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.     

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.     

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.     

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.     

LNA nucleotides improve cleavage efficiency of singular and binary hammerhead ribozymes

Variants of trans-acting hammerhead ribozymes were modified with Locked Nucleic Acid (LNA) nucleotides to reduce their size, to improve access to their RNA target and to explore combinational properties of binary constructs. Using low Mg(2+) concentrations and low substrate and ribozyme concentrations, it was found that insertion of LNA monomers into the substrate binding arms allowed these to be shortened and results in a very active enzyme under both single and multiple turnover conditions. Incorporation of a mix of LNA and DNA residues further increased the multiple turnover cleavage activity. At high Mg(2+) concentrations or high substrate and ribozyme concentrations, the enhancing effect of LNA incorporation was even more prominent. Using LNA in the stem of Helix II diminished cleavage activity, but allowed deletion of the tetra-loop and thus separating the ribozyme into two molecules with each half binding to the substrate. Efficient, binary hammerhead ribozymes were pursued in a combinatorial approach using a 6-times 5 library, which was analysed concerning the best combinations, buffer conditions and fragment ratios.     

2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes.

The design, synthesis and study of internally fluorescent hammerhead (HH) ribozymes, where changes in fluorescence parameters directly reflect the progress of the ribozyme's cleavage chemistry, are described. The approach relies on a HH substrate modified at position 1.1, proximal to the cleavage site, with 2-aminopurine (2AP), an intensely fluorescent adenosine isoster. The incorporation of 2AP, an unnatural nucleoside, does not interfere with the ribozyme folding and catalysis. Since 2AP is highly sensitive to environmental changes, its fluorescence is dramatically altered upon ribozyme-mediated cleavage of the substrate. This generates a measurable signal that directly reflects the progress of the ribozyme's reaction in real time. Identical pseudo first order rate constants are obtained for HH constructs using both continuous fluorescence monitoring and radioactive labeling. This rapid and real-time monitoring facilitates the study of ribozyme activity under different conditions (e.g., ionic strength, pH, etc.), and provides a useful assay to rapidly screen potential inhibitors. Three hitherto unknown HH inhibitors are presented and compared to neomycin B and chlortetracycline, two previously studied HH inhibitors. All three new small molecules, neo-acridine, guanidino-neomycin B, and [Delta-(Eilatin)Ru(bpy)(2)](2+), prove to be better inhibitors than neomycin B or chlortetracycline. Investigating HH inhibition under different ionic strengths reveals that the binding of neo-acridine, [Delta-(Eilatin)Ru(bpy)(2)](2+), and chlortetracycline to the HH involves hydrophobic interactions as their RNA affinities are largely unaffected by increasing salt concentrations. In contrast, neomycin B loses more than 50-fold of its inhibitory ability as the NaCl concentration is increased from 50 to 500mM.     

Comparative analysis of cleavage rates after systematic permutation of the NUX consensus target motif for hammerhead ribozymes.

A trans-cleaving asymmetric hammerhead ribozyme directed against an AUC decreases target motif within an RNA specific for human immunodeficiency virus type 1 (HIV-1) was generated. The AUC decreases motif of the target RNA was permutated in order to generate all 12 variants of an NUX decreases consensus target motif, wherein N = A, C, G or U and X = A, C or U. Four asymmetric hammerhead ribozymes differing in the nucleotide that is complementary to N were generated, of which each was specific for three of the 12 target motifs. The residual sequence context within helices I and III remained unchanged. All 12 combinations resulted in cleavage of the target RNA. Using single-turnover conditions, the detectable cleavage rate constants at 37 degrees C were determined, which varied considerably depending on the NUX decreases motif. The NUC decreases motifs were cleaved more efficiently, with AUC decreases being cleaved best. Comparison with previous studies indicates that the sequence context of the NUX decreases motif plays a major role for the detectable cleavage activity.     

Morphing the minimal and full-length hammerhead ribozymes: implications for the cleavage mechanism

The hammerhead ribozyme is a small, intensively studied catalytic RNA, and has been used as a prototype for understanding how RNA catalysis works. In 2003, the importance of a set of tertiary contacts that appear in natural sequences of the hammerhead RNA was finally understood. The presence of these contact regions in stems I and II in 'full-length hammerhead ribozymes' is accompanied by an up to 1000-fold catalytic rate enhancement, indicating a profound structural effect upon the active site. Although the new structure resolved most of what appeared to be irreconcilable differences with mechanistic studies in solution, it did so in a way that is simultaneously reconcilable with earlier crystallographic mechanistic studies, within the limits imposed by the truncated sequence of the minimal hammerhead. Here we present an analysis of the correspondence between the full-length and minimal hammerhead crystal structures, using adiabatic morphing calculations that for the first time test the hypothesis that the minimal hammerhead structure occasionally visits the active conformation, both in solution and in the crystalline state in a sterically allowed manner, and argue that this is the simplest hypothesis that consistently explains all of the experimental observations.     

Action of metal ions directly involved in the cleavage reaction of hammerhead ribozymes.

Recently, hammerhead ribozyme-mediated cleavage was analyzed as a function of the concentration of La3+ ions in the presence of a fixed concentration of Mg2+ ions so that the role could be monitored of metal ions that are directly involved in the cleavage reaction. The resultant bell-shaped curve for activation of cleavage was used to support the proposed double-metal-ion mechanism of catalysis. However, other studies demonstrated that binding of a metal ion to the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A9 and N7 of nucleotide G10.1 is critical for efficient catalysis. In order to clarify the effect of this metal ion, we chemically synthesized hammerhead ribozyme (7-deaza-R34) that included a minimal modification, namely, an N7-deazaguanine residue in place of G10.1.     

Restriction in the cleavage activity of hammerhead ribozymes ensures ongoing evolution in prebiotic RNA world

Self-cleaving infectious RNAs found in many plant viruses and viroids can also cleave intrans and form hammerhead type secondary structure. It has been observed that the cleavage site must contain the triplet GUC. Also, in other cases, the sequence XUY holds good where X = A, C, G, U and Y = A, C, U but not G. The high electronegative nature of guanosine holds the key to its resistance to cleavage which does not allow hybrid formation between the ribozyme and substrate strands. Guanosine resistance to cleavage might have been the starting thrust for the evolution of a translational initiation codon from XUG. A hypothesis is proposed in this regard and its evolutionary consequences are discussed briefly. Presented at the National Symposium on Evolution of Life.     

Identification of the hammerhead ribozyme metal ion binding site responsible for rescue of the deleterious effect of a cleavage site phosphorothioate

The hammerhead ribozyme crystal structure identified a specific metal ion binding site referred to as the P9/G10.1 site. Although this metal ion binding site is approximately 20 A away from the cleavage site, its disruption is highly deleterious for catalysis. Additional published results have suggested that the pro-R(P) oxygen at the cleavage site is coordinated by a metal ion in the reaction's transition state. Herein, we report a study on Cd(2+) rescue of the deleterious phosphorothioate substitution at the cleavage site. Under all conditions, the Cd(2+) concentration dependence can be accounted for by binding of a single rescuing metal ion. The affinity of the rescuing Cd(2+) is sensitive to perturbations at the P9/G10.1 site but not at the cleavage site or other sites in the conserved core. These observations led to a model in which a metal ion bound at the P9/G10.1 site in the ground state acquires an additional interaction with the cleavage site prior to and in the transition state. A titration experiment ruled out the possibility that a second tight-binding metal ion (< 10 microM) is involved in the rescue, further supporting the single metal ion model. Additionally, weakening Cd(2+) binding at the P9/G10.1 site did not result in the biphasic binding curve predicted from other models involving two metal ions. The large stereospecific thio-effects at the P9/G10.1 and the cleavage site suggest that there are interactions with these oxygen atoms in the normal reaction that are compromised by replacement of oxygen with sulfur. The simplest interpretation of the substantial rescue by Cd(2+) is that these atoms interact with a common metal ion in the normal reaction. Furthermore, base deletions and functional group modifications have similar energetic effects on the transition state in the Cd(2+)-rescued phosphorothioate reaction and the wild-type reaction, further supporting the model that a metal ion bridges the P9/G10.1 and the cleavage site in the normal reaction (i.e., with phosphate linkages rather than phosphorothioate linkages). These results suggest that the hammerhead undergoes a substantial conformational rearrangement to attain its catalytic conformation. Such rearrangements appear to be general features of small functional RNAs, presumably reflecting their structural limitations.     

Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes.

The chemical synthesis is described of oligoribonucleotides containing a single phosphorothioate linkage of defined Rp and Sp configuration. The oligoribonucleotides were used as substrates in the study of the mechanism of cleavage of an RNA hammerhead domain having the phosphorothioate group at the cleavage site. Whereas the Rp isomer was cleaved only very slowly in the presence of magnesium ion, the rate of cleavage of the Sp isomer was only slightly reduced from that of the unmodified phosphodiester. This finding gives further evidence for the hypothesis that the magnesium ion is bound to the pro-R oxygen in the transition state of the hammerhead cleavage reaction. Also, inversion of configuration at phosphorus is confirmed for a two-stranded hammerhead.     

Sequence-specific cleavage of hepatitis X RNA in cis and trans by novel monotarget and multitarget hammerhead motif-containing ribozymes

We constructed two monoribozymes and a diribozyme against the conserved region of the X RNA of hepatitis B virus (HBV). All the ribozymes (Rzs) possessed sequence-specific cleavage activities under standard and simulated physiologic conditions. Specific cleavage was also obtained when the same Rzs were placed in cis configuration with respect to X gene in multiple combinations. Rz-expressing cells were able to specifically interfere with the functional expression of X RNA and protein production in a liver-specific cell line HepG2. Potential applications of these novel Rzs are discussed.     

Rational evolution of Cd2+-specific DNAzymes with phosphorothioate modified cleavage junction and Cd2+ sensing

In vitro selection of RNA-cleaving DNAzymes is a powerful method for isolating metal-specific DNA. A few successful examples are known, but it is still difficult to target some thiophilic metals such as Cd²⁺ due to limited functional groups in DNA. While using modified bases expands the chemical functionality of DNA, a single phosphorothioate modification might boost its affinity for thiophilic metals without complicating the selection process or using bases that are not commercially available. In this work, the first such in vitro selection for Cd²⁺ is reported. After using a blocking DNA and negative selections to rationally direct the library outcome, a highly specific DNAzyme with only 12 nucleotides in the catalytic loop is isolated. This DNAzyme has a cleavage rate of 0.12 min⁻¹ with 10 μM Cd²⁺ at pH 6.0. The R_p form of the substrate is cleaved ∼100-fold faster than the S_p form. The DNAzyme is most active with Cd²⁺ and its selectivity against Zn²⁺ is over 100 000-fold. Its application in detecting Cd²⁺ is also demonstrated. The idea of introducing single modifications in the fixed region expands the scope of DNA/metal interactions with minimal perturbation of DNA structure and property.     

Optimization of hammerhead ribozymes for the cleavage of S100A4 (CAPL) mRNA

Previously, suppression of the S100A4 mRNA by an endogenously expressed ribozyme in osteosarcoma cells was shown to inhibit their metastasis in rats. As a prelude to performing similar studies with exogenous, synthetic ribozymes, we compared a series of hammerhead ribozymes targeted against different sites in the mRNA. The ribozymes differed only in the 7-base flanking sequences complementary to the substrate and were protected against nucleases by chemical modification. Cleavage efficiency varied widely and was not obviously related to the predicted secondary structure of the target RNA. The most active ribozyme of the series was chosen for further optimization. Lengthening its flanking sequences was counterproductive and reduced cleavage even when using excess ribozyme. Using excess substrate (multiple-turnover kinetics), cleavage was fastest with the (6+8) ribozyme having 6 nucleotides (nt) in stem III and 8 nt in stem I. Although these stems strongly influence ribozyme performance, their optimization is still empirical. Faster cleavage was obtained by adding facilitator oligonucleotides to ribozymes with shorter stems of (6+6) and (5+5) nt. Stimulation was particularly strong in the case of the (5+5) ribozyme, which was poorly active by itself. The enhancement caused by different facilitator oligonucleotides paralleled their expected ability to hybridize to RNA as a function of length and chemical modification.