Sky1p, a recently identified SR protein kinase, is a key regulator of polyamine transport. Yeast cells deleted for SKY1 developed tolerance to toxic levels of spermine, while overexpression of Sky1p in wild-type cells increased their sensitivity to spermine. Expression of the wild-type Sky1p but not of a catalytically inactive mutant restored sensitivity to spermine. SKY1 disruption results in dramatically reduced uptake of spermine, spermidine, and putrescine. In addition to spermine tolerance, sky1Δ cells exhibit increased tolerance to lithium and sodium ions but somewhat increased sensitivity to osmotic shock. The observed halotolerance suggests potential regulatory interaction between the transport of polyamines and inorganic ions, as suggested in the case of the Ptk2p, a recently described regulator of polyamine transport. We demonstrate that these two kinases act in two different signaling pathways. While deletion or overexpression of SKY1 did not significantly affect Pma1p activity, the ability of overexpressed Sky1p, Ptk1p, and Ptk2p to increase sensitivity to LiCl depends on the integrity of PPZ1 but not of ENA1.     

Polyamine analogues: potent inducers of nucleosomal array oligomerization and inhibitors of yeast cell growth

Polyamines are naturally occurring intracellular polycations that are essential for viability and growth of eukaryotes. Dysregulation of polyamine metabolism is a hallmark of cancer and the carcinogenic process, and consequently development of polyamine analogues has emerged as a viable strategy for therapeutic intervention. Previously, we showed that the naturally occurring polyamines spermidine and spermine were quite effective at inducing the oligomerization of nucleosomal arrays in vitro, suggesting that polyamines may play a key role in regulating higher order chromatin structures in vivo. Here, we analyse the ability of a number of synthetic polyamine analogues to potentiate formation of higher order chromatin structures in vitro. We find that a class of long-chain polyamines called oligoamines are potent inducers of nucleosomal array oligomerization in vitro and that these same polyamine analogues rapidly block yeast cell growth.     

Ribozymes in the age of molecular therapeutics

Ribozymes are RNA molecules capable of sequence-specific cleavage of other RNA molecules. Since the discovery of the first group I intron ribozyme in 1982, new classes of ribozymes, each with their own unique reaction, target site specifications, and potential applications, have been identified. These include hammerhead, hairpin, hepatitis delta, varkud satellite, groups I and II intron, and RNase P ribozymes, as well as the ribosome and spliceosome. Meanwhile, ribozyme engineering has enabled the in vitro selection of synthetic ribozymes with unique properties. This, along with advances in ribozyme delivery methods and expression systems, has led to an explosion in the potential therapeutic applications of ribozymes, whether for anti-cancer or anti-viral therapy, or for gene repair.     

Characterization of the inducible polyamine transporter in bovine lymphocytes

The polyamine uptake system in bovine lymphocytes was activated by concanavalin A. The system was common to putrescine, spermidine and spermine. The Kt values for uptake activities of putrescine, spermidine and spermine were 3.7 microM, 0.38 microM and 0.23 microM in that order. The uptake activity was inhibited by carbonyl cyanide m-chlorophenylhydrazone, gramicidin D or valinomycin in the presence of 20 mM K+ suggesting that polyamine uptake depends on the membrane potential. The uptake activity appeared 10 h after addition of concanavalin A, and the maximum was reached at 28 h indicating that induction of the polyamine transporter precedes the initiation of DNA synthesis. Addition of polyamine antimetabolites, such as alpha-difluoromethylornithine and ethylglyoxal bis(guanylhydrazone), to the medium enhanced at least eightfold the induction of the polyamine transporter. The induction was repressed by addition of 50 microM spermidine or spermine, but not putrescine. We propose here that the induction of the membrane-potential-dependent polyamine transporter is regulated by the intracellular level of spermidine and spermine.     

The many faces of the hairpin ribozyme: structural and functional variants of a small catalytic RNA

The hairpin ribozyme is a small catalytic RNA that has been reengineered resulting in a number of variants with extended or even new functions. Thus, manipulation of the hairpin ribozyme structure has allowed for activity control by external effectors, namely oligonucleotides, flavine mononucleotide, and adenine. Hairpin ribozyme-derived twin ribozymes that mediate RNA fragment exchange reactions as well as self-processing hairpin ribozymes were designed. Furthermore, several hairpin ribozyme variants have been engineered for knock down of specific RNA substrates by adapting the substrate-binding domain to the specific target sequence. This review will focus on hairpin ribozymes possessing structural extensions/variations and thus functionally differing from the parent hairpin ribozyme.     

Mechanistic and structural analysis of human spermidine/spermine N1-acetyltransferase

The N1-acetylation of spermidine and spermine by spermidine/spermine acetyltransferase (SSAT) is a crucial step in the regulation of the cellular polyamine levels in eukaryotic cells. Altered polyamine levels are associated with a variety of cancers as well as other diseases, and key enzymes in the polyamine pathway, including SSAT, are being explored as potential therapeutic drug targets. We have expressed and purified human SSAT in Escherichia coli and characterized its kinetic and chemical mechanism. Initial velocity and inhibition studies support a random sequential mechanism for the enzyme. The bisubstrate analogue, N1-spermine-acetyl-coenzyme A, exhibited linear, competitive inhibition against both substrates with a true Ki of 6 nM. The pH-activity profile was bell-shaped, depending on the ionization state of two groups exhibiting apparent pKa values of 7.27 and 8.87. The three-dimensional crystal structure of SSAT with bound bisubstrate inhibitor was determined at 2.3 A resolution. The structure of the SSAT-spermine-acetyl-coenzyme A complex suggested that Tyr140 acts as general acid and Glu92, through one or more water molecules, acts as the general base during catalysis. On the basis of kinetic properties, pH dependence, and structural information, we propose an acid/base-assisted reaction catalyzed by SSAT, involving a ternary complex.     

Nuclear localization of human spermine oxidase isoforms - possible implications in drug response and disease etiology

The recent discovery of the direct oxidation of spermine via spermine oxidase (SMO) as a mechanism through which specific antitumor polyamine analogues exert their cytotoxic effects has fueled interest in the study of the polyamine catabolic pathway. A major byproduct of spermine oxidation is H2O2, a source of toxic reactive oxygen species. Recent targeted small interfering RNA studies have confirmed that SMO-produced reactive oxygen species are directly responsible for oxidative stress capable of inducing apoptosis and potentially mutagenic DNA damage. In the present study, we describe a second catalytically active splice variant protein of the human spermine oxidase gene, designated SMO5, which exhibits substrate specificities and affinities comparable to those of the originally identified human spermine oxidase-1, SMO/PAOh1, and, as such, is an additional source of H2O2. Importantly, overexpression of either of these SMO isoforms in NCI-H157 human non-small cell lung carcinoma cells resulted in significant localization of SMO protein in the nucleus, as determined by confocal microscopy. Furthermore, cell lines overexpressing either SMO/PAOh1 or SMO5 demonstrated increased spermine oxidation in the nucleus, with accompanying alterations in individual nuclear polyamine concentrations. This increased oxidation of spermine in the nucleus therefore increases the production of highly reactive H2O2 in close proximity to DNA, as well as decreases nuclear spermine levels, thus altering the protective roles of spermine in free radical scavenging and DNA shielding, and resulting in an overall increased potential for oxidative DNA damage in these cells. The results of these studies therefore have considerable significance both with respect to targeting polyamine oxidation as an antineoplastic strategy, and in regard to the potential role of spermine oxidase in inflammation-induced carcinogenesis.     

Polyamine metabolism in the Microsporidia

Members of the phylum Microspora are all obligate intracellular parasites. Little is known concerning metabolic pathways in these parasites, some of which pose serious problems in immunocompromised patients. We investigated polyamine metabolism in the systemic pathogen Enterocytozoon cuniculi using intact pre-emergent spores, and cell-free preparations. We found both polyamine synthetic and interconversion pathways to be operative, as evidenced by conversion of ornithine into polyamines, and production of spermidine from spermine by pre-emergent spores. Recent developments in the antitumour field have highlighted the ability of bis-ethylated polyamine analogues to reduce polyamine levels and block growth of tumour cells. In light of enhanced polyamine uptake in Enc. cuniculi, we have begun to study bis-aryl 3-7-3 and bis-ethyl oligoamine analogues as leads for chemotherapy of microsporidia.     

Investigation of adenosine base ionization in the hairpin ribozyme by nucleotide analog interference mapping.

Tertiary structure in globular RNA folds can create local environments that lead to pKa perturbation of specific nucleotide functional groups. To assess the prevalence of functionally relevant adenosine-specific pKa perturbation in RNA structure, we have altered the nucleotide analog interference mapping (NAIM) approach to include a series of a phosphorothioate-tagged adenosine analogs with shifted N1 pKa values. We have used these analogs to analyze the hairpin ribozyme, a small self-cleaving/ligating RNA catalyst that is proposed to employ a general acid-base reaction mechanism. A single adenosine (A10) within the ribozyme active site displayed an interference pattern consistent with a functionally significant base ionization. The exocyclic amino group of a second adenosine (A38) contributes substantially to hairpin catalysis, but ionization of the nucleotide does not appear to be important for activity. Within the hairpin ribozyme crystal structure, A10 and A38 line opposite edges of a solvent-excluded cavity adjacent to the 5'-OH nucleophile. The results are inconsistent with the model of ribozyme chemistry in which A38 acts as a general acid-base catalyst, and suggest that the hairpin ribozyme uses an alternative mechanism to achieve catalytic rate enhancement that utilizes functional groups within a solvent-excluded cleft in the ribozyme active site.     

Thermodynamics and kinetics of the hairpin ribozyme from atomistic folding/unfolding simulations

We report a set of atomistic folding/unfolding simulations for the hairpin ribozyme using a Monte Carlo algorithm. The hairpin ribozyme folds in solution and catalyzes self-cleavage or ligation via a specific two-domain structure. The minimal active ribozyme has been studied extensively, showing stabilization of the active structure by cations and dynamic motion of the active structure. Here, we introduce a simple model of tertiary-structure formation that leads to a phase diagram for the RNA as a function of temperature and tertiary-structure strength. We then employ this model to capture many folding/unfolding events and to examine the transition-state ensemble (TSE) of the RNA during folding to its active “docked” conformation. The TSE is compact but with few tertiary interactions formed, in agreement with single-molecule dynamics experiments. To compare with experimental kinetic parameters, we introduce a novel method to benchmark Monte Carlo kinetic parameters to docking/undocking rates collected over many single molecular trajectories. We find that topology alone, as encoded in a biased potential that discriminates between secondary and tertiary interactions, is sufficient to predict the thermodynamic behavior and kinetic folding pathway of the hairpin ribozyme. This method should be useful in predicting folding transition states for many natural or man-made RNA tertiary structures.