Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain.

Several mixed DNA/RNA and 2'-O-methylribonucleotide/RNA analogues derived from the "hammerhead" domain of RNA catalysis have been prepared to study the minimum ribonucleotide requirement for catalytic activity. Oligodeoxyribonucleotides containing from seven to as few as four ribonucleotides are active in cleaving a substrate RNA. Predominantly deoxyribonucleotide-containing analogues have kcat values 20-300 and kcat/KM values approximately 100-2000 times lower than those of all-RNA ribozyme. In the case of predominantly 2'-O-methyl analogues, at least five ribonucleotides are needed to assure catalytic activity. In addition, both predominantly deoxyribonucleotide and 2'-O-methyl oligomers are at least 3 orders of magnitude more stable than an all-RNA ribozyme in incubations with RNase A and a yeast extract. These results suggest that the ribophosphate backbone is not a strict requirement for ribozyme-type catalysis. The identification of the four required ribonucleotides in the hammerhead catalytic domain provides valuable information for the rational design of chemical species having ribonuclease activities.     

Mixed DNA/RNA polymers are cleaved by the hammerhead ribozyme.

A series of chemically synthesized oligodeoxyribonucleotides containing one or two ribonucleotides (DNA/RNA mixed polymers) at and/or adjacent to the cleavage site of the substrate can be cleaved by the "hammerhead" ribozyme. In comparison with the all-RNA substrate, the predominantly deoxyribonucleotide substrates have (1) lower optimal temperatures of cleavage, (2) approximately 6-fold higher Km's and 7-fold lower kcat's at 30 degrees C, and (3) 15-fold higher Km's and 8-fold lower kcat's at 37 degrees C. The extent to which the RNA substrate cleavage is inhibited in the presence of an all-DNA (KI = 13 microM) and an RNA substrate analogue with a dC at the cleavage site (KI = 0.96 microM) supports the contention that the formation of the ribozyme-substrate complex with the predominantly deoxyribonucleotide substrates (D substrates) is impaired. The weaker binding of D substrates was confirmed by thermal denaturation and determination of the Tm of the complex. Analysis of the kinetic data also suggests that the conformation of the catalytic core of the ribozyme-substrate complex differs from that of the all-RNA complex, a change that results from the presence of a DNA/RNA heteroduplex in the complex.     

A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability.

Hammerhead ribozymes cleave RNA substrates containing the UX sequence, where X = U, C or A, embedded within sequences which are complementary to the hybridising 'arms' of the ribozyme. In this study we have replaced the RNA in the hybridising arms of the ribozyme with DNA, and the resulting ribozyme is many times more active than its precursor. In turnover-kinetics experiments with a 13-mer RNA substrate, the kcat/Km ratios are 10 and 150 microM-1min-1 for the RNA- and DNA-armed ribozymes, respectively. The effect is due mainly to differences in kcat. In independent experiments where the cleavage step is rate-limiting, the DNA-armed ribozyme cleaves the substrate with a rate constant more than 3 times greater than the all-RNA ribozyme. DNA substrates containing a ribocytidine at the cleavage site have been shown to be cleaved less efficiently than their all-RNA analogues; again however, the DNA-armed ribozyme is more effective than the all-RNA ribozyme against such DNA substrates. These results demonstrate that there are no 2'-hydroxyl groups in the arms of the ribozyme that are required for cleavage; and that the structure of the complex formed by the DNA-armed ribozyme with its substrate is more favourable for cleavage than that formed by the all-RNA ribozyme and its substrate.     

Photocrosslinking detects a compact, active structure of the hammerhead ribozyme

The hammerhead ribozyme has been intensively studied for approximately 15 years, but its cleavage mechanism is not yet understood. Crystal structures reveal a Y-shaped molecule in which the cleavage site is not ideally aligned for an S(N)2 reaction and no RNA functional groups are positioned appropriately to perform the roles of acid and base or other functions in the catalysis. If the ribozyme folds to a more compact structure in the transition state, it probably does so only transiently. We have used photocrosslinking as a tool to trap hammerhead ribozyme-substrate complexes in various stages of folding. Results suggest that the two substrate residues flanking the cleavage site approach and stack upon two guanosines (G8 and G12) in domain 2, moving 10-15 A closer to domain 2 than they appear in the crystal structure. Most crosslinks obtained with the nucleotide analogues positioned in the ribozyme core are catalytically inactive; however, one cobalt(III) hexaammine-dependent crosslink of an unmodified ribozyme retains catalytic activity and confirms the close stacking of cleavage site residue C17 with nucleotide G8 in domain 2. These findings suggest that residues involved in the chemistry of hammerhead catalysis are likely located in that region containing G8 and G12.     

Kinetics of intermolecular cleavage by hammerhead ribozymes.

The hammerhead catalytic RNA effects cleavage of the phosphodiester backbone of RNA through a transesterification mechanism that generates products with 2'-3'-cyclic phosphate and 5'-hydroxyl termini. A minimal kinetic mechanism for the intermolecular hammerhead cleavage reaction includes substrate binding, cleavage, and product release. Elemental rate constants for these steps were measured with six hammerhead sequences. Changes in substrate length and sequence had little effect on the rate of the cleavage step, but dramatic differences were observed in the substrate dissociation and product release steps that require helix-coil transitions. Rates of substrate binding and product dissociation correlated well with predictions based on the behavior of simple RNA duplexes, but substrate dissociation rates were significantly faster than expected. Ribozyme and substrate alterations that eliminated catalytic activity increased the stability of the hammerhead complex. These results suggest that substrate destabilization may play a role in hammerhead catalysis.     

HIV-1 TAR as anchoring site for optimized catalytic RNAs

Ribozymes have a great potential for developing specific gene silencing molecules. One of the main limitations to ensure the efficient application of ribozymes is to achieve effective binding to the target. Stem-loop domains support efficient formation of the kissing complex between natural antisense molecules and their target sequence. We have characterized catalytic antisense RNA hybrid molecules composed of a hammerhead ribozyme and a stem-loop antisense domain. A series of artificial RNA substrates containing the TAR-RNA stem-loop and a target for the hammerhead ribozyme were constructed and challenged with a catalytic antisense RNA carrying the TAR complementary stem-loop. The catalytic antisense RNA cleaves each of these substrates significantly more efficiently than the parental hammerhead ribozyme. Deletion of the TAR domain in the substrate abolishes the positive effect. These results suggest that the enhancement is due to the interaction of both complementary stem-loop motifs. A similar improvement was corroborated when targeting the LTR region of HIV-1 with either hammerhead- and hairpin-based catalytic antisense RNAs. Our results indicate that the TAR domain can be used as an anchoring site to facilitate the access of ribozymes to their specific target sequences within TAR-containing RNAs. Finally, we propose the addition of stable stem-loop motifs to the ribozyme domain as a rational way for constructing catalytic antisense RNAs.     

Nuclear magnetic resonance studies of the hammerhead ribozyme domain. Secondary structure formation and magnesium ion dependence

Proton nuclear magnetic resonance (n.m.r.) experiments were used to probe base-pair formation in several hammerhead RNA enzyme (ribozyme) domains. The hammerhead domains consist of a 34 nucleotide ribozyme bound to a complementary 13 nucleotide non-cleavable DNA substrate. Three hammerhead domains were studied that differ in the sequence and stability of one of the helices involved in recognition of the substrate by the ribozyme. The n.m.r. data show a 1:1 stoichiometry for the ribozyme-substrate complexes. The imino proton resonances in the hammerhead complexes were assigned by two-dimensional nuclear Overhauser effect experiments. These data confirm the presence of two of the three helical regions in the hammerhead domain, predicted from phylogenetic data; and are also consistent with the formation of the third helix. Since a divalent cation is required for efficient catalytic activity of the hammerhead domain, the magnesium ion dependence of the n.m.r. spectra was studied for two of the hammerhead complexes. One of the complexes showed very large spectral changes upon addition of magnesium ions. However, the complex that has the most C.G base-pairs in one of the recognition helices shows essentially no spectral (and therefore presumably structural) changes upon addition of magnesium. These data are consistent with a model where the magnesium binding site already exists in the magnesium-free complex, suggesting that the magnesium ion serves primarily a catalytic, and not a structural, role under the conditions used here.     

Enhancement of the cleavage rates of DNA-armed hammerhead ribozymes by various divalent metal ions.

In order to characterize structure-function relationships, the kinetic behavior of chimeric RNA/DNA ribozyme was compared with that of all RNA ribozyme. Determined kcat values were proven to represent the chemical-cleavage step and not the product-dissociation step. In agreement with the finding by Dahm and Uhlenbeck [Biochemistry 30, 9464-9469 (1991)], various metal ions, including Co2+ and Ca2+ with the ionic radius of 0.65 and 1.0 A, respectively, could support hammerhead cleavage for both types of ribozyme. Measurements of kinetic parameters in the presence of various divalent metal ions revealed that DNA arms always enhanced kcat values. Chemical-probing data using dimethylsulfate indicated that the catalytic-loop structures of all-RNA and chimeric ribozymes were nearly identical with the exception of enhanced termination of primer extension reactions at C3 in the case of the chimeric ribozyme. These observations and others demonstrate that DNA substitution in non-catalytic-loop regions increases chemical-cleavage activity, possibly with an accompanying very subtle change in the structure.     

Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.     

Relationship between 2'-hydroxyls and magnesium binding in the hammerhead RNA domain: a model for ribozyme catalysis.

The use of deoxyribonucleotide substitution in RNA (mixed RNA/DNA polymers) permits an evaluation of the role of 2'-hydroxyl groups in ribozyme catalysis. Specific deoxyribonucleotide substitution at G9 and A13 of the ribozyme decreases the catalytic activity (kcat) of the ribozyme by factors of 14 and 20, respectively. The reduction of the reaction rate concomitant with the absence of these 2'-OHs or the 2'-OH of the substrate U7 position can be partially compensated by increasing the Mg2+ concentration above 10 mM. The KMg of the all-RNA ribozyme is 5.3 mM, and the lack of either of the three influential 2'-OHs increases this value by a factor of approximately 3. These and other reaction constants for the ribozyme and the deoxy-substituted analogues have been determined by assuming a three-step mechanism. The data presented here provide the basis for the formulation of a molecular model of ribozyme activity.     

An RNA chaperone activity of non-specific RNA binding proteins in hammerhead ribozyme catalysis.

We have previously shown that a protein derived from the p7 nucleocapsid (NC) protein of HIV type-1 increases kcat/Km and kcat for cleavage of a cognate substrate by a hammerhead ribozyme. Here we show directly that the increase in kcat/Km arises from catalysis of the annealing of the RNA substrate to the ribozyme and the increase in kcat arises from catalysis of dissociation of the RNA products from the ribozyme. A peptide polymer derived from the consensus sequence of the C-terminal domain of the hnRNP A1 protein (A1 CTD) provides similar enhancements. Although these effects apparently arise from non-specific interactions, not all non-specific binding interactions led to these enhancements. NC and A1 CTD exert their effects by accelerating attainment of the thermodynamically most stable species throughout the ribozyme catalytic cycle. In addition, NC protein is shown to resolve a misfolded ribozyme-RNA complex that is otherwise long lived. These in vitro results suggest that non-specific RNA binding proteins such as NC and hnRNP proteins may have a biological role as RNA chaperones that prevent misfolding of RNAs and resolve RNAs that have misfolded, thereby ensuring that RNA is accessible for its biological functions.     

Selected classes of minimised hammerhead ribozyme have very high cleavage rates at low Mg2+ concentration.

In vitro selection was used to enrich for highly efficient RNA phosphodiesterases within a size-constrained (18 nt) ribonucleotide domain. The starting population (g0) was directed in trans against an RNA oligonucleotide substrate immobilised to an avidin-magnetic phase. Four rounds of selection were conducted using 20 mM Mg2+to fractionate the population on the basis of divalent metal ion-dependent phosphodiesterase activity. The resulting generation 4 (g4) RNA was then directed through a further two rounds of selection using low concentrations of Mg2+. Generation 6 (g6) was composed of sets of active, trans cleaving minimised ribozymes, containing recognised hammerhead motifs in the conserved nucleotides, but with highly variable linker domains (loop II-L.1-L.4). Cleavage rate constants in the g6 population ranged from 0.004 to 1.3 min-1at 1 mM Mg2+(pH 8.0, 37 degrees C). Selection was further used to define conserved positions between G(10.1) and C(11.1) required for high cleavage activity at low Mg2+concentration. At 10 mM MgCl2the kinetic phenotype of these molecules was comparable to a hammerhead ribozyme with 4 bp in helix II. At low Mg2+concentration, the disparity in cleavage rate constants increases in favour of the minimised ribozymes. Favourable kinetic traits appeared to be a general property for specific selected linker sequences, as the high rates of catalysis were transferable to a different substrate system.     

Characterization of deoxy- and ribo-containing oligonucleotide substrates in the hammerhead self-cleavage reaction

Deoxynucleotides were introduced into a substrate fragment of the hammerhead RNA self-cleaving domain. A substrate lacking the 2' hydroxyl adjacent to the cleavage site showed no detectable cleavage under a variety of reaction conditions. Competition experiments indicate that this fragment binds to the ribozyme with an affinity similar to the all RNA fragment, suggesting that the attacking 2' hydroxyl does not substantially contribute to the binding of substrate to ribozyme. Similar competition experiments with the all DNA substrate indicate a much lower affinity for the ribozyme perhaps due to the lack of other 2' hydroxyls. A substrate containing all deoxy residues except for a ribonucleotide at the cleavage site was also shown to be active.     

Unexpected anisotropy in substrate cleavage rates by asymmetric hammerhead ribozymes.

RNA substrates which form relatively short helices I and III with hammerhead ribozymes are generally cleaved more rapidly than substrates which create longer binding helices. We speculated that for optimum cleavage rates, one of the helices needed to be relatively weak. To identify this helix, a series of ribozymes and substrates of varying lengths were made such that in the complex, helices I and III consisted of 5 and 10 bp respectively or vice versa. In two independent systems, substrates in the complexes with the shorter helix I and longer helix III were cleaved one to two orders of magnitude more rapidly than those in the complexes with the longer helix I and shorter helix III. Similar results were obtained whether the numbers of base pairs in helices I and III were limited either by the length of the hybridizing arms of the ribozyme or the length of the substrate. The phenomenon was observed for both all-RNA and DNA armed ribozymes. Thus, a relatively short helix I is required for fast cleavage rates in pre-formed hammer-head ribozyme-substrate complexes. When helix III has 10 bp, the optimum length for helix I is approximately 5 bp.     

The synthesis and functional evaluation of RNA and DNA polymers having the sequence of Escherichia coli tRNA(fMet)

Stepwise, solid-phase chemical synthesis has provided long RNA and DNA polymers related to the sequence of Escherichia coli tRNA(fMet). The 34-ribonucleotide oligomer corresponding to the sequence of the 5'-half tRNA molecule has been synthesized and then characterized by gel purification, terminal nucleotide determinations and sequence analysis. This 34-nucleotide oligomer serves as an acceptor in the RNA-ligase-catalyzed reaction with a phosphorylated 43-ribonucleotide oligomer corresponding to the sequence of the 3'-half molecule of tRNA(fMet). The DNA molecule having the sequence of tRNA(fMet) is a 76-deoxyribonucleotide oligomer with a 3'-terminal riboadenosine residue and all U residues replaced by T. These polymers have been compared with an oligodeoxyribonucleotide lacking all 2'-hydroxyl groups except for the 3'-terminal 2'-OH, an oligoribonucleotide lacking modified nucleosides and E. coli tRNA(fMet). The all-RNA 77-nucleotide oligomer can be aminoacylated by E. coli methionyl-tRNA synthetase preparation from E. coli with methionine and threonylated in the A37 position using a yeast extract. In agreement with work by Khan and Roe using tDNA(Phe) and tDNA(Lys), the rA77-DNA(fMet) can be aminoacylated, and preliminary evidence suggests that it can be threonylated to a small extent. Kinetic data support the notion that aminoacylation of tRNA(fMet) does not depend on the presence of 2'-hydroxyl groups with the exception of that in the 3'-terminal nucleotide.     

High hydrostatic pressure approach proves RNA catalytic activity without magnesium

High hydrostatic pressure (HHP) technique was used to evaluate a mechanism of RNA hydrolysis with RNA. We showed that hammerhead ribozyme specifically cleaves RNA substrate at HHP in the absence of Mg(2+). A deoxyribozyme "10-23" was active in the same conditions. These results pointed out that the hydrolytic activity of nucleic acid depends on proper tertiary structure of a complex with a substrate. They prove that magnesium ion is not directly involved in catalysis process. On that basis we show the mechanism of RNA hydrolysis catalyzed with nucleic acids at HHP.     

Generation and application of asymmetric hammerhead ribozymes.

Most researchers who intend to suppress a particular gene are interested primarily in the application of ribozyme technology rather than its mechanistic details. This article provides some background information and describes a straightforward strategy to generate and test a special design of a ribozyme: the asymmetric hammerhead ribozyme. This version of a hammerhead ribozyme carries at its 5' end the catalytic domain and at its 3' end a relatively long antisense flank that is complementary to the target RNA. Asymmetric hammerhead ribozymes can be constructed via polymerase chain reaction amplification, and rules are provided on how to select the DNA oligonucleotides required for this reaction. In addition to details on construction, we describe how to test asymmetric hammerhead ribozymes for association with the target RNA in vitro, so that RNA constructs can be selected and optimized for fast hybridization with their target RNA. This test can allow one to minimize association problems caused by the secondary structure of the target RNA. Additionally, we describe the in vitro cleavage assay and the determination of the cleavage rate constant. Testing for efficient cleavage is also a prerequisite for reliable and successful application of the technology. A carefully selected RNA will be more promising when eventually used for target suppression in living cells.     

NMR spectroscopic characterization of a model RNA duplex reflecting the core sequence of hammerhead ribozymes

ABSTRACT

Hammerhead ribozymes are a model system for studying molecular mechanism of RNA catalysis. Physicochemical data-driven mechanistic studies are an indispensable step towards understanding the catalysis of hammerhead ribozymes. Here we characterized a model RNA duplex with catalytically important sheared-type G12-A9 base pair and A9-G10.1 metal ion-binding motif in hammerhead ribozymes. By using high magnetic field NMR, all base proton signals, including catalytic residues, were unambiguously assigned. We further characterized structural features of this RNA molecule and found that it reflects the structural features of the A9-G10.1 motif of hammerhead ribozymes. Therefore, this RNA molecule is suitable for extracting an intrinsic physicochemical properties of catalytically important residues.     

Three conserved guanosines approach the reaction site in native and minimal hammerhead ribozymes

Native hammerhead ribozymes contain RNA domains that enable high catalytic activity under physiological conditions, where minimal hammerheads show little activity. However, little is known about potential differences in native versus minimal ribozyme folding. Here, we present results of photocross-linking analysis of native and minimal hammerheads containing photoreactive nucleobases 6-thioguanosine, 2,6-diaminopurine, 4-thiouridine, and pyrrolocytidine, introduced at specific sites within the catalytic core. Under conditions where catalytic activity is observed, the two substrate nucleobases spanning the cleavage site approach and stack upon G8 and G12 of the native hammerhead, two conserved nucleobases that show similar behavior in minimal constructs, have been implicated in general acid-base catalysis, and are >15 A from the cleavage site in the crystal structures. Pyrrolocytidine at cleavage site position 17 forms an efficient crosslink to G12, and the crosslinked RNA retains catalytic activity. Multiple cross-linked species point to a structural rearrangement within the U-turn, positioning residue G5 in the vicinity of cleavage site position 1.1. Intriguing crosslinks were triggered by nucleotide analogues at positions distal to the crosslinked residues; for example, 6-thioguanosine at position 5 induced a crosslink between G12 and C17, suggesting an intimate functional communication among these three nucleobases. Together, these results support a model in which the native hammerhead folds to an active structure similar to that of the minimal ribozyme, and significantly different from the crystallographic structures.