RNA aptamer-mediated interference of HCV replication by targeting the CRE-5BSL3.2 domain.

The RNA genome of hepatitis C virus (HCV) contains multiple conserved structural RNA domains that play key roles in essential viral processes. A conserved structural component within the 3' end of the region coding for viral RNA-dependent RNA polymerase (NS5B) has been characterized as a functional cis-acting replication element (CRE). This study reports the ability of two RNA aptamers, P-58 and P-78, to interfere with HCV replication by targeting the essential 5BSL3.2 domain within this CRE. Structure-probing assays showed the binding of the aptamers to the CRE results in a structural reorganization of the apical portion of the 5BSL3.2 stem-loop domain. This interfered with the binding of the NS5B protein to the CRE and induced a significant reduction in HCV replication (≈50%) in an autonomous subgenomic HCV replication system. These results highlight the potential of this CRE as a target for the development of anti-HCV therapies and underscore the potential of antiviral agents based on RNA aptamer molecules.

Inhibition of hepatitis C virus internal ribosome entry site-mediated translation by an RNA targeting the conserved IIIf domain.

Hepatitis C virus (HCV) translation initiation depends on an internal ribosome entry site (IRES). We previously identified an RNA molecule (HH363-10) able to bind and cleave the HCV IRES region. This paper characterizes its capacity to interfere with IRES function. Inhibition assays showed that it blocks IRES activity both in vitro and in a human hepatoma cell line. Although nucleotides involved in binding and cleavage reside in separate regions of the inhibitor HH363-10, further analysis demonstrated the strongest effect to be an intrinsic feature of the entire molecule; the abolishment of either of the two activities resulted in a reduction in its function. Probing assays demonstrate that HH363-10 specifically interacts with the conserved IIIf domain of the pseudoknot structure in the IRES, leading to the inhibition of the formation of translationally competent 80S particles. The combination of two inhibitory activities targeting different sequences in a chimeric molecule may be a good strategy to avoid the emergence of resistant viral variants.

Targets and tools: recent advances in the development of anti-HCV nucleic acids.

Hepatitis C virus (HCV), the major etiological agent of transfusion-associated non-A, non-B hepatitis, is a severe health problem affecting up to 3% of the world population. Since its identification in 1989, enormous efforts have been made to characterize the viral cycle. However, many details regarding the virus' penetration of hepatocytes, its replication and translation, and the assembling of virions remain unknown, mostly because of a lack of an efficient culture system. This has also hampered the development of fully effective antiviral drugs. Current treatments based on the combination of interferon and ribavirin trigger a sustained virological response in only 40% of infected individuals, thus the development of alternative therapeutic strategies is a major research goal. Nucleic acid based therapeutic agents may be of some potential in hepatitis C treatment. In recent years, much effort has gone into the improvement of DNA and RNA molecules as specific gene silencing tools. This review summarizes the state of the art in the development of new HCV therapies, paying special attention to those involving antisense oligonucleotides, aptamers, ribozymes, decoys and siRNA inhibitors. The identification of potential viral targets is also discussed.

Selection of targets and the most efficient hairpin ribozymes for inactivation of mRNAs using a self-cleaving RNA library.

The identification of proficient target sites within long RNA molecules, as well as the most efficient ribozymes for each, is a major concern for the use of ribozymes as gene suppressers. In vitro selection methods using combinatorial libraries are powerful tools for the rapid elucidation of interactions between macromolecules, and have been successfully used for different types of ribozyme study. This paper describes a new method for selecting effective target sites within long RNAs using a combinatorial library of self-cleaving hairpin ribozymes that includes all possible specificities. The method also allows the identification of the most appropriate ribozyme for each identified site. Searching for targets within the lacZ gene with this strategy yielded a clearly accessible site. Sequence analysis of ribozymes identified two variants as the most appropriate for this site. Both selected ribozymes showed significant inhibitory activity in the cell milieu.

Self-initiation of translation of mRNAs devoid of translational initiators in Escherichia coli.

Recent studies have shown that the canonical SD-anti-SD interaction is dispensable for the initiation of translation of certain mRNAs in Escherichia coli. In this study the cat and tetR genes were modified to either destroy complementarity to E. coli 16S RNA or completely delete their 5' non-translated regions. Thus a series of cat- and tetR-derived genes were constructed, cloned under a strong constitutive promoter and expressed in E. coli cells. The efficiency of expression was evaluated by the yield of CAT (for the cat gene) and cell viability in increasing concentrations of antibiotic (for both cat and tetR genes). The obtained results show that the mRNAs transcribed from both series of reporter genes (cat and tetR) were active in vivo. Their activity was preserved even in the cases when the length of their 5' non-translated leader sequences was reduced to one nucleotide for the cat gene and eight nucleotides for the tetR gene. The yield of protein obtained with the latter constructs was detectable and sufficient for bacteria to survive at 50-100 microg/ml chloramphenicol and 20 microg/ml tetracycline, respectively.

Comparative kinetic analysis of structural variants of the hairpin ribozyme reveals further potential to optimize its catalytic performance.

The hairpin ribozyme derived from the minus strand of the satellite RNA associated with the tobacco ringspot virus is one of the small catalytic RNAs that has been shown to catalyze trans-cleavage reactions. There is much interest in designing hairpin ribozymes with improved catalytic activity for the development of new therapeutic agents. Extensive mutagenesis studies as well as in vitro selection experiments have been performed to define the structure and optimize its catalytic activity. This communication describes a comparative kinetic analysis of four structural variants, introduced, either alone, or in combination, into the hairpin ribozyme. We have shown that extension of the helix 2 from 4 to 6 bp resulted in a significant decrease in K(M). Furthermore, the combination of this extension with the simultaneous stabilization of helix 4, led to a more than two-fold increase in the catalytic efficiency. This variant showed a 15-fold reduction in the K(M) value in respect to the wild-type ribozyme. This could be of great interest for the in vivo application of this catalytic motif. The 9-bp enlargement of helix 4 implied about a three-fold improvement in the catalytic activity. Similarly, the U39C substitution brought up the efficiency of the ribozyme slightly. However, introduction of nucleotides at the hinge region between A and B domains reduced the catalytic activity. This reduction was gradually increased with the number of nucleotides. Results obtained with variants carrying more than one modification always agreed with the ones obtained from each single variant.

Specificity of the hairpin ribozyme. Sequence requirements surrounding the cleavage site.

Substrate sequence requirements of the hairpin ribozyme have been partially defined by both mutational and in vitro selection experiments. It was considered that the best targets were those that included the N downward arrowGUC sequence surrounding the cleavage site. In contrast to previous studies that failed to evaluate all possible combinations of these nucleotides, we have performed an exhaustive analysis of the cleavage of 64 substrate variants. They represent all possible sequence combinations of the J2/1 nucleotides except the well established G(+1). No cleavage was observed with 24 sequences. C(+2) variants showed little or no cleavage, whereas U(+2) substrates were all cleavable. The maximal cleavage rate was obtained with the AGUC substrate. Cleavage rates of sequences HGUC (H = A, C, or U), GGUN, GGGR (R = A or G), AGUU, and UGUA were up to 5 times lower than the AGUC one. This shows that other sequences besides NGUC could also be considered as good targets. A second group of sequences WGGG (W = A or U), UGUK (K = G or U), MGAG (M = A or C), AGUA, and UGGA were cleaved between 6 and 10 times less efficiently. Furthermore, the UGCU sequence of a noncleavable viral target was mutated to AGUC resulting in a proficiently cleavable substrate by its cognate hairpin ribozyme. This indicates that our conclusions may be extrapolated to other hairpin ribozymes with different specificity.

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.

Determination of HCV RNA concentration by direct quantitation of the products from a single RT-PCR.

A novel method for the estimation of HCV RNA levels in vivo was developed, based on competitive RT-PCR. The use of the Tth DNA polymerase and 5' 32P-labeled antisense primer respectively reduced cross-contamination and permitted the direct quantification of viral loads by the analysis of the radioactivity of PCR products derived from a clinical sample and a competitive deleted template, separated previously on a polyacrilamide gel. A HCV fragment (H) and a competitive (deltaH) RNA templates were synthesized for optimizing the method. The minimal starting RNA detectable by RT-PCR was 40 copies. RT-PCR performed with ratios deltaH/H ranging from 1/1 to 1/20 revealed different relative percentages of both H and deltaH products, changing from 90% of deltaH product when the ratio was 1/1 to 5%, when it was 1/20. Regression analysis was adjusted to a linear model and served to further estimate HCV RNA loads from clinical samples. HCV RNA quantitation was carried out in 19 patients. Higher viral loads were related to type 1b infection and persistence of HCV RNA after interferon therapy. This method is simple, reproducible and useful for rapid estimation of HCV RNA load in vivo.

The Capsicum L3 gene-mediated resistance against the tobamoviruses is elicited by the coat protein.

The L3 gene is responsible for the hypersensitive response in Capsicum plants against infection by tobamoviruses. The resistance conferred by this gene is one of the most effective so far described against tobamoviruses. Certain isolates of pepper mild mottle virus (PMMV) are the only tobamoviruses able to overcome the L3 resistance. Chimeric viral genomes between PMMV-S (to which L3 plants are hypersensitive) and PMMV-I (an L3 resistance-breaking isolate) led us to conclude that sequence variation within the coat protein gene of both isolates determines their different virulence in L3L3 plants. Furthermore, the results indicate that a single amino acid substitution, Asn to Met, at position 138 of the PMMV-I coat protein is sufficient to induce the hypersensitive response and localization of viral infection in C. chinense plants. Finally, the use of a mutant coding for a truncated coat protein (maintaining the Met138 coding sequence at the RNA level) demonstrates that a functional coat protein is required for elicitation of the L3 gene-mediated resistance.

Novel system for analysis of group I 3' splice site reactions based on functional trans-interaction of the P1/P10 reaction helix with the ribozyme's catalytic core.

A group I intron from a bacterial tRNA precursor has been converted into an RNA enzyme that catalyzes the efficient polymerization of oligoribonucleotide analogs of tRNA exons using a reaction scheme consisting of multiple cycles of reverse and forward exon ligation reactions. Here, we present results showing that this system represents a novel and useful tool for the analysis of 3' splice site reactions of group I ribozymes. First, analysis of variant substrates containing base substitutions in group I secondary structure elements P1, P9.0 and P10 confirms that exon polymerization is dependent on these structures, and therefore constitutes an appropriate and relevant model system for studying the exon ligation step of splicing. Second, to probe interactions between the intron's catalytic core and the bases and backbone of the P1/P10 reaction helix, two successful strategies for separating the internal guide sequence from the intron core were devised. One such strategy uses a construct in which the reaction helix interacts functionally with the catalytic core using only tertiary contacts. Further stabilization of this interaction through the inclusion of a 7 bp intermolecular P2 helix generates increased reaction efficiency. Third, when provided with two reaction helices, the ribozyme synthesizes mixed polymers through a mechanism that involves sequential binding and release of the duplexes. Fourth, in these reactions, turnover of the external guide sequence requires unwinding and annealing of the P2 helix, suggesting that P2 unwinding may occur during group I splicing. These results provide novel experimental tools to probe the relatively poorly understood 3' splice site reactions of group I introns, and may be relevant to ribozyme-catalyzed assembly and recombination of oligomers in prebiotic scenarios.

Novel RNA polymerization reaction catalyzed by a group I ribozyme.

We have converted a bacterial tRNA precursor containing a 205 nt self-splicing group I intron into a RNA enzyme that catalyzes polymerization of an external RNA substrate. The reaction involves transesterification steps analogous to both the forward and reverse exon ligation steps of group I splicing; as such it depends entirely on 3' splice site reactions. The RNA substrate is a 20 nt analogue of the ligated exons (E1.E2), whose 3' end resembles the 3' terminus of the intron RNA enzyme (IVS). The splice junction of the substrate is attacked by the 3' end of the intron, then the molecule displaces the original 3' terminal guanosine so that the new 3' terminus is brought into the active site and used as the attacking nucleophile in the next reaction. Polymerization occurs via a series of covalent enzyme-linked intermediates of the structure IVS.(E2)n, where n = 1 to > or = 18. The 5' exon accumulates during the course of the reaction and can attack the covalent intermediates to produce elongation products of structure E1.(E2)n, regenerating the intron RNA enzyme in unchanged form. In this manner, the enzyme converts 20 nt oligoribonucleotides into polyribonucleotides up to at least 180 nt by 10 nt increments. These results have significant implications for the evolution of RNA-based self-replicating systems.

2'-Hydroxyl groups important for exon polymerization and reverse exon ligation reactions catalyzed by a group I ribozyme.

The functional importance of ribose moieties in both exons and in intron sequences proximal to the 3' splice site of a group I intron has been analyzed using a novel exon polymerization reaction. The ribozyme is a modified version of a self-splicing bacterial tRNA intron (I) that attacks a 20-nucleotide synthetic ligated exon substrate (E1.E2), yielding E1 and I.E2 by reverse exon ligation. A series of repetitive reactions then polymerize E2 on the 3' end of the intron; attack by E1 subsequently generates E1.(E2)n. Systematic deoxyribonucleotide substitution within E1.E2 was used to probe the function of 2'-hydroxyl groups in each exon and the 3'-terminal nucleotides of the intron. We find that ribose at the splice junction (U-1) and at the two adjacent positions with E1 (A-2, C-3) is important for reverse exon ligation. Within E2, deletion of 2'-hydroxyl groups of the nucleotides that form P10 does not affect reactivity. In contrast, ribose at the 3' end of the intron is essential for reverse exon ligation, and the presence of a 2'-OH group in each of the nucleotides comprising P9.0[3'] contributes to reaction efficiency. These results support a model in which specific 2'-hydroxyl groups at and adjacent to the reaction sites form tertiary contacts that serve to stabilize interactions with the catalytic core of the ribozyme. Furthermore, they suggest that the mechanism by which guanosine at the 3' end of the intron is activated for reverse exon ligation is the same as that by which guanosine mononucleotide is activated in the first step of splicing.

Four ribose 2'-hydroxyl groups essential for catalytic function of the hairpin ribozyme.

The hairpin ribozyme catalyzes site-specific cleavage of an RNA substrate using a magnesium-dependent transphosphorylation mechanism. Here, we describe experiments designed to test the importance of ribose 2'-hydroxyl groups for ribozyme function. Ribozymes for this work were synthesized in two segments using solid-phase RNA phosphoramidite chemistry. 2'-Deoxyribonucleotides were systematically introduced at each of the 50 positions within the ribozyme, and the catalytic activity of the resulting mixed RNA-DNA polymers was measured. Deletion of the 2'-hydroxyl group at each of four sites (A10, G11, A24, and C25) was found to result in severe inhibition of cleavage activity (kcat/KM decreased by 100- to 1000-fold), although KM measurements and mobility-shift assays showed that substrate binding was not affected. Identical results were obtained upon substitution of these ribonucleotides with 2'-O-methyl derivatives. Inhibition by 2'-modified sugars at G11 or A24 was rescued by increased Mg2+ concentrations, suggesting that these 2'-hydroxyls may function in magnesium binding. Our results demonstrate that the 2'-hydroxyl groups at A10, G11, A24, and C25 provide essential functions for catalysis, possibly forming important tertiary contacts or magnesium coordination sites that are necessary for active site architecture.

Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme.

In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme-substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild-type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme-substrate complex are derived.

Ionic requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme.

Metal ion requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme have been analyzed. RNA cleavage is observed when Mg2+, Sr2+, or Ca2+ are added to a 40 mM Tris-HCl buffer, indicating that these divalent cations were capable of supporting the reaction. No reaction was observed when other ions (Mn2+, Co2+, Cd2+, Ni2+, Ba2+, Na+, K+, Li+, NH4+, Rb+, and Cs+) were tested. In the absence of added metal ions, spermidine can induce a very slow ribozyme-catalyzed cleavage reaction that is not quenched by chelating agents (EDTA and EGTA) that are capable of quenching the metal-dependent reaction. Addition of Mn2+ to a reaction containing 2 mM spermidine increases the rate of the catalytic step by at least 100-fold. Spermidine also reduces the magnesium requirement for the reaction and strongly stimulates activity at limiting Mg2+ concentrations. There are no special ionic requirements for formation of the initial ribozyme-substrate complex--analysis of complex formation using native gels and kinetic assays shows that the ribozyme can bind substrate in 40 mM Tris-HCl buffer. Complex formation is inhibited by both Mn2+ and Co2+. Ionic requirements for the ribozyme-catalyzed ligation reaction are very similar to those for the cleavage reaction. We propose a model for catalysis by the hairpin ribozyme that is consistent with these findings. Formation of an initial ribozyme-substrate complex occurs without the obligatory involvement of divalent cations. Ions (e.g., Mg2+) can then bind to form a catalytically proficient complex, which reacts and dissociates.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates.

Substrate recognition by the hairpin ribozyme has been proposed to involve two short intermolecular helices, termed helix 1 and helix 2. We have used a combination of three methods (cleavage of mismatched substrates, in vitro selection, and site-specific mutational analysis) to systematically determine the substrate recognition rules for this RNA enzyme. Assays measuring substrate cleavage in trans under multiple turnover conditions were conducted using the wild-type ribozyme and substrates containing mismatches in all sites potentially recognized by the ribozyme. Molecules containing single- and multiple-base mismatches in helix 2 at sites distant from the cleavage site (g-4c, u-5a, g-4c: u-5a) were cleaved with reduced efficiency, whereas those with mismatches proximal to the cleavage site (c-2a, a-3c, c-2a: a-3c) were not cut. Analogous results were obtained for helix 1, where mismatches distal from the cleavage site (u+7a, u+8a, u+9a, u+7a: u+8a: u+9a) were used much more efficiently than those proximal to the cleavage site (c+4a, u-5a, g+6c, c+4a: u+5a: g+6c). In vitro selection experiments were carried out to identify active variants from populations of molecules in which either helix 1 or helix 2 was randomized. Results constitute an artificial phylogenetic data base that proves base-pairing of nucleotides at five positions within helix 1 and three positions within helix 2 and reveals a significant sequence bias at 3 bp (c+4.G6, c-2.G11, and a-3.U12). This sequence bias was confirmed at two sites by measuring relative cleavage rates of all 16 possible dinucleotide combinations at base pairs c+4.G6 and c-2.G11.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro selection and evolution of RNA: applications for catalytic RNA, molecular recognition, and drug discovery.

In vitro selection and in vitro evolution methods represent powerful tools for isolating functional RNA molecules, and are proving to have wide applications in biology. Selection in the absence of living cells is possible because some RNA molecules possess a selectable "phenotype" (catalytic activity or ligand binding) as well as a "genotype" (nucleotide sequence). This review discusses the basic principles of in vitro selection technology and the application of these methods to isolate RNA molecules with interesting and novel properties. Selection techniques have been used to analyze the structure and function of catalytic RNA molecules (ribozymes), and to isolate novel catalytic structures not found in nature. They are also useful for studying protein-RNA interactions and for isolating RNA molecules that bind specifically to peptides and other ligands. The isolation of RNA molecules with new binding functionalities (aptamers) for both large and small molecules has exciting potential for discovery of new drugs and diagnostic reagents.