Binding and cleavage of nucleic acids by the "hairpin" ribozyme

The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+.     

The role of magnesium ions and 2'-hydroxyl groups in the VS ribozyme-substrate interaction

The minimal substrate of the trans-cleaving Neurospora VS ribozyme has a stem-loop structure and interacts with the ribozyme by RNA tertiary interactions that remain only partially defined. The magnesium ion dependence of the catalytic parameters of a trans-cleaving VS-derived ribozyme were studied. The turnover number of the catalytic RNA was found to depend on the binding of at least three magnesium ions, with an apparent magnesium ion dissociation constant of 16mM, but K(M) was observed to be metal ion independent in the millimolar range. To address the role of 2'-hydroxyl groups of the VS substrate RNA in interactions with the ribozyme, 23 altered substrates, each with a single 2'-deoxyribonucleoside substitution, were synthesised and their kinetic properties in the VS ribozyme reaction were analysed. The removal of five 2'-hydroxyl groups, at positions G620, A621, U628, C629 and G630 inhibited the reaction, whereas at two sites, G623 and A639, reaction was stimulated by the modification. Substitution of G620 with a 2'-deoxynucleoside was expected to inhibit the reaction, in line with the critical role of this 2'-hydroxyl group in the transesterification reaction. Altered substrates in which a 2'-O-methyl nucleoside replaced A621, U628, C629 and G630 were prepared and characterised. Although removal of the hydroxyl group of A621 inhibited the turnover number of the ribozyme significantly, this activity was recovered upon 2'-O-methyl adenosine substitution, suggesting that the 2'-oxygen atom of this nucleoside forms an important contact within the ribozyme active site. A cluster of residues within the loop region of the substrate, were more modestly affected by 2'-deoxynucleoside substitution. In two cases, magnesium binding was impaired, suggesting that stem-loop I is a possible magnesium ion binding site.     

Mutations in a nonconserved sequence of the Tetrahymena ribozyme increase activity and specificity

The RNA substrate-binding site of the Tetrahymena ribozyme is connected to the catalytic core by the joining region J1/2. Although J1/2 is not conserved among group I introns, small insertions or deletions in this sequence have dramatic effects, enhancing the turnover number and sequence specificity of ribozyme-catalyzed RNA cleavage. Measurements of rate constants for individual steps in the reaction have revealed the basis of these improvements. Ironically, the higher turnover and specificity both result from decreased affinity for RNA, rather than better cleavage. These results provide evidence that the nonconserved J1/2 sequence positions the RNA substrate to optimize tertiary interactions and ensure cleavage at the position corresponding to the 5' splice site. The wild-type RNA is well adapted to its biological function, and its limitations in multiple turnover can be corrected by mutation.     

A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability.

Hammerhead ribozymes cleave RNA substrates containing the UX sequence, where X = U, C or A, embedded within sequences which are complementary to the hybridising 'arms' of the ribozyme. In this study we have replaced the RNA in the hybridising arms of the ribozyme with DNA, and the resulting ribozyme is many times more active than its precursor. In turnover-kinetics experiments with a 13-mer RNA substrate, the kcat/Km ratios are 10 and 150 microM-1min-1 for the RNA- and DNA-armed ribozymes, respectively. The effect is due mainly to differences in kcat. In independent experiments where the cleavage step is rate-limiting, the DNA-armed ribozyme cleaves the substrate with a rate constant more than 3 times greater than the all-RNA ribozyme. DNA substrates containing a ribocytidine at the cleavage site have been shown to be cleaved less efficiently than their all-RNA analogues; again however, the DNA-armed ribozyme is more effective than the all-RNA ribozyme against such DNA substrates. These results demonstrate that there are no 2'-hydroxyl groups in the arms of the ribozyme that are required for cleavage; and that the structure of the complex formed by the DNA-armed ribozyme with its substrate is more favourable for cleavage than that formed by the all-RNA ribozyme and its substrate.     

Characterization of a local folding event of the Tetrahymena group I ribozyme: effects of oligonucleotide substrate length, pH, and temperature on the two substrate binding steps

Binding of the Tetrahymena group I ribozyme's oligonucleotide substrate occurs in two steps: P1 duplex formation with the ribozyme's internal guide sequence which forms an "open complex" is followed by docking of the P1 duplex into tertiary interactions within the catalytic core which forms a "closed complex". By systematically varying substrate length, pH, and temperature, we have identified conditions under which P1 duplex formation, P1 docking, or the chemical cleavage step limits the rate of the ribozyme reaction. This has enabled characterization of the individual steps as a function of substrate length, pH, and temperature, leading to several conclusions. (1) The rate constant for formation of the open complex is largely independent of substrate length, pH, and temperature, analogous to that of duplex formation in solution. This extends previous results suggesting that open complex formation entails mainly secondary structure formation and strengthens the view that the second binding step, P1 docking, represents a simple transition from secondary to tertiary structure in the context of an otherwise folded RNA. (2) The temperature dependence of the rate constant for P1 docking yields a negative activation entropy, in contrast to the positive entropy change previously observed for the docking equilibrium. This suggests a model in which tertiary interactions are not substantially formed in the transition state for P1 docking. (3) Shortening the substrate by three residues decreases the equilibrium constant for P1 docking by 200-fold, suggesting that the rigidity imposed by full-length duplex formation facilitates formation of tertiary interactions. (4) Once docked, shortened substrates are cleaved at rates within 3-fold of that for the full-length substrate. Thus, all the active site interactions required to accelerate the chemical cleavage event are maintained with shorter substrates. (5) The rate constant of approximately 10(3) min(-1) obtained for P1 docking is significantly faster than the other steps previously identified in the tertiary folding of this RNA. Nevertheless, P1 docking presumably follows other tertiary folding steps because the P1 duplex docks into a core that is formed only upon folding of the rest of the ribozyme.     

Mutational inhibition of ligation in the hairpin ribozyme: substitutions of conserved nucleobases A9 and A10 destabilize tertiary structure and selectively promote cleavage

The hairpin ribozyme acts as a reversible, site-specific endoribonuclease that ligates much more rapidly than it cleaves cognate substrate. While the reaction pathway for ligation is the reversal of cleavage, little is known about the atomic and electrostatic details of the two processes. Here, we report the functional consequences of molecular substitutions of A9 and A10, two highly conserved nucleobases located adjacent to the hairpin ribozyme active site, using G, C, U, 2-aminopurine, 2,6-diaminopurine, purine, and inosine. Cleavage and ligation kinetics were analyzed, tertiary folding was monitored by hydroxyl radical footprinting, and interdomain docking was studied by native gel electrophoresis. We determined that nucleobase substitutions that exhibit significant levels of interference with tertiary folding and interdomain docking have relatively large inhibitory effects on ligation rates while showing little inhibition of cleavage. Indeed, one variant, A10G, showed a fivefold enhancement of cleavage rate and no detectable ligation, and we suggest that this property may be uniquely well suited to intracellular targeted RNA cleavage applications. Results support a model in which formation of a kinetically stable tertiary structure is essential for ligation of the hairpin ribozyme, but is not necessary for cleavage.     

Crystal structure of the leadzyme at 1.8 A resolution: metal ion binding and the implications for catalytic mechanism and allo site ion regulation

The leadzyme is a small ribozyme, derived from in vitro selection, which catalyzes site specific, Pb(2+)-dependent RNA cleavage. Pb(2+) is required for activity; Mg(2+) inhibits activity, while many divalent and trivalent ions enhance it. The leadzyme structure consists of an RNA duplex interrupted by a trinucleotide bulge. Here, crystal structures determined to 1.8 A resolution, both with Mg(2+) as the sole divalent counterion and with Mg(2+) and Sr(2+) (which mimics Pb(2+) with respect to binding but not catalysis), reveal the metal ion interactions with both the ground state and precatalytic conformations of the leadzyme. Mg(H(2)O)(6)(2+) ions bridge complementary strands of the duplex at multiple locations by binding tandem purines of one RNA strand in the major groove. At one site, Mg(H(2)O)(6)(2+) ligates the phosphodiester backbone of the trinucleotide bulge in the ground state conformation, but not in the precatalytic conformation, suggesting (a) Mg(2+) may inhibit leadzyme activity by stabilizing the ground state and (b) metal ions which displace Mg(2+) from this site may activate the leadzyme. Binding of Sr(2+) to the presumed catalytic Pb(2+) site in the precatalytic leadzyme induces local structural changes in a manner that would facilitate alignment of the catalytic ribose 2'-hydroxyl with the scissile bond for cleavage. These data support a model wherein binding of a catalytic ion to a precatalytic conformation of the leadzyme, in conjunction with the flexibility of the trinucleotide bulge, may facilitate structural rearrangements around the scissle phosphodiester bond favoring configurations that allow bond cleavage.     

Recognition elements for 5' exon substrate binding to the Candida albicans group I intron

A group I intron precursor and ribozyme were cloned from the large subunit rRNA of the human pathogen Candida albicans. Both the precursor and ribozyme are functional as determined from in vitro assays. Comparisons of dissociation constants for oligonucleotide binding to the ribozyme and to a hexanucleotide mimic of its internal guide sequence lead to a model for recognition of the 5' exon substrate by this intron. In particular, tertiary contacts with the P1 helix that help align the splice site include three 2'-hydroxyl groups, a G.U pair that occurs at the intron's splice junction, and a G.A pair. The free energy contribution that each interaction contributes to tertiary binding is determined. When the G.A pair is replaced with a G-C pair, tertiary interactions to 5' exon mimic 2'-hydroxyl groups are significantly weakened. When the G.A pair is replaced with a G.U pair, tertiary interactions are retained and binding is 10-fold tighter. These results expand our knowledge of substrate recognition by group I introns, and also provide a basis for rational design of oligonucleotide-based therapeutics for targeting group I introns by binding enhancement by tertiary interactions and suicide inhibition strategies.     

Efficient cleavage of pre-tRNAs by E. coli RNAse P RNA requires the 2'-hydroxyl of the ribose at the cleavage site.

RNAse P cleaves pre-tRNAs to liberate 5'-flanks and 5'-matured, 5'-phosphorylated tRNAs. It is not evident if the 2'-hydroxyls of the ribose moieties in the substrate are involved in the reaction. To study their influence in two different pre-tRNAs, we have modified specifically the 2'-hydroxyl groups at the cleavage site and in neighbouring positions. We have shown that these hydroxyls are important but not essential for the processing of these substrates by E. coli RNase P RNA (M1 RNA). The reduction in the catalytic efficiency was moderate for 2'-deoxy and severe for 2'-methoxy substitutions at the cleavage site. Additional effects of modifications in neighbouring positions were smaller. Based on our data we suggest that the modifications do not interfere with binding of the substrate, whereas they prevent an optimal steric arrangement for the hydrolysis reaction.     

The P5abc peripheral element facilitates preorganization of the tetrahymena group I ribozyme for catalysis

Phylogenetic comparisons and site-directed mutagenesis indicate that group I introns are composed of a catalytic core that is universally conserved and peripheral elements that are conserved only within intron subclasses. Despite this low overall conservation, peripheral elements are essential for efficient splicing of their parent introns. We have undertaken an in-depth structure-function analysis to investigate the role of one of these elements, P5abc, using the well-characterized ribozyme derived from the Tetrahymena group I intron. Structural comparisons using solution-based free radical cleavage revealed that a ribozyme lacking P5abc (E(DeltaP5abc)) and E(DeltaP5abc) with P5abc added in trans (E(DeltaP5abc).P5abc) adopt a similar global tertiary structure at Mg(2+) concentrations greater than 20 mM [Doherty, E. A., et al. (1999) Biochemistry 38, 2982-90]. However, free E(DeltaP5abc) is greatly compromised in overall oligonucleotide cleavage activity, even at Mg(2+) concentrations as high as 100 mM. Further characterization of E(DeltaP5abc) via DMS modification revealed local structural differences at several positions in the conserved core that cluster around the substrate binding sites. Kinetic and thermodynamic dissection of individual reaction steps identified defects in binding of both substrates to E(DeltaP5abc), with > or =25-fold weaker binding of a guanosine nucleophile and > or =350-fold weaker docking of the oligonucleotide substrate into its tertiary interactions with the ribozyme core. These defects in binding of the substrates account for essentially all of the 10(4)-fold decrease in overall activity of the deletion mutant. Together, the structural and functional observations suggest that the P5abc peripheral element not only provides stability but also positions active site residues through indirect interactions, thereby preferentially stabilizing the active ribozyme structure relative to alternative less active states. This is consistent with the view that peripheral elements engage in a network of mutually reinforcing interactions that together ensure cooperative folding of the ribozyme to its active structure.     

Conformational heterogeneity at position U37 of an all-RNA hairpin ribozyme with implications for metal binding and the catalytic structure of the S-turn

The hairpin ribozyme is an RNA enzyme that performs site-specific phosphodiester bond cleavage between nucleotides A-1 and G+1 within its cognate substrate. Previous functional studies revealed that the minimal hairpin ribozyme exhibited "gain-of-function" cleavage properties resulting from U39C or U39 to propyl linker (C3) modifications. Furthermore, each "mutant" displayed different magnesium-dependence in its activity. To investigate the molecular basis for these gain-of-function variants, crystal structures of minimal, junctionless hairpin ribozymes were solved in native (U39), and mutant U39C and U39(C3) forms. The results revealed an overall molecular architecture comprising two docked internal loop domains folded into a wishbone shape, whose tertiary interface forms a sequestered active site. All three minimal hairpin ribozymes bound Co(NH(3))(6)(3+) at G21/A40, the E-loop/S-turn boundary. The native structure also showed that U37 of the S-turn adopts both sequestered and exposed conformations that differ by a maximum displacement of 13 A. In the sequestered form, the U37 base packs against G36, and its 2'-hydroxyl group forms a water mediated hydrogen bond to O4' of G+1. These interactions were not observed in previous four-way-junction hairpin ribozyme structures due to crystal contacts with the U1A splicing protein. Interestingly, the U39C and U39(C3) mutations shifted the equilibrium conformation of U37 into the sequestered form through formation of new hydrogen bonds in the S-turn, proximal to the essential nucleotide A38. A comparison of all three new structures has implications for the catalytically relevant conformation of the S-turn and suggests a rationale for the distinctive metal dependence of each mutant.     

The hairpin ribozyme

The hairpin ribozyme is a member of a family of small RNA endonucleases, which includes hammer-head, human hepatitis delta virus, Neurospora VS, and the lead-dependent catalytic RNAs. All these catalytic RNAs reversibly cleave the phosphodiester bond of substrate RNA to generate 5'-hydroxyl and 2',3'-cyclic phosphate termini. Whereas the reaction products from family members are similar, large structural and mechanistic differences exist. Structurally the hairpin ribozyme has two principal domains that interact to facilitate catalysis. The hairpin ribozyme uses a catalytic mechanism that does not require metals for cleavage or ligation of substrate RNA. In this regard it is presently unique among RNA catalysts. Targeting rules for cleavage of substrate have been determined and required bases for catalysis have been identified. The hairpin ribozyme has been developed and used for gene therapy and was the first ribozyme to be approved for human clinical trials.     

Guiding ribozyme cleavage through motif recognition: the mechanism of cleavage site selection by a group ii intron ribozyme

The mechanism by which group II introns cleave the correct phosphodiester linkage was investigated by studying the reaction of mutant substrates with a ribozyme derived from intron ai5gamma. While fidelity was found to be quite high in most cases, a single mutation on the substrate (+1C) resulted in a dramatic loss of fidelity. When this mutation was combined with a second mutation that induces a bulge in the exon binding site 1/intron binding site 1 (EBS1/IBS1) duplex, the base-pairing register of the EBS1/IBS1 duplex was shifted and the cleavage site moved to a downstream position on the substrate. Conversely, when mismatches were incorporated at the EBS1/IBS1 terminus, the duplex was effectively truncated and cleavage occurred at an upstream site. Taken together, these data demonstrate that the cleavage site of a group II intron ribozyme can be tuned at will by manipulating the thermodynamic stability and structure of the EBS1/IBS1 pairing. The results are consistent with a model in which the cleavage site is not designated through recognition of specific nucleotides (such as the 5'-terminal residue of EBS1). Instead, the ribozyme detects a structure at the junction between single and double-stranded residues on the bound substrate. This finding explains the puzzling lack of phylogenetic conservation in ribozyme and substrate sequences near group II intron target sites.     

Molecular recognition by the Candida albicans group I intron: tertiary interactions with an imino G.A pair facilitate binding of the 5' exon and lower the KM for guanosine

A G.A pair at position -5 in the P1 helix of the Candida albicans ribozyme contributes to tertiary binding of the 5' exon substrate [Disney, M. D., Haidaris, C. G., and Turner, D. H. (2001) Biochemistry 40, 6507-6519]. Here, the G in the G.A pair is replaced with inosine (I) in both semisynthetic ribozymes and oligonucleotide mimics of the internal guide sequence. Comparisons of oligonucleotide binding affinity for these and other sequences indicate that the G.A pair is in an imino conformation where the exocyclic amine of G contributes approximately 1.4 kcal/mol to tertiary interactions that help dock the ribozyme's P1 helix. Furthermore, replacement of the G.A pair with a G-C pair produces less favorable interactions with the 2'-hydroxyl group at the -3 position and a less favorable K(M) for pG in a ribozyme-catalyzed transesterification reaction. These results are also consistent with the G.A pair promoting docking of the P1 helix into the catalytic core. Evidently, tertiary interactions with the exocyclic amino group of a G in a single G.A pair can increase the equilibrium constant for tertiary folding of RNA by roughly 10-fold at 37 degrees C. Results with a G.U or G.G pair replacing the G.A pair at the -5 position suggest similar tertiary interactions with these pairs.     

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.     

Characterization of the Endoribonuclease Active Site of Human Apurinic/Apyrimidinic Endonuclease 1

Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalian enzyme in DNA base excision repair that cleaves the DNA phosphodiester backbone immediately 5′ to abasic sites. Recently, we identified APE1 as an endoribonuclease that cleaves a specific coding region of c-myc mRNA in vitro, regulating c-myc mRNA level and half-life in cells. Here, we further characterized the endoribonuclease activity of APE1, focusing on the active-site center of the enzyme previously defined for DNA nuclease activities. We found that most site-directed APE1 mutant proteins (N68A, D70A, Y171F, D210N, F266A, D308A, and H309S), which target amino acid residues constituting the abasic DNA endonuclease active-site pocket, showed significant decreases in endoribonuclease activity. Intriguingly, the D283N APE1 mutant protein retained endoribonuclease and abasic single-stranded RNA cleavage activities, with concurrent loss of apurinic/apyrimidinic (AP) site cleavage activities on double-stranded DNA and single-stranded DNA (ssDNA). The mutant proteins bound c-myc RNA equally well as wild-type (WT) APE1, with the exception of H309N, suggesting that most of these residues contributed primarily to RNA catalysis and not to RNA binding. Interestingly, both the endoribonuclease and the ssRNA AP site cleavage activities of WT APE1 were present in the absence of Mg²⁺, while ssDNA AP site cleavage required Mg²⁺ (optimally at 0.5-2.0 mM). We also found that a 2′-OH on the sugar moiety was absolutely required for RNA cleavage by WT APE1, consistent with APE1 leaving a 3′-PO₄²⁻ group following cleavage of RNA. Altogether, our data support the notion that a common active site is shared for the endoribonuclease and other nuclease activities of APE1; however, we provide evidence that the mechanisms for cleaving RNA, abasic single-stranded RNA, and abasic DNA by APE1 are not identical, an observation that has implications for unraveling the endoribonuclease function of APE1 in vivo.     

Identification and characterization of a divalent metal ion-dependent cleavage site in the hammerhead ribozyme.

We describe a new RNA cleavage motif, found in the hammerhead ribozyme. Cleavage occurs between nucleotides G8 and A9, yielding a free 5'-hydroxyl group and a 2',3'-cyclic phosphate. This cleavage is dependent upon divalent metal ions and is the first evidence for a metalloribozyme known to show preference for Zn(2+). Cleavage is also observed in the presence of Ni(2+), Co(2+), Mn(2+), Cd(2+), and Pb(2+), while negligible cleavage was detected in the presence of the alkaline-earth metal ions Mg(2+), Ca(2+), Sr(2+), and Ba(2+). A linear relationship between the logarithm of the rate and pH was observed for the Zn(2+)-dependent cleavage, which is indicative of proton loss in the cleavage mechanism, either prior to or in the rate-determining step. We postulate that a zinc hydroxide complex, bound to the known A9/G10.1 metal ion binding site, abstracts the proton from the 2'-hydroxyl group of G8, which attacks the A9 phosphate and initiates cleavage. This hypothesis is supported by a previously reported crystal structure [Murray, J. B., Terwey, D. P., Maloney, L., Karpeisky, A., Usman, N., Beigelman, L., and Scott, W. G. (1998) Cell 92, 665-673], which shows the conformation required for RNA cleavage and proximity of the 2'-hydroxyl group to the metal ion complex.     

Functional group requirements in the probable active site of the VS ribozyme

The VS ribozyme catalyses the site-specific cleavage of a phosphodiester linkage by a transesterification reaction that entails the attack of the neighbouring 2'-oxygen with departure of the 5'-oxygen. We have previously suggested that the A730 loop is an important component of the active site of the ribozyme, and that A756 is especially important in the cleavage reaction. Functional group modification experiments reported here indicate that the base of A756 is more important than its ribose for catalysis. A number of changes to the base, including complete ablation, lead to cleavage rates that are reduced 1000-fold, while removal of the 2'-hydroxyl group from the ribose results in tenfold slower cleavage. 2-Aminopurine fluorescence experiments indicate that this 2'-hydroxyl group is important for the structure of the A730 loop. Catalytic activity is especially sensitive to changes involving the exocyclic amine of A756; by contrast, the cleavage activity is only weakly sensitive to modification at the 7-position of the purine nucleus. These results suggest that the Watson-Crick edge of the adenine base is important in ribozyme function. We sought to test the possibility of a direct role of the nucleobase in the chemistry of the cleavage reaction. Addition of imidazole base in the medium failed to restore the activity of a ribozyme from which the nucleobase of A756 was removed. However, no restoration was obtained with exogenous adenine base either, indicating that the cavity that might result from ablation of the base was closed.     

Mutational analysis of the antigenomic trans-acting delta ribozyme: the alterations of the middle nucleotides located on the P1 stem

Our previous report on delta ribozyme cleavage using a trans -acting antigenomic delta ribozyme and a collection of short substrates showed that the middle nucleotides of the P1 stem, the substrate binding site, are essential for the cleavage activity. Here we have further investigated the effect of alterations in the P1 stem on the kinetic and thermodynamic parameters of delta ribozyme cleavage using various ribozyme variants carrying single base mutations at putative positions reported. The kinetic and thermodynamic values obtained in mutational studies of the two middle nucleotides of the P1 stem suggest that the binding and active sites of the delta ribozyme are uniquely formed. Firstly, the substrate and the ribozyme are engaged in the formation of a helix, known as the P1 stem, which may contain a weak hydrogen bond(s) or a bulge. Secondly, a tertiary interaction involving the base moieties in the middle of the P1 stem likely plays a role in defining the chemical environment. As a con-sequence, the active site might form simultaneously or subsequently to the binding site during later steps of the pathway.     

Addition of an extra substrate binding site and partial destabilization of stem structures in HDV ribozyme give rise to high sequence-specificity for its target RNA

Because the substrate binding site (P1) of HDV ribozyme consists of only seven nucleotides, cleavage of undesired RNA is likely to occur when applied for a specific long RNA target such as mRNA. To overcome this problem, we designed modified trans-acting HDV ribozymes with an extra substrate-binding site (P5) in addition to the original binding site (P1). By inserting an additional seven base-pair stem (P5 stem) into the J1/2 single-stranded region of the ribozyme core system and partial destabilization of the P2 or P4 stem, we succeeded in preparation of new HDV ribozymes that can cleave the target RNA depending on the formation of P5 stem. Moreover, the ribozyme with a six-nucleotide P1 site was able to distinguish the substrate RNA with a complete match from that with a single mismatch in the P1 region. These results suggest that the HDV ribozyme system is useful for the application in vivo.