Peptide nucleic acid (PNA) is capable of enhancing hammerhead ribozyme activity with long but not with short RNA substrates.

Long RNA substrates are inefficiently cleaved by hammerhead ribozymes in trans. Oligonucleotide facilitators capable of affecting the ribozyme activity by interacting with the substrates at the termini of the ribozyme provide a possibility to improve ribozyme mediated cleavage of long RNA substrates. We have examined the effect of PNA as facilitator in vitro in order to test if even artificial compounds have facilitating potential. Effects of 12mer PNA- (peptide nucleic acid), RNA- and DNA-facilitators of identical sequence were measured with three substrates containing either 942, 452 or 39 nucleotides. The PNA facilitator enhances the ribozyme activity with both, the 942mer and the 452mer substrate to a slightly smaller extent than RNA and DNA facilitators. This effect was observed up to PNA facilitator:substrate ratios of 200:1. The enhancement becomes smaller as the PNA facilitator:substrate ratio exceeds 200:1. With the 39mer substrate, the PNA facilitator decreases the ribozyme activity by more than 100-fold, even at PNA facilitator:substrate ratios of 1:1. Although with long substrates the effect of the PNA facilitator is slightly smaller than the effect of identical RNA or DNA facilitators, PNA may be a more practical choice for potential applications in vivo because PNA is much more resistant to degradation by cellular enzymes.     

Oligonucleotide facilitators enhance the catalytic activity of RNA-cleaving DNA enzymes.

From in vitro selection studies, DNA structures have been found that cleave target RNA sequence specifically and show a certain similarity to the well-investigated hammerhead ribozymes. Such DNA enzymes are more resistant to nuclease-mediated degradation than RNA enzymes. On the other hand, their cleavage activity is lower than the activity of hammerhead ribozymes. In the present study, we improved the activity of DNA enzymes by adding oligonucleotide facilitators complementary to the 5' and the 3' ends of the substrate to the cleavage reaction. DNA enzyme activity in vitro was monitored under multiple turnover conditions using short RNA model substrates. We have shown that oligonucleotide facilitators strongly enhance the multiple turnover activity of the DNA enzyme reaction. In one of our model systems with a suitable facilitator combination, we were able to observe a more than 200-fold enhancement of the k(cat)/Km value. The comparison of two DNA enzyme-substrate systems showed that the principal effects of the facilitators were independent of the substrate sequence. However, the degree of facilitator effect was noticeably dependent on the basic catalytic efficiency of DNA enzymes. Furthermore, the efficiency of the DNA enzyme reaction with facilitator was compared with the reaction of a DNA enzyme with a stem sequence extended by the sequence of the facilitator. The multiple turnover activity of such a "long DNA enzyme" is higher than the activity of the short DNA enzyme without facilitators. However, when compared with the multiple turnover reactions of the short DNA enzyme with facilitator, the reaction with the long DNA enzyme is considerably slower. The results obtained with our model systems demonstrate that oligonucleotide facilitators enable DNA enzymes to act as effective multiple turnover catalysts by cleavage of RNA substrates.     

Energetic contribution of non-essential 5' sequence to catalysis in a hepatitis delta virus ribozyme

Hepatitis delta virus (HDV) ribozymes employ multiple catalytic strategies to achieve overall rate enhancement of RNA cleavage. These strategies include general acid-base catalysis by a cytosine side chain and involvement of divalent metal ions. Here we used a trans-acting form of the antigenomic ribozyme to examine the contribution of the 5' sequence in the substrate to HDV ribozyme catalysis. The cleavage rate constants increased for substrates with 5' sequence alterations that reduced ground-state binding to the ribozyme. Quantitatively, a plot of activation free energy of chemical conversion versus Gibb's free energy of substrate binding revealed a linear relationship with a slope of -1. This relationship is consistent with a model in which components of the substrate immediately 5' to the cleavage site in the HDV ribozyme-substrate complex destabilize ground-state binding. The intrinsic binding energy derived from the ground-state destabilization could contribute up to 2 kcal/mol toward the total 8.5 kcal/mol reduction in activation free energy for RNA cleavage catalyzed by the HDV ribozyme.     

Enhancement of ribozyme catalytic activity by a contiguous oligodeoxynucleotide (facilitator) and by 2''-O-methylation.

RNA catalysts (ribozymes) designed to cleave sequences unique to viral RNA's might be developed as therapeutics. For this purpose, they would require high catalytic efficiency and resistance to nucleases. Reported here are two approaches that can be used in combination to improve these properties. First, catalytic efficiency can be improved by oligonucleotides (facilitators) that bind to the substrate contiguously with the 3'-end of the ribozyme. Second, 2'-O-methylation of flanking sequences of the ribozyme increases catalytic activity as well as resistance to nucleases. In combination with a facilitator oligodeoxynucleotide, the cleavage rate was increased 20 fold over that of the unmodified ribozyme.     

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.     

Cross-linking experiments reveal the presence of novel structural features between a hepatitis delta virus ribozyme and its substrate

The kinetic pathway of a trans-acting delta ribozyme includes an essential structural rearrangement involving the P1 stem, a stem that is formed between the substrate and the ribozyme. We performed cross-linking experiments to determine the substrate position within the catalytic center of an antigenomic, trans-acting, delta ribozyme. Substrates that included a 4-thiouridine either in position -1, +4, or +8 (i.e., adjacent to the cleavage site, or located either in the middle of or at the 3'-end of the P1 stem, respectively) were synthesized and shown to be efficiently cleaved. Examination of the cross-linking conditions, the use of various mutated ribozymes, as well as the probing and characterization of the resulting ribozyme-substrate complexes, revealed several new features of the molecular mechanism: (1) the close proximity of several bases between nucleotides of the substrate and ribozyme; (2) the active ribozyme-substrate complex folds in a manner that docks the middle of the P1 stem on the P3 stem, while concomitantly the scissile phosphate is in close proximity to the catalytic cytosine; and, (3) some complexes appear to be compatible with being active intermediates along the folding pathway, while others seem to correspond to misfolded structures. To provide a model representation of these data, a three-dimensional structure of the delta ribozyme was developed using several RNA bioinformatic software packages.     

Length variation of helix III in a hammerhead ribozyme and its influence on cleavage activity

The previously described HIV-1 directed hammerhead ribozyme 2as-Rz12 can form with its target RNA 2s helices I and III of 128 and 278 base pairs (bp). A series of derivatives was made in which helix III was truncated to 8, 5, 4, 3, and 2 nucleotides (nt). These asymmetric hammerhead ribozymes were tested for in vitro cleavage and for inhibition of HIV-1 replication in human cells. Truncation of helix III to 8 bp did not affect the in vitro cleavage potential of the parental catalytic antisense RNA 2as-Rz12. Further truncation of helix III led to decreased cleavage rates, with no measurable cleavage activity for the 2 bp construct. All catalytically active constructs showed complex cleavage kinetics. Three kinetic subpopulations of ribozyme-substrate complexes could be discriminated that were cleaved with fast or slow rates or not at all. Gel purification of preformed ribozyme-substrate complexes led to a significant increase in cleavage rates. However, the complex cleavage pattern remained. In mammalian cells, the helix III-truncated constructs showed the same but no increased inhibitory effect of the comparable antisense RNA on HIV-1 replication.     

The antisense sequence of the HIV-1 TAR stem-loop structure covalently linked to the hairpin ribozyme enhances its catalytic activity against two artificial substrates

This work is an in vitro study of the efficiency of catalytic antisense RNAs whose catalytic domain is the wild-type sequence of the hairpin ribozyme, derived from the minus strand of the tobacco ringspot virus satellite RNA. The sequence in the target RNA recognized by the antisense molecule was the stem-loop structure of the human immunodeficiency virus-1 (HIV-1) TAR region. This region was able to form a complex with its antisense RNA with a binding rate of 2 x 10(4) M(-1)s(-1). Any deletion of the antisense RNA comprising nucleotides of the stem-loop resulted in a decrease in binding rate. Sequences 3' of the stem in the sense RNA also contributed to binding. This stem-loop TAR-antisense segment, covalently linked to a hairpin ribozyme, enhanced its catalytic activity. The highest cleavage rate was obtained when the stem-loop structure was present in both ribozyme and substrate RNAs and they were complementary. Similarly, an extension at the 5'-end of the hairpin ribozyme increased the cleavage rate when its complementary sequence was present in the substrate. Inclusion of the stem-loop at the 3'-end and the extension at the 5'-end of the hairpin ribozyme abolished the positive effect of both antisense units independently. These results may help in the design of hairpin ribozymes for gene silencing.     

Interaction between hammerhead ribozyme and RNA substrates measured by a surface plasmon resonance biosensor

Dynamic interactions between hammerhead ribozymes and RNA substrates were measured using the surface plasmon resonance (SPR) technology. Two in vitro transcribed substrates (non-cleavable and cleavable) were immobilised on streptavidin-coated dextran matrices and subsequently challenged with non-related yeast tRNA or two hammerhead ribozymes, both of which had previously been shown to exhibit functional binding and cleavage of complementary target RNAs. The target-binding domain of one of the ribozymes was fully complementary to a 16-ribonucleotide stretch on the immobilised substrates, while the other ribozyme had a nine-ribonucleotide complementarity. The two ribozymes could readily be differentiated with regard to affinity. Cleavage could be measured, using the ribozyme with full target complementarity to the cleavable substrate. In contrast, the ribozyme with lower affinity lacked cleavage activity. We suggest that SPR will be useful for investigations of ribozyme-substrate interactions.     

Cleavage of Short RNAs Containing Higher Ordered Structures by Hammerhead Ribozymes

Abstract

Cleavage of two types of secondary structure-forming substrates by their cognate hammerhead ribozymes were studied by measuring their kinetic parameters. A substrate with a self-complementary structure (GGUCCUAGGA, CL-3) was slowly cleaved by a two-stranded ribozyme. An isomer having no complementary sequence (GGUCGUAGCA, CL-3N) was cleaved more than 10 times faster than the self-complementary substrate. A newly designed ribozyme which contained a stable loop and stem cleaved the self-complementary decamer 40 times faster than the two-stranded ribozyme. A 15 mer which derived from a ras mRNA was found to have an intermolecular base pairs and was used to design more efficient ribozymes. Gel mobility shift assay was employed to investigate the binding properties of substrates to ribozymes. Investigations of the thermodynamic stability of the ribozyme-substrate complex are essential in the design of ribozymes that efficiently cleave RNA.

     

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.     

Facilitation of hammerhead ribozyme catalysis by the nucleocapsid protein of HIV-1 and the heterogeneous nuclear ribonucleoprotein A1.

In order to improve the activity of hammerhead ribozymes in vivo, we have analyzed the effect of several prototypical RNA binding proteins on the ribozyme cleavage reaction: bacteriophage T4 gene 32 protein (gp32), hnRNP A1 (A1) and the nucleocapsid protein of HIV-1 (NCp7). We show that, while gp32 has no effect on the cleavage reaction, A1 and NCp7 affect different steps of the reaction. Moreover, some of these effects depend upon the ribozyme-substrate hybrid length. A1 and NCp7 inhibit the reaction of the least stable ribozyme-substrate complexes, which have 12 bp of duplex. NCp7, but not A1, inhibits the cleavage of substrates that have long ribozyme-substrate duplexes (17 or 20 bp), while cleavage of complexes having shorter duplexes (13 or 14 bp) is not affected. NCp7 and A1 enhance the turnover of ribozymes by increasing the rate of product dissociation, but only when both cleavage products are bound with < or = 7 bp. A1 and NCp7 enhance ribozyme binding to long substrates, such as mRNAs, the structure of which otherwise limits ribozyme binding. Therefore, the effects of A1 or NCp7 on the different steps of the cleavage reaction define a length of the ribozyme-substrate duplex which allows enhancement of the rate of binding and product release without inhibiting the cleavage step. Interestingly, this duplex length (14 bases, or 7 on each side of the cleavage site) is identical for A1 and NCp7. Since A1 is thought to interact with most, if not all mRNAs in vivo, it may enhance the intracellular activity of ribozymes targeted against any mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Cleavage of oligoribonucleotides by a ribozyme derived from the hepatitis delta virus RNA sequence.

A self-cleaving RNA sequence from hepatitis delta virus was modified to produce a ribozyme capable of catalyzing the cleavage of RNA in an intermolecular (trans) reaction. The delta-derived ribozyme cleaved substrate RNA at a specific site, and the sequence specificity could be altered with mutations in the region of the ribozyme proposed to base pair with the substrate. A substrate target size of approximately 8 nucleotides in length was identified. Octanucleotides containing a single ribonucleotide immediately 5' to the cleavage site were substrates for cleavage, and cleavage activity was significantly reduced only with a guanine base at that position. A deoxyribose 5' to the cleavage site blocked the reaction. These data are consistent with a proposed secondary structure for the self-cleaving form of the hepatitis delta virus ribozyme in which a duplex forms with sequences 3' to the cleavage site, and they support a proposed mechanism in which cleavage involves attack on the phosphorus at the cleavage site by the adjacent 2'-hydroxyl group.     

Specificity of hammerhead ribozyme cleavage.

To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths.     

Examination of the folding pathway of the antigenomic hepatitis delta virus ribozyme reveals key interactions of the L3 loop

With the goal of gaining insight into the tertiary structure of the hepatitis delta virus ribozyme, cross-linking experiments using 4-thiouridine residues introduced in either the 5'-end portion of the substrate, or at seven strategic positions within the ribozyme, were performed. Mapping of the newly formed covalent bonds in cross-linked species obtained under various conditions, as well as using several mutated ribozymes, permitted monitoring of the formation of the ribozyme-substrate complex as the ribozyme proceeded along the folding pathway. In order to aid visualization of the tertiary structure transformation, an in silico animation of the "on" folding pathway was developed. In combination with those of the cleavage assays of structured substrates, these data shed light on the key contribution of the L3 loop in the formation of an active tertiary complex.