Properties of antigenomic hepatitis delta virus ribozyme cis- and trans- analogs

A series of permuted variants of antigenomic HDV ribozyme and trans-acting variants were constructed. The catalytic activity study of the ribozymes has shown that all the variants were capable of self-cleaving with equally biphasic kinetics. Ribonuclease and Fe(II)-EDTA cleavage have provided evidence that all designed ribozymes fold according to the pseudoknot model and the conformations of the initial and cleaved ribozyme are different. A scheme of HDV ribozyme self-cleavage reaction was suggested. The role of hydrogen bonds in the reaction was evaluated by substitution of ribose in the ribozyme for deoxyribose. It was found that the 2'-OH group of U23 and C27 is critical for the reaction to occur; the 2'-OH group of U32 and U39 is important, while 2'-OH groups of other nucleotides of loop 3, stem 4 and stem 1 are unimportant for the cleavage activity.     

The kinetics and magnesium requirements for the folding of antigenomic delta ribozymes

Using an oligonucleotide hybridization assay to gain insight into the folding of delta ribozymes, we demonstrate a correlation between their folding and catalytic behavior. Together with recent structural information on the crystal structure of self-cleaved genomic delta ribozyme, in which the L3 loop interacts with J1/4 to form the newly proposed stem P1.1, we conclude that it is likely that the P1.1 stem forms only in the presence of Mg(2+). This stem can be detected in both the self-cleaved and trans-acting delta ribozymes. When the trans-acting version of antigenomic delta ribozyme was studied, it is demonstrated that its L3 loop requires magnesium and, apparently, formation of the P1 stem for the subsequently formation of the P1.1 stem. Most importantly, the kinetics were monitored, and provide a significant addition to our understanding of ribozyme tertiary structure formation prior to the chemical cleavage step. Using previous kinetic data and our new findings, we discuss the rate-limiting characteristics of delta ribozyme folding.     

A nested double pseudoknot is required for self-cleavage activity of both the genomic and antigenomic hepatitis delta virus ribozymes.

The crystal structure of a genomic hepatitis delta virus (HDV) ribozyme 3' cleavage product predicts the existence of a 2 bp duplex, P1.1, that had not been previously identified in the HDV ribozymes. P1.1 consists of two canonical C-G base pairs stacked beneath the G.U wobble pair at the cleavage site and would appear to pull together critical structural elements of the ribozyme. P1.1 is the second stem of a second pseudoknot in the ribozyme, making the overall fold of the ribozyme a nested double pseudoknot. Sequence comparison suggests the potential for P1.1 and a similar fold in the antigenomic ribozyme. In this study, the base pairing requirements of P1.1 for cleavage activity were tested in both the genomic and antigenomic HDV ribozymes by mutagenesis. In both sequences, cleavage activity was severely reduced when mismatches were introduced into P1.1, but restored when alternative base pairing combinations were incorporated. Thus, P1.1 is an essential structural element required for cleavage of both the genomic and antigenomic HDV ribozymes and the model for the antigenomic ribozyme secondary structure should also be modified to include P1.1.     

Delta ribozyme has the ability to cleave in transan mRNA.

We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy.     

A circular trans-acting hepatitis delta virus ribozyme.

A circular trans-acting ribozyme designed to adopt the motif of the hepatitis delta virus (HDV) trans-acting ribozyme was produced. The circular form was generated in vitro by splicing a modified group I intron precursor RNA in which the relative order of the 5' and 3' splice sites, flanking the single HDV-like ribozyme sequence-containing exon, is reversed. Trans-cleavage activity of the circular HDV-like ribozyme was comparable to linear permutations of HDV ribozymes containing the same core sequence, and was shown not to be due to linear contaminants in the circular ribozyme preparation. In nuclear and cytoplasmic extracts from HeLa cells, the circular ribozyme had enhanced resistance to nuclease degradation relative to a linear form of the ribozyme, suggesting that circularization may be a viable alternative to chemical modification as a means of stabilizing ribozymes against nuclease degradation.     

Delta ribozyme benefits from a good stability in vitro that becomes outstanding in vivo

The stability of a trans-acting delta ribozyme was studied under various conditions. Although in vitro (i.e., in the presence of protein extracts) this delta ribozyme appears to be only slightly more stable than a hammerhead ribozyme, in vivo (i.e., after cell transfection) it exhibits an outstanding stability that manifests itself in the calculated half-life of over 100 h regardless of the means of transfection. The P2 stem, which includes both the 5' and 3' ends, is shown to play a critical role in this stability. Direct mutagenesis of the most nuclease susceptible nucleotides failed to generate a more stable ribozyme that retained the same catalytic potential. Clearly, delta ribozyme appears to be well adapted to the human cell environment, and is therefore ideal for the development of a gene-inactivation system.     

A pH-sensitive RNA tertiary interaction affects self-cleavage activity of the HDV ribozymes in the absence of added divalent metal ion

Self-cleavage of the genomic and antigenomic ribozymes from hepatitis delta virus (HDV) requires divalent cation for optimal activity. Recently, the HDV genomic ribozyme has been shown to be active in NaCl in the absence of added divalent metal ion at low pH (apparent pKa 5.7). However, we find that the antigenomic ribozyme is 100 to 1000-fold less active under similar conditions. With deletion of a three-nucleotide sequence (C41-A42-A43) unique to the genomic ribozyme, the rate constant for cleavage decreased substantially, while activity of the antigenomic ribozyme was enhanced by introducing a CAA sequence. From the crystal structure, it has been proposed that C41 in this sequence is protonated. To investigate a possible connection between activity at low pH and protonation of C41, mutations were made that were predicted to either eliminate protonation or alter the nature of the tertiary interaction upon protonation. In the absence of added Mg2+, these mutations reduced activity and eliminated the observed pH-rate dependence. Thermal denaturation studies revealed a pH-sensitive structural feature in the genomic ribozyme, while unfolding of the mutant ribozymes was pH-independent. We propose that, in the absence of added Mg2+, protonation of C41 contributes to enhanced activity of the HDV genomic ribozyme at low pH.     

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.     

RNase P ribozymes selected in vitro to cleave a viral mRNA effectively inhibit its expression in cell culture

An in vitro selection procedure was used to select RNase P ribozyme variants that efficiently cleaved the sequence of the mRNA encoding thymidine kinase of herpes simplex virus 1. Of the 45 selected variants sequenced, 25 ribozymes carried a common mutation at nucleotides 224 and 225 of RNase P catalytic RNA from Escherichia coli (G(224)G(225) --> AA). These selected ribozymes exhibited at least 10 times higher cleavage efficiency (k(cat)/K(m)) than that derived from the wild type ribozyme. Our results suggest that the mutated A(224)A(225) are in close proximity to the substrate and enhance substrate binding of the ribozyme. When these ribozyme variants were expressed in herpes simplex virus 1-infected cells, the levels of thymidine kinase mRNA and protein were reduced by 95-99%. Our study provides the first direct evidence that RNase P ribozyme variants isolated by the selection procedure can be used for the construction of gene-targeting ribozymes that are highly effective in tissue culture. These results demonstrate the potential for using RNase P ribozymes as gene-targeting agents against any mRNA sequences, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.     

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.     

Ribozymes of the hepatitis delta virus: recent findings on their structure, mechanism of catalysis and possible applications.

Although the delta ribozymes have been studied for more than ten years the most important information concerning their structure and mechanism of catalysis were only obtained very recently. The crystal structure of the genomic delta ribozyme turns out to be an excellent example of the extraordinary properties of RNA molecules to fold into uniquely compact structures. Details of the X-ray structure have greatly stimulated further studies on the folding of the ribozymes into functionally active molecules as well as on the mechanism of RNA catalysis. The ability of the delta ribozymes to carry out general acid-base catalysis by nucleotide side chains has been assumed in two proposed mechanisms of self-cleavage. Recently, considerable progress has been also made in characterizing the catalytic properties of trans-acting ribozyme variants that are potentially attractive tools in the strategy of directed RNA degradation.     

Engineered RNase P ribozymes increase their cleavage activities and efficacies in inhibiting viral gene expression in cells by enhancing the rate of cleavage and binding of the target mRNA

Engineered RNase P ribozymes are promising gene-targeting agents that can be used in both basic research and clinical applications. We have previously selected ribozyme variants for their activity in cleaving an mRNA substrate from a pool of ribozymes containing randomized sequences. In this study, one of the variants was used to target the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). The variant exhibited enhanced cleavage and substrate binding and was at least 30 times more efficient in cleaving TK mRNA in vitro than the ribozyme derived from the wild type sequence. Our results provide the first direct evidence to suggest that a point mutation at nucleotide 95 of RNase P catalytic RNA from Escherichia coli (G(95) --> U(95)) increases the rate of cleavage, whereas another mutation at nucleotide 200 (A(200) --> C(200)) enhances substrate binding of the ribozyme. A reduction of about 99% in TK expression was observed in cells expressing the variant, whereas a 70% reduction was found in cells expressing the ribozyme derived from the wild type sequence. Thus, the RNase P ribozyme variant is highly effective in inhibiting HSV-1 gene expression. Our study demonstrates that ribozyme variants increase their cleavage activity and efficacy in blocking gene expression in cells through enhanced substrate binding and rate of cleavage. These results also provide insights into the mechanism of how RNase P ribozymes efficiently cleave an mRNA substrate and, furthermore, facilitate the development of highly active RNase P ribozymes for gene-targeting applications.     

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.     

Cytoplasmic delivery of ribozymes leads to efficient reduction in alpha-lactalbumin mRNA levels in C127I mouse cells.

Ribozymes targeted to five sites along the alpha-lactalbumin (alpha-lac) mRNA were delivered to the cytoplasm of mouse C127I mammary cells using the T7-vaccinia virus delivery system and the amount of alpha-lac mRNA was monitored 24-48 h post-transfection. Three target sites were selected in the alpha-lac coding region (nucleotides 15, 145 and 361) and two were located in the 3' non-coding region (nucleotides 442 and 694). Acting in trans and at a target:ribozyme ratio of 1:1000, ribozymes targeting sites 361 and 694 reduced alpha-lac mRNA by > 80%; another two ribozymes (targeting nucleotides 442 and 145) reduced mRNA levels by 80 and 60% respectively; the fifth ribozyme (targeting nucleotide 15, near the AUG) was largely ineffective. The kinetic activity (kcat) of each ribozyme in vitro was somewhat predictive of the activity of the two ribozymes that targeted nucleotides 361 and 694, but was not predictive of the in vivo activity of the other three ribozymes. Down-regulation of the intracellular levels of alpha-lac paralleled the ribozyme-dependent reduction achieved for mRNA. For site 442, the reduction in both mRNA and protein was attributed to the catalytic activity of the ribozyme rather than to the antisense effects of the flanking arms, because delivery of an engineered (catalytically-inactive) variant had no effect on mRNA levels and a minimal effect on the level of alpha-lac present in the cell.     

Characterization in vitro and in vivo of hammerhead ribozymes directed against murine tumor necrosis factoralpha.

A hammerhead ribozyme directed against murine TNFalpha (mTNFalpha) mRNA has been constructed. In vitro studies showed that this ribozyme was released from the parent molecule by flanking cis-acting hammerhead and hairpin ribozymes. This same anti-mTNFalpha ribozyme specifically cleaved both synthetically derived substrate RNA and mTNFalpha mRNA within a pool of total cellular RNA. Endogenous delivery of this anti-mTNFalpha ribozyme via the self-cleaving cassette reduced mTNFalpha mRNA and protein levels in lipopolysaccharide (LPS)-stimulated, stably transfected murine macrophage RAW 264.7 cells. When complexed to liposomes and exogenously delivered to mouse peritoneal macrophages, the same ribozyme, with and without the cis-acting ribozymes, reduced mTNFalpha protein levels. However, an irrelevant ribozyme delivered in an identical fashion was also effective at reducing mTNFalpha protein levels. These data suggest that anti-mTNFalpha ribozymes can be constructed which efficiently cleave mTNFalpha mRNA, but irrelevant RNA/liposome complexes also effectively limit TNFalpha mRNA expression and can mimic functional ribozyme activity under in vitro conditions.