Cleavage Activity Of Ribozyme Research Articles

Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs

The naturally occurring structure and biological functions of RNA are correlated, which includes hammerhead ribozymes. We proposed new variants of hammerhead ribozymes targeting conserved structural motifs of segment 5 of influenza A virus (IAV) (+)RNA. The variants carry structural and chemical modifications aiming to improve the RNA cleavage activity of ribozymes. We introduced an additional hairpin motif and attempted to select ribozyme-target pairs with sequence features that enable the potential formation of the trans-Hoogsteen interactions that are present in full-length, highly active hammerhead ribozymes. We placed structurally defined guanosine analogs into the ribozyme catalytic core. Herein, the significantly improved synthesis of 2′-deoxy-2′-fluoroarabinoguanosine derivatives is described. The most potent hammerhead ribozymes were applied to chimeric short hairpin RNA (shRNA)-ribozyme plasmid constructs to improve the antiviral activity of the two components. The modified hammerhead ribozymes showed moderate cleavage activity. Treatment of IAV-infected Madin-Darby canine kidney (MDCK) cells with the plasmid constructs resulted in significant inhibition of virus replication. Real-time PCR analysis revealed a significant (80%–88%) reduction in viral RNA when plasmids carriers were used. A focus formation assay (FFA) for chimeric plasmids showed inhibition of virus replication by 1.6–1.7 log10 units, whereas the use of plasmids carrying ribozymes or shRNAs alone resulted in lower inhibition.

Engineering a ribozyme cleavage-induced split fluorescent aptamer complementation assay.

Hammerhead ribozymes are self-cleaving RNA molecules capable of regulating gene expression in living cells. Their cleavage performance is strongly influenced by intra-molecular loop–loop interactions, a feature not readily accessible through modern prediction algorithms. Ribozyme engineering and efficient implementation of ribozyme-based genetic switches requires detailed knowledge of individual self-cleavage performances. By rational design, we devised fluorescent aptamer-ribozyme RNA architectures that allow for the real-time measurement of ribozyme self-cleavage activity in vitro. The engineered nucleic acid molecules implement a split Spinach aptamer sequence that is made accessible for strand displacement upon ribozyme self-cleavage, thereby complementing the fluorescent Spinach aptamer. This fully RNA-based ribozyme performance assay correlates ribozyme cleavage activity with Spinach fluorescence to provide a rapid and straightforward technology for the validation of loop–loop interactions in hammerhead ribozymes.

Studies of viomycin, an anti-tuberculosis antibiotic: copper(ii) coordination, DNA degradation and the impact on delta ribozyme cleavage activity

Viomycin is a basic peptide antibiotic, which is among the most effective agents against multidrug-resistant tuberculosis. In this paper we provide the characteristics of its acid base properties, coordination preferences towards the Cu(ii) ions, as well as the reactivity of the resulting complexes against plasmid DNA and HDV ribozyme. Careful coordination studies throughout the wide pH range allow for the characterisation of all the Cu(ii)-viomycin complex species. The assignment of proton chemical shifts was achieved by NMR experiments, while the DTF level of theory was applied to support molecular structures of the studied complexes. The experiments with the plasmid DNA reveal that at the physiological levels of hydrogen peroxide the Cu(ii)-viomycin complex is more aggressive against DNA than uncomplexed metal ions. Moreover, the degradation of DNA by viomycin can be carried out without the presence of transition metal ions. In the studies of antigenomic delta ribozyme catalytic activity, viomycin and its complex are shown to modulate the ribozyme functioning. The molecular modelling approach allows the indication of two different locations of viomycin binding sites to the ribozyme.

RNA Thermodynamic Structural Entropy.

Conformational entropy for atomic-level, three dimensional biomolecules is known experimentally to play an important role in protein-ligand discrimination, yet reliable computation of entropy remains a difficult problem. Here we describe the first two accurate and efficient algorithms to compute the conformational entropy for RNA secondary structures, with respect to the Turner energy model, where free energy parameters are determined from UV absorption experiments. An algorithm to compute the derivational entropy for RNA secondary structures had previously been introduced, using stochastic context free grammars (SCFGs). However, the numerical value of derivational entropy depends heavily on the chosen context free grammar and on the training set used to estimate rule probabilities. Using data from the Rfam database, we determine that both of our thermodynamic methods, which agree in numerical value, are substantially faster than the SCFG method. Thermodynamic structural entropy is much smaller than derivational entropy, and the correlation between length-normalized thermodynamic entropy and derivational entropy is moderately weak to poor. In applications, we plot the structural entropy as a function of temperature for known thermoswitches, such as the repression of heat shock gene expression (ROSE) element, we determine that the correlation between hammerhead ribozyme cleavage activity and total free energy is improved by including an additional free energy term arising from conformational entropy, and we plot the structural entropy of windows of the HIV-1 genome. Our software RNAentropy can compute structural entropy for any user-specified temperature, and supports both the Turner’99 and Turner’04 energy parameters. It follows that RNAentropy is state-of-the-art software to compute RNA secondary structure conformational entropy. Source code is available at https://github.com/clotelab/RNAentropy/; a full web server is available at http://bioinformatics.bc.edu/clotelab/RNAentropy, including source code and ancillary programs.

Inhibition of telomerase activity by HDV ribozyme in cancers

BackgroundTelomerase plays an important role in cell proliferation and carcinogenesis and is believed to be a good target for anti-cancer drugs. Elimination of template function of telomerase RNA may repress the telomerase activity.MethodsA pseudo-knotted HDV ribozyme (g.RZ57) directed against the RNA component of human telomerase (hTR) was designed and synthesized. An in vitro transcription plasmid and a eukaryotic expression plasmid of ribozyme were constructed. The eukaryotic expression plasmid was induced into heptocellular carcinoma 7402 cells, colon cancer HCT116 cells and L02 hepatocytes respectively. Then we determine the cleavage activity of ribozyme against human telomerase RNA component (hTR) both in vitro and in vivo, and detect telomerase activity continuously.ResultsHDV ribozyme showed a specific cleavage activity against the telomerase RNA in vitro. The maximum cleavage ratio reached about 70.4%. Transfection of HDV ribozyme into 7402 cells and colon cancer cells HCT116 led to growth arrest and the spontaneous apoptosis of cells, and the telomerase activity dropped to 10% of that before.ConclussionHDV ribozyme (g.RZ57) is an effective strategy for gene therapy.

Direct selection for ribozyme cleavage activity in cells

Selection may prove to be a powerful tool for the generation of functional RNAs for in vivo genetic regulation. However, traditional in vitro selection schemes do not mimic physiological conditions, and in vivo selection schemes frequently use small pool sizes. Here we describe a hybrid in vitro/in vivo selection scheme that overcomes both of these disadvantages. In this new method, PCR-amplified expression templates are transfected into mammalian cells, transcribed hammerhead RNAs self-cleave, and the extracted, functional hammerhead ribozyme species are specifically amplified for the next round of selection. Using this method we have selected a number of cis-cleaving hammerhead ribozyme variants that are functional in vivo and lead to the inhibition of gene expression. More importantly, these results have led us to develop a quantitative, kinetic model that can be used to assess the stringency of the hybrid selection scheme and to direct future experiments.

Expression and in vitro cleavage activity of anti-caspase-7 hammerhead ribozymes.

To prepare hammerhead ribozymes against mouse caspase-7 and identify their cleavage activity in vitro, in order to select a ribozyme with specific cleavage activity against mouse caspase-7 as a potential gene therapy for apoptosis-related diseases. Anti-caspase-7 ribozymes targeting sites 333 and 394 (named Rz333 and Rz394) were designed by computer software, and their DNA sequences encoding ribozymes were synthesized. Caspase-7 DNA sequence was acquired by RT-PCR. Ribozymes and caspase-7 DNA obtained by in vitro transcription were cloned into pBSKneo U6' and pGEM-T vectors, respectively. The cleavage activity of ribozymes against mouse caspase-7 was identified by cleavage experiments in vitro. Rz333 and Rz394 were designed and their DNA sequences were synthesized respectively. The expression vector of caspase-7 and plasmids containing Rz333 and Rz394 were reconstructed successfully. Ribozymes and caspase-7 mRNA were expressed by in vitro transcription. In vitro cleavage experiment showed that 243-nt and 744-nt segments were produced after caspase-7 mRNA was mixed with Rz333 in equivalent, and the cleavage efficiency was 67.98%. No cleaved segment was observed when caspase-7 mRNA was mixed with Rz394. Rz333 can site-specific cleave mouse caspase-7 mRNA, and it shows a potential for gene therapy of apoptosis-related diseases by down-regulating gene expression of caspase-7.

Regulation of ribozyme cleavage activity by oligonucleotides.

Here, we describe allosteric ribozymes, which are activated by the addition of a short regulator oligonucleotide. The allosteric hammerhead ribozyme, which contains a single-stranded loop instead of stem II, exhibited minimal cleavage of the target RNA; however, it becomes active by the addition of oligonucleotides that bind to the ribozyme. We have also carried out in vitro selection to obtain an allosteric hairpin ribozyme, which is activated with a short oligonucleotide. The ribozyme contains the characteristic hairpin loop with the structure that changes upon regulator oligonucleotide binding. Since both regulations are sequence-specific, ribozymes containing different sequences can be positively and independently controlled by a specific oligonucleotide. Furthermore, these allosteric hairpin loops have the ability to control the activities of other functional RNAs.

Importance of specific nucleotides in the folding of the natural form of the hairpin ribozyme.

The hairpin ribozyme in its natural context consists of two loops in RNA duplexes that are connected as arms of a four-way helical junction. Magnesium ions induce folding into the active conformation in which the two loops are in proximity. In this study, we have investigated nucleotides that are important to this folding process. We have analyzed the folding in terms of the cooperativity and apparent affinity for magnesium ions as a function of changes in base sequence and functional groups, using fluorescence resonance energy transfer. Our results suggest that the interaction between the loops is the sum of a number of component interactions. Some sequence variants such as A10U, G+1A, and C25U exhibit loss of cooperativity and reduced affinity of apparent magnesium ion binding. These variants are also very impaired in ribozyme cleavage activity. Nucleotides A10, G+1, and C25 thus appear to be essential in creating the conformational environment necessary for ion binding. The double variant G+1A/C25U exhibits a marked recovery of both folding and catalytic activity compared to either individual variant, consistent with the proposal of a triple-base interaction among A9, G+1, and C25 [Pinard, R., Lambert, D., Walter, N. G., Heckman, J. E., Major, F., and Burke, J. M. (1999) Biochemistry 38, 16035-16039]. However, substitution of A9 leads to relatively small changes in folding properties and cleavage activity, and the double variant G+1DAP/C25U (DAP is 2,6-diaminopurine), which could form an isosteric triple-base interaction, exhibits folding and cleavage activities that are both very impaired compared to those of the natural sequence. Our results indicate an important role for a Watson--Crick base pair between G+1 and C25; this may be buttressed by an interaction with A9, but the loss of this has less significant consequences for folding. 2'-Deoxyribose substitution leads to folding with reduced magnesium ion affinity in the following order: unmodified RNA > dA9 > dA10 > dC25 approximately dA10 plus dC25. The results are interpreted in terms of an interaction between the ribose ring of C25 and the ribose and base of A10, in agreement with the proposal of Ryder and Strobel [Ryder, S. P., and Strobel, S. A. (1999) J. Mol. Biol. 291, 295-311]. In general, there is a correlation between the ability to undergo ion-induced folding and the rate of ribozyme cleavage. An exception to this is provided by G8, for which substitution with uridine leads to severe impairment of cleavage but folding characteristics that are virtually unaltered from those of the natural species. This is consistent with a direct role for the nucleobase of G8 in the chemistry of cleavage.

Replacement of the Phosphodiester Bond Between U4 and G5 in the U-Turn of a Chemically Modified Hammerhead Ribozyme by an Amide Bond

The phosphodiester bond between U4 and G5 in the U-turn of a chemically modified hammerhead ribozyme was substituted by an amide backbone without compromising the ribozyme's cleavage activity. Furthermore, the modified ribozyme proved to be completely stable against endonucleolytic digestion at this position.

The kinetic mechanism of the hairpin ribozyme in vivo: influence of RNA helix stability on intracellular cleavage kinetics

The relationship between hairpin ribozyme structure, and cleavage and ligation kinetics, and equilibria has been characterized extensively under a variety of reaction conditions in vitro. We developed a quantitative assay of hairpin ribozyme cleavage activity in yeast to learn how structure-function relationships defined for RNA enzymes in vitro relate to RNA-mediated reactions in cells. Here, we report the effects of variation in the stability of an essential secondary structure element, H1, on intracellular cleavage kinetics. H1 is the base-paired helix formed between ribozyme and 3′ cleavage product RNAs. H1 sequences with fewer than three base-pairs fail to support full activity in vitro or in vivo, arguing against any significant difference in the stability of short RNA helices under in vitro and intracellular conditions. Under standard conditions in vitro that include 10 mM MgCl2, the internal equilibrium between cleavage and ligation of ribozyme-bound products favors ligation. Consequently, ribozymes with stable H1 sequences display sharply reduced self-cleavage rates, because cleavage is reversed by rapid re-ligation of bound products. In contrast, ribozymes with as many as 26 base-pairs in H1 continue to self-cleave at maximum rates in vivo. The failure of large products to inhibit cleavage could be explained if intracellular conditions promote rapid product dissociation or shift the internal equilibrium to favor cleavage. Model experiments in vitro suggest that the internal equilibrium between cleavage and ligation of bound products is likely to favor cleavage under intracellular ionic conditions.

Sequential Folding of the Genomic Ribozyme of the Hepatitis Delta Virus: Structural Analysis of RNA Transcription Intermediates

The structures of the model oligoribonucleotides that mimic the consecutive stages in the transcription of genomic HDV ribozyme have been analyzed by the Pb 2+-induced cleavage method, partial digestion with specific nucleases and chemical probing. In the transcription intermediates, the P1 and P4 helical segments are found to be present in the final folded forms in which they exist in the full-length transcript. However, the region corresponding to the central hairpin forms another thermodynamically stable hairpin structure. Its correct folding requires the presence of a ribozyme 3′-terminal sequence and the formation of helix P2. This confirms the ribozyme structure of the pseudoknot type and points to the crucial role of helix P2 in its overall folding. Moreover, we show that the J4/2 region can be specifically cleaved in the presence of selected divalent metal ions in the full-length transcript, but not in a shorter one lacking six 3′-terminal nucleotides, which cannot form the pseudoknotted structure. Thus, a particular RNA conformation around that cleavage site is required for specific hydrolysis, and the J4/2 region seems to be involved in the formation of a general metal ion binding site. Recently, it has been proposed that, in the antigenomic ribozyme, a four nucleotide sequence within the J1/2 region may contribute to the folding pathway, being part of a mechanism responsible for controlling ribozyme cleavage activity. Our study shows that in the genomic ribozyme the central hairpin region may contribute to a similar mechanism, providing a barrier to the formation of an active structure in the ribozyme folding pathway.

Substitution of the 2′-Hydroxyl Group at Position 2.1 by an Amino Group Interferes with Mg2+ Binding and Efficient Cleavage by Hammerhead Ribozyme

Recently we have demonstrated that hammerhead ribozymes can be fully substituted with 2′-amino pyrimidines without detriment to the catalytic activity, provided that positions 2.2 and/or 2.1 are not modified. We now report on the potential molecular mechanisms by which 2′-amino groups at these positions inhibit the ribozyme cleavage activity. In the presence of Mg2+, the 2′-amino modification at positions 2.2 and/or 2.1 had no significant effect on substrate binding. Detailed analysis of the ribozyme initial cleavage rates in the presence of various Mg2+ concentrations indicated that Mg2+ binding is inhibited by the 2′-amino group at position 2.1. Furthermore, preannealed substrate molecules to the modified ribozyme are not effectively cleaved upon Mg2+ addition, indicating an alteration of the ribozyme cleavage step. Surprisingly, the cleavage activity of the modified ribozymes was substantially increased when Mg2+ ions were replaced by the thiophilic Mn2+ ions, whereas only a moderate cleavage enhancement occurred with its unmodified version. Taken together, our findings indicate that changes in the sugar at position 2.1 alter Mg2+-promoting ribozyme cleavage.

High Cleavage Activity and Stability of Hammerhead Ribozymes with a Uniform 2′-Amino Pyrimidine Modification

The uniform 2′-pyrimidine modifications have been found to inhibit ribozyme cleavage activity. However, in the present study we show that a good cleavage activity can be achieved for the 2′-amino modified ribozymes when their sequences were designed to contain only a few pyrimidines in helix I. In particular, ribozymes with no pyrimidines in helix I cleaved their target RNAs with almost the same efficacy as their unmodified versions. Interestingly, selective 2′-amino modification at positions 2.1 and 2.2 reduced the ribozyme cleavage activity by 8-fold, suggesting that the 2′-amino groups at these two positions may interfere with the formation of the ribozyme active conformation. In addition, uniformly modified ribozymes showed a remarkable stability in serum. Taken together these results should facilitate the design of stable ribozymes with sustained cleavage activity.

Cleavage of collagen RNA transcripts by hammerhead ribozymes in vitro is mutation-specific and shows competitive binding effects.

We report here the in vitro use of hammerhead ribozymes as an approach to the gene therapy of osteogenesis imperfecta (OI). Our strategy for the treatment of this dominant genetic disorder is based on selective reduction of the level of the mRNA transcripts from the mutant allele. We studied the in vitro cleavage activity of five different hammerhead ribozymes targeted against synthetic transcripts of two naturally occurring human collagen mutations and against a point mutation introduced into a construct containing a portion of the mouse COL1A1 gene. This is the first demonstration that ribozyme cleavage is absolutely dependent on the presence of the ribozyme cleavage site introduced by the disease-causing mutation. Cleavage specificity and activity were unchanged when the cleavage site was located in transcripts of progressively longer length. Cleavage efficiency depended directly on the ratio of ribozyme/substrate, as well as on the time and temperature of incubation. We investigated the competitive effects of both total RNA and normal synthetic transcripts on ribozyme cleavage activity. The ribozyme was able to localize and cleave its specific target even in the presence of a vast excess of total RNA. However, cleavage efficiency was linearly inhibited by the presence of a non- cleavable competitor substrate which contained a ribozyme binding site identical to the site present in the cleavable target. Although this competition could be eliminated by introducing a mismatch into one ribozyme binding arm, the presence of the mismatch decreased ribozyme cleavage efficiency. The mutation- specificity of ribozyme cleavage demonstrated in this work provides support for in vivo studies aimed at ribozyme development as a treatment for dominant negative genetic disorders.

Purine Functional Groups in Essential Residues of the Hairpin Ribozyme Required for Catalytic Cleavage of RNA

Synthetic chemistry techniques have been used to study the functional group requirements of the essential purine residues in hairpin ribozyme cleavage. Three-stranded ribozymes were prepared that had functional group deletions or alterations at single purine sites within loops A and B of the hairpin, and the kinetics of cleavage were compared to those of the unmodified ribozyme. Adenosine analogues used were purine riboside and N7-deazaadenosine, and guanosine analogues used were inosine, N7-deazaguanosine, and O6-methylguanosine. In many cases, introduction of one of these analogues caused substantial loss of ribozyme cleavage activity. Most of the impairments of activity were found to be due to changes in kcat rather than in KM. The losses corresponded in magnitude to loss of at least one hydrogen bond, and the results were rationalized in terms of removal of potential cross-strand hydrogen bonds as well as potential hydrogen bonds between loops A and B. A new secondary structure model for loop B was proposed. Finally, the magnesium ion dependence of cleavage was studied for the modified ribozymes and compared to that of the unmodified ribozyme. It is proposed that magnesium binds in the ground state to the N7-positions of G + 1 and A43 and in the transition state to the N7-position at A9. The results provide further evidence for the folding of the two arms of the hairpin so that in the active conformation loops A and B approach closely to form a specific three-dimensional structure with a magnesium ion (or ions) placed between the loops, making contacts in the ground state and in the transition state.

Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter.

RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human meti tRNA-derived pol III promoter > 100-fold by modifying the 3' terminus of the transcripts to hybridize to the 5' terminus. This terminal duplex includes the 8 nt leader sequence present in the primary wild-type meti tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed 'TRZ', was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs.

A multifunctional expression vector for an anti-HIV-1 ribozyme that produces a 5'- and 3'-trimmed trans-acting ribozyme, targeted against HIV-1 RNA, and cis-acting ribozymes that are designed to bind to and thereby sequester trans-activator proteins such as Tat and Rev.

We previously constructed a multiribozyme expression vector by combining cis- and trans-acting ribozymes and we showed that several ribozymes, each directed against a different target in the HIV genome and acting independently in a 'shotgun' manner, markedly increased the efficiency of cleavage of HIV RNA in vitro [Ohkawa et al., Proc. Natl Acad. Sci. USA 90, 11302 (1993)]. However, the cis-acting ribozymes that had trimmed the 5' and 3' ends of each trans-acting ribozyme were designed merely to await for degradation by RNases when they were used in vivo. Since several trans-activator proteins are essential for viral replication of HIV-1, we wondered whether a decoy function could be coupled with the cleavage activity of ribozymes. We therefore introduced the TAR or the RRE sequence into the stem II region of each cis-acting ribozyme. When the activity of each resulting cis-acting ribozyme that had been endowed with the decoy function was examined in vitro, it was found to retain almost full trimming activity. Moreover, cis-acting ribozymes with either the TAR or the RRE sequence were shown to be able to trap Tat or Rev protein successfully. It is, therefore, possible to endow the stem II region with a specific protein-binding function without the loss of ribozyme function. Thus, cis-acting ribozymes, endowed with the decoy function, can first trim the 5' and 3' ends of each trans-acting ribozyme and are then still available for trapping trans-activator proteins possibly prior to their degradation by RNases when they are to be used in vivo. Furthermore, it is also expected that the reduction in production of HIV RNA that is achieved by sequestering the trans-activator proteins might provide the trans-acting ribozymes, targeted to HIV RNA, with a better chance of eliminating the remaining HIV RNA.

Ribozyme-Mediated Cleavage of an HIV-1gagRNA: The Effects of Nontargeted Sequences and Secondary Structure on Ribozyme Cleavage Activity

Catalytic antisense RNAs, or ribozymes, have great potential as inhibitors of gene expression and as antiviral therapeutic agents. The major advantage of ribozymes versus standard antisense RNAs is their catalytic capability, enabling these RNAs to cleave multiple substrates. We have been investigating the antiviral activity of ribozymes targeted to the HIV-1 genome. The successful use of these antisense agents in an intracellular milieu requires stabilization of the ribozymes by flanking, non-base-pairing sequences, or some modification of the sugar-phosphate backbone. We describe a systematic investigation of the effects of flanking, non-base-pairing sequences on the catalytic activity of an anti-HIV-1 gag ribozyme embedded in radically different transcripts. Amazingly, these complex ribozyme-containing transcripts maintain substantial catalytic activity. Finally, we describe a bacterial gene fusion system that has potential for the large scale production of catalytically active ribozymes.