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A number of small, self-cleaving ribozyme classes have been identified including the hammerhead, hairpin, hepatitis delta virus (HDV), Varkud satellite (VS), glmS, twister, hatchet, pistol, and twister sister ribozymes. Within the active sites of these ribozymes, myriad functional groups contribute to catalysis. There has been extensive structure-function analysis of individual ribozymes, but the extent to which catalytic devices are shared across different ribozyme classes is unclear. As such, emergent catalytic principles for ribozymes may await discovery. Identification of conserved catalytic devices can deepen our understanding of RNA catalysis specifically and of enzymic catalysis generally. To probe similarities and differences amongst ribozyme classes, active sites from more than 80 high-resolution crystal structures of self-cleaving ribozymes were compared computationally. We identify commonalities amongst ribozyme classes pertaining to four classic catalytic devices: deprotonation of the 2′OH nucleophile (γ), neutralization of the non-bridging oxygens of the scissile phosphate (β), neutralization of the O5′ leaving group (δ), and in-line nucleophilic attack (α). In addition, we uncover conservation of two catalytic devices, each of which centers on the activation of the 2′OH nucleophile by a guanine: one to acidify the 2′OH by hydrogen bond donation to it (γ′) and one to acidify the 2′OH by releasing it from non-productive interactions by competitive hydrogen bonding (γ′′). Our findings reveal that the amidine functionalities of G, A, and C are especially important for these strategies, and help explain absence of U at ribozyme active sites. The identified γ′ and γ′′ catalytic strategies help unify the catalytic strategies shared amongst catalytic RNAs and may be important for large ribozymes, as well as protein enzymes that act on nucleic acids.

Protein mutants are studied in a variety of contexts in the life sciences. However, individual mutations need to be generated in order to transcribe and translate the respective protein variants. Here, we introduce a novel strategy for controlling the incorporation of different amino acids in response to an amber stop codon by utilizing switchable designer transfer RNAs in Escherichia coli .

RNA possesses great potential for expanding the toolbox currently available to synthetic biologists. Here, the modulation of the Hepatitis Delta Virus ribozyme's activity with a series of rationally designed aptamers and effector RNA oligonucleotides is described. The ribozyme was initially fused with an 18-nucleotide hairpin structure that abolished its self-cleaving activity. The binding of a 14-mer oligonucleotide to the hairpin rescued the self-cleavage in a concentration-dependent manner. This modified ribozyme was inserted into the 5' UTR of a reporter gene, and the resulting construct was used to demonstrate that it is possible to modulate the ribozyme activity in cellulo with the oligonucleotide. Subsequently, ribozymes possessing specialized aptamers respecting other logic gates were also successfully designed and found to be functional in vitro. To our knowledge, this is the first example of HDV ribozyme regulation by oligonucleotides, as well as the first allosteric regulation of HDV ribozyme in mammalian cells.

We have investigated the role of a single-stranded RNA junction, J1/2, that connects the substrate-containing P1 duplex to the remainder of the Tetrahymena group I ribozyme. Single-turnover kinetics, fluorescence anisotropy, and single-molecule fluorescence resonance energy transfer studies of a series of J1/2 mutants were used to probe the sequence dependence of the catalytic activity, the P1 dynamics, and the thermodynamics of docking of the P1 duplex into the ribozyme's catalytic core. We found that A29, the center A of three adenosine residues in J1/2, contributes 2 orders of magnitude to the overall ribozyme activity, and double-mutant cycles suggested that J1/2 stabilizes the docked state of P1 over the undocked state via a tertiary interaction involving A29 and the first base pair in helix P2 of the ribozyme, A31·U56. Comparative sequence analysis of this group I intron subclass suggests that the A29 interaction sets one end of a molecular ruler whose other end specifies the 5'-splice site and that this molecular ruler is conserved among a subclass of group I introns related to the Tetrahymena intron. Our results reveal substantial functional effects from a seemingly simple single-stranded RNA junction and suggest that junction sequences may evolve rapidly to provide important interactions in functional RNAs.

The hepatitis delta virus (HDV) ribozyme and related RNAs are widely dispersed in nature. This RNA is a small nucleolytic ribozyme that self-cleaves to generate products with a 2',3'-cyclic phosphate and a free 5'-hydroxyl. Although small ribozymes are dependent on divalent metal ions under biologically relevant buffer conditions, they function in the absence of divalent metal ions at high ionic strengths. This characteristic suggests that a functional group within the covalent structure of small ribozymes is facilitating catalysis. Structural and mechanistic analyses have demonstrated that the HDV ribozyme active site contains a cytosine with a perturbed pK(a) that serves as a general acid to protonate the leaving group. The reaction of the HDV ribozyme in monovalent cations alone never approaches the velocity of the Mg(2+)-dependent reaction, and there is significant biochemical evidence that a Mg(2+) ion participates directly in catalysis. A recent crystal structure of the HDV ribozyme revealed that there is a metal binding pocket in the HDV ribozyme active site. Modeling of the cleavage site into the structure suggested that this metal ion can interact directly with the scissile phosphate and the nucleophile. In this manner, the Mg(2+) ion can serve as a Lewis acid, facilitating deprotonation of the nucleophile and stabilizing the conformation of the cleavage site for in-line attack of the nucleophile at the scissile phosphate. This catalytic strategy had previously been observed only in much larger ribozymes. Thus, in contrast to most large and small ribozymes, the HDV ribozyme uses two distinct catalytic strategies in its cleavage reaction.

The hepatitis delta virus (HDV) ribozyme and HDV-like ribozymes are self-cleaving RNAs found throughout all kingdoms of life. These RNAs fold into a double-nested pseudoknot structure and cleave RNA, yielding 2',3'-cyclic phosphate and 5'-hydroxyl termini. The active site nucleotide C75 has a pK(a) shifted2 pH units toward neutrality and has been implicated as a general acid/base in the cleavage reaction. An active site Mg(2+) ion that helps activate the 2'-hydroxyl for nucleophilic attack has been characterized biochemically; however, this ion has not been visualized in any previous structures. To create a snapshot of the ribozyme in a state poised for catalysis, we have crystallized and determined the structure of the HDV ribozyme bound to an inhibitor RNA containing a deoxynucleotide at the cleavage site. This structure includes the wild-type C75 nucleotide and Mg(2+) ions, both of which are required for maximal ribozyme activity. This structure suggests that the position of C75 does not change during the cleavage reaction. A partially hydrated Mg(2+) ion is also found within the active site where it interacts with a newly resolved G.U reverse wobble. Although the inhibitor exhibits crystallographic disorder, we modeled the ribozyme-substrate complex using the conformation of the inhibitor strand observed in the hammerhead ribozyme. This model suggests that the pro-R(P) oxygen of the scissile phosphate and the 2'-hydroxyl nucleophile are inner-sphere ligands to the active site Mg(2+) ion. Thus, the HDV ribozyme may use a combination of metal ion Lewis acid and nucleobase general acid strategies to effect RNA cleavage.

Self-cleaving RNAs have recently been identified in mammalian genomes. A small ribozyme related in structure to the hepatitis delta virus (HDV) ribozyme occurs in a number of mammals, including chimpanzees and humans, within an intron of the CPEB3 gene. The catalytic mechanisms for the CPEB3 and HDV ribozymes appear to be similar, generating cleavage products with 5'-hydroxyl and 2',3'-cyclic phosphate termini; nonetheless, the cleavage rate reported for the CPEB3 ribozyme is more than 6000-fold slower than for the fastest HDV ribozyme. Herein, we use full-length RNA and cotranscriptional self-cleavage assays to compare reaction rates among human CPEB3, chimp CPEB3, and HDV ribozymes. Our data reveal that a single base change of the upstream flanking sequence, which sequesters an intrinsically weak P1.1 pairing in a misfold, increases the rate of the wild-type human CPEB3 ribozyme by approximately 250-fold; thus, the human ribozyme is intrinsically fast-reacting. Secondary structure determination and native gel analyses reveal that the cleaved population of the CPEB3 ribozyme has a single, secondary structure that closely resembles the HDV ribozyme. In contrast, the precleavage population of the CPEB3 ribozyme appears to have a more diverse secondary structure, possibly reflecting misfolding with the upstream sequence and dynamics intrinsic to the ribozyme. Prior identification of expressed sequence tags (ESTs) in human cells indicated that cleavage activity of the human ribozyme is tissue-specific. It is therefore possible that cellular factors interact with regions upstream of the CPEB3 ribozyme to unmask its high intrinsic reactivity.

The HDV ribozyme self-cleaves by a chemical mechanism involving general acid-base catalysis to generate a 2′,3′-cyclic phosphate and a 5′-hydroxyl termini. Biochemical studies from several laboratories have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pKa shifted > 2 pH units toward neutrality [Gong, B., Chen, J. H., Chase, E., Chadalavada, D. M., Yajima, R., Golden, B. L., Bevilacqua, P. C., and Carey, P. R. (2007) J. Am. Chem. Soc. 129, 13335–13342.], while in crystal structures, it is well-positioned for proton transfer. However no crystallographic evidence for a hydrated magnesium poised to serve as a general base in the reaction has been observed in high-resolution crystal structures of various reaction states and mutants. Herein, we use solution kinetic experiments and parallel Raman crystallographic studies to examine the effects of pH on rate and Mg2+-binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously-unobserved hydrated magnesium ion interacts with the N7 of the cleavage site G•U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochemical data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochemical data.

The hammerhead ribozyme from Schistosoma mansoni is the best characterized of the natural hammerhead ribozymes. Biophysical, biochemical, and structural studies have shown that the formation of the loop-loop tertiary interaction between stems I and II alters the global folding, cleavage kinetics, and conformation of the catalytic core of this hammerhead, leading to a ribozyme that is readily cleaved under physiological conditions. This study investigates the ligation kinetics and the internal equilibrium between cleavage and ligation for the Schistosoma hammerhead. Single turnover kinetic studies on a construct where the ribozyme cleaves and ligates substrate(s) in trans showed up to 23% ligation when starting from fully cleaved products. This was achieved by an approximately 2000-fold increase in the rate of ligation compared to a minimal hammerhead without the loop-loop tertiary interaction, yielding an internal equilibrium that ranges from 2 to 3 at physiological Mg2+ ion concentrations (0.1-1 mM). Thus, the natural Schistosoma hammerhead ribozyme is almost as efficient at ligation as it is at cleavage. The results here are consistent with a model where formation of the loop-loop tertiary interaction leads to a higher population of catalytically active molecules and where formation of this tertiary interaction has a much larger effect on the ligation than the cleavage activity of the Schistosoma hammerhead ribozyme.

The DEAD-box protein CYT-19 functions in the folding of several group I introns in vivo and a diverse set of group I and group II RNAs in vitro. Recent work using the Tetrahymena group I ribozyme demonstrated that CYT-19 possesses a second RNA-binding site, distinct from the unwinding active site, which enhances unwinding activity by binding nonspecifically to the adjacent RNA structure. Here, we probe the region of CYT-19 responsible for that binding by constructing a C-terminal truncation variant that lacks 49 amino acids and terminates at a domain boundary, as defined by limited proteolysis. This truncated protein unwinds a six-base-pair duplex, formed between the oligonucleotide substrate of the Tetrahymena ribozyme and an oligonucleotide corresponding to the internal guide sequence of the ribozyme, with near-wild-type efficiency. However, the truncated protein is activated much less than the wild-type protein when the duplex is covalently linked to the ribozyme or single-stranded or double-stranded extensions. Thus, the active site for RNA unwinding remains functional in the truncated CYT-19, but the site that binds the adjacent RNA structure has been compromised. Equilibrium binding experiments confirmed that the truncated protein binds RNA less tightly than the wild-type protein. RNA binding by the compromised site is important for chaperone activity, because the truncated protein is less active in facilitating the folding of a group I intron that requires CYT-19 in vivo. The deleted region contains arginine-rich sequences, as found in other RNA-binding proteins, and may function by tethering CYT-19 to structured RNAs, so that it can efficiently disrupt exposed, non-native structural elements, allowing them to refold. Many other DExD/H-box proteins also contain arginine-rich ancillary domains, and some of these domains may function similarly as nonspecific RNA-binding elements that enhance general RNA chaperone activity.

The hammerhead ribozyme has been intensively studied for approximately 15 years, but its cleavage mechanism is not yet understood. Crystal structures reveal a Y-shaped molecule in which the cleavage site is not ideally aligned for an S N2 reaction and no RNA functional groups are positioned appropriately to perform the roles of acid and base or other functions in the catalysis. If the ribozyme folds to a more compact structure in the transition state, it probably does so only transiently. We have used photocrosslinking as a tool to trap hammerhead ribozyme-substrate complexes in various stages of folding. Results suggest that the two substrate residues flanking the cleavage site approach and stack upon two guanosines (G8 and G12) in domain 2, moving 10-15 A closer to domain 2 than they appear in the crystal structure. Most crosslinks obtained with the nucleotide analogues positioned in the ribozyme core are catalytically inactive; however, one cobalt(III) hexaammine-dependent crosslink of an unmodified ribozyme retains catalytic activity and confirms the close stacking of cleavage site residue C17 with nucleotide G8 in domain 2. These findings suggest that residues involved in the chemistry of hammerhead catalysis are likely located in that region containing G8 and G12. The hammerhead ribozyme is a member of a class of small catalytic RNAs (including the hairpin, hepatitis ‰, Neuro- spora VS, and newly discovered bacterial GlmS ribozyme) (1), which carry out reversible cleavage of RNA, generating products containing 2',3'-cyclic phosphate and 5'-OH termini. The hammerhead cleavage reaction results in inversion of the configuration of the phosphate and thus is presumed to occur by deprotonation of the 2'-OH group, followed by in- line SN2 attack of the 2'-O - upon the phosphate (2, 3). The cleavage rate of hammerhead ribozymes increases in log-linear fashion with increasing pH, and thus, early investigations centered on the likelihood of a catalytic magnesium acting at the cleavage site (4). However, it was discovered that the cleavage reaction could be mediated (albeit at around a 10-40-fold reduced rate) by high concentrations of monovalent ions (5-7). These findings suggest that divalent ions serve primarily to promote folding of the ribozyme. A directly coordinated divalent ion might play a role in driving formation of the active ribozyme structure and/or in providing a favorable electrostatic envi- ronment at the active site. On the other hand, the activity of the ribozyme in monovalent ions suggests that a general acid-base mechanism may be utilized in the reaction and that important roles in the catalysis may be carried out by functional groups of the RNA itself.

The "RNA world" hypothesis proposes that early in the evolution of life, before the appearance of DNA or protein, RNA was responsible both for encoding genetic information and for catalyzing biochemical reactions. Ribo-organisms living in the RNA world would have replicated their RNA genomes by using an RNA polymerase ribozyme. Efforts to provide experimental support for the RNA world hypothesis have focused on producing such a polymerase, and in vitro evolution methods have led to the isolation of a polymerase ribozyme that catalyzes primer extension which is accurate and general, but slow. To understand the reaction of this ribozyme, we developed a method of measuring polymerase processivity that is particularly useful in the case of an inefficient polymerase. This method allowed us to demonstrate that the polymerase ribozyme, despite its inefficiency, is partially processive. It is currently limited by a low affinity for the primer-template duplex, but once it successfully binds the primer-template duplex in the productive alignment, it catalyzes an extension reaction that is so rapid that it can occur multiple times during the short span of a single binding event. This finding contributes to the understanding of one of the more sophisticated activities yet to be generated de novo in the laboratory and sheds light on the parameters to be targeted for further optimization.

The hepatitis delta virus (HDV), an infectious human pathogen affecting millions of people worldwide, leads to intensified disease symptoms, including progression to liver cirrhosis upon coinfection with its helper virus, HBV. Both the circular RNA genome of HDV and its complementary antigenome contain a common cis-cleaving catalytic RNA motif, the HDV ribozyme, which plays a crucial role in viral replication. Previously, the crystal structure of the product form of the cis-acting genomic HDV ribozyme has been determined, and the precursor form has been suggested to be structurally similar. In contrast, solution studies by fluorescence resonance energy transfer (FRET) on a trans-cleaving form of the ribozyme have shown significant global conformational changes upon catalysis, while 2-aminopurine (AP) fluorescence assays have detected concomitant local conformational changes in the catalytic core. Here, we augment these studies by using terbium(III) to probe the structure of the trans-acting HDV ribozyme at nucleotide resolution. We observe significant structural differences between the precursor and product forms, especially in the P1.1 helix and the trefoil turn in the single-stranded region connecting P4 and P2 (termed J4/2) of the catalytic core. We show, using terbium(III) footprinting and sensitized luminescence spectroscopy as well as steady-state, time-resolved, and gel-mobility FRET assays on a systematic set of substrates, that the substrate sequence immediately 5 ' to the cleavage site significantly modulates these local as well as resultant global structural differences. Our results suggest a structural basis for the previously observed impact of the 5' substrate sequence on catalytic activity.

We demonstrate that a group I intron-derived ribozyme from the opportunistic pathogen Pneumocystis carinii can bind an RNA in trans and excise from within it an internal segment, resulting in the splicing of the remaining ends of the RNA back together (the trans excision-splicing reaction). The reaction is intramolecular with regard to substrate. The ribozyme targets its substrate by base pairing with two or three noncontiguous regions on the substrate, and the reaction occurs through a nucleotide cofactor independent mechanism. The excised segment can be as long as 28 nucleotides, or more, and as little as one nucleotide. The potential usefulness of this reaction is demonstrated by engineering a ribozyme that excises the triplet-repeat expansion region from a truncated myotonic dystrophy protein kinase transcript mimic in vitro.

The Hepatitis Delta Virus (HDV) ribozyme was the first RNA enzyme proposed to use a proton-transfer mechanism for catalysis. Previous biochemical evidence suggested that the genomic HDV ribozyme promotes cis-cleavage using cytosine 75 whose pK(a) is perturbed within the active site. Here we present further biochemical evidence for the involvement of C75 in proton transfer, as well as evidence to support a plausible mechanism for C75 pK(a) perturbation. Nucleotide analogue interference mapping (NAIM) experiments with C analogues having altered N3 pK(a)s demonstrate the importance of C75 ionization in the HDV cis-cleavage reaction. pH-dependent interference rescue with C analogues having enhanced N3 acidity indicates that C75 is the only cytidine residue that must be protonated for ribozyme activity. Furthermore, interference analysis with pseudoisocytidine, a charge-neutral mimic of a C with a protonated N3, shows a pattern consistent with proton transfer, possibly from the C75 N3 to the 5'-oxyanion leaving group during the cis-cleavage reaction. Strong pH-independent inhibition of ribozyme function also occurs at C75 with a C analogue that lacks the N4 amino group, implicating the exocyclic amine in critical interactions in the active site. Interactions with the amino group may play an important role in perturbing the C75 N3 pK(a). Protonation of C41 has been proposed to be important for ribozyme activity; however, no interference at C41 was observed in this analogue series, which argues against a functional role for C41 protonation. These data support a model wherein C75 of the genomic HDV ribozyme acts as a general acid during its cis-cleavage reaction, and provide a glimpse into how RNAs, in a manner similar to protein enzymes, might employ local environmental electronic modulation to catalyze reactions.

The bacterial RNase P ribozyme is a site-specific endonuclease that catalyzes the removal of pre-tRNA leader sequences to form the 5' end of mature tRNA. While several specific interactions between enzyme and substrate that direct this process have been determined, nucleotides on the ribozyme that interact directly with functional groups at the cleavage site are not well-defined. To identify individual nucleotides in the ribozyme that are in close proximity to the pre-tRNA cleavage site, we introduced the short-range photoaffinity cross-linking reagent 6-thioguanosine (s6G) at position +1 of tRNA and position -1 in a tRNA bearing a one-nucleotide leader sequence [tRNA(G-1)] and examined cross-linking in representatives of the two structural classes of bacterial RNase P RNA (from Escherichia coli and Bacillus subtilis). These photoagent-modified tRNAs bind with similar high affinity to both ribozymes, and the substrate bearing a single s6G upstream of the cleavage (-1) site is cleaved accurately. Interestingly, s6G at position +1 of tRNA cross-links with high efficiency to homologous positions in J5/15 in both E. coli and B. subtilis RNase P RNAs, while s6G at position -1 of tRNA(G-1) cross-links to homologous nucleotides in J18/2. Both cross-links are detected over a range of ribozyme and substrate concentrations, and importantly, ribozymes cross-linked to position -1 of tRNA(G-1) accurately cleave the covalently attached substrate. These data indicate that the conserved guanosine at the 5' end of tRNA is adjacent to A248 (E. coli) of J5/15, while the base upstream of the substrate phosphate is adjacent to G332 (E. coli) of J18/2 and, along with available biochemical data, suggest that these nucleotides play a direct role in binding the substrate at the cleavage site.

The ability of delta ribozyme to catalyze the cleavage of an 11-mer RNA substrate was examined under both single- and multiple-turnover conditions. In both cases only small differences in the kinetic parameters were observed in the presence of either magnesium or calcium as cofactor. Under multiple-turnover conditions, the catalytic efficiency of the ribozyme (kcat/KM) was higher at 37 degreesC than at 56 degreesC. The cleavage reaction seems to be limited by the product release step at 37 degreesC and by the chemical cleavage step at 56 degreesC. We observed substrate inhibition at high concentrations of the 11-mer substrate. Cleavage rate constants were determined with a structural derivative characterized by an ultrastable L4 tetraloop. The kinetic parameters (kcat and KM) and dissociation constant (Kd) were almost identical for both ribozymes, suggesting that the stability of the L4 loop has a negligible impact on the catalytic activities of the examined ribozymes. Various cleavage inhibition and gel-shift assays with analogues, substrate, and both active and inactive ribozymes were performed. The 2'-hydroxyl group adjacent to the scissile phosphate was shown to be involved in binding with the ribozyme, while the essential cytosine residue of the J4/2 junction was shown to contribute to substrate association. We clearly show that substrate binding to the delta ribozyme is not restricted to the formation of a helix located downstream of the cleavage site. Using these results, we postulate a kinetic pathway involving a conformational transition step essential for the formation of the active ribozyme/substrate complex.

Group II intron ai5 gamma was reconstructed into a multiple-turnover ribozyme that efficiently cleaves small oligonucleotide substrates in-trans. This construct makes it possible to investigate sequence specificity, since second-order rate constants (kcat/K(m), or the specificity constant) can be obtained and compared with values for mutant substrates and with other ribozymes. The ribozyme used in this study consists of intron domains 1 and 3 connected in-cis, together with domain 5 as a separate catalytic cofactor. This ribozyme has mechanistic features similar to the first step of reverse-splicing, in which a lariat intron attacks exogenous RNA and DNA substrates, and it therefore serves as a model for the sequence specificity of group II intron mobility. To quantitatively evaluate the sequence specificity of this ribozyme, the WT kcat/Km value was compared to individual kcat/Km values for a series of mutant substrates and ribozymes containing single base changes, which were designed to create mismatches at varying positions along the two ribozyme-substrate recognition helices. These mismatches had remarkably large effects on the discrimination index (1/relative kcat/K(m)), resulting in values > 10,000 in several cases. The delta delta G++ for mismatches ranged from 2 to 6 kcal/mol depending on the mismatch and its position. The high specificity of the ribozyme is attributable to effects on duplex stabilization (1-3 kcal/mol) and unexpectedly large effects on the chemical step of reaction (0.5-2.5 kcal/mol). In addition, substrate association is accompanied by an energetic penalty that lowers the overall binding energy between ribozyme and substrate, thereby causing the off-rate to be faster than the rate of catalysis and resulting in high specificity for the cleavage of long target sequences (> or = 13 nucleotides).

The recent increase in the population of immunocompromised patients has led to an insurgence of opportunistic human fungal infections. The lack of effective treatments against some of these pathogens makes it important to develop new therapeutic strategies. One such strategy is to target key RNAs with antisense compounds. We report the development of a model system for studying the potential for antisense targeting of group I self-splicing introns in fungal pathogens. The group I intron from the large ribosomal subunit RNA of mouse-derived Pneumocystis carinii has been isolated and characterized. This intron self-splices in vitro. A catalytically active ribozyme, P-8/4x, has been constructed from this intron to allow measurement of dissociation constants for potential antisense agents. At 37 degrees C, in 50 mM Hepes (25 mM Na+), 15 mM MgCl2, and 135 mM KCl at pH 7.5, the exogenous 5' exon mimic r(AUGACU) binds about 60 000 times more tightly to this ribozyme than to r(GGUCAU), a mimic of its complementary binding site on the ribozyme. This enhanced binding is due to tertiary interactions. This tertiary stabilization is increased by single deoxynucleotide substitutions in the exon mimic at every position except for the internal A, which is essentially unchanged. Thus 2' OH groups of the 5' exon mimic do not form stabilizing tertiary interactions with the P-8/4x ribozyme, in contrast to the Tetrahymena L-21 ScaI ribozyme. Furthermore, at 37 degrees C, the exogenous 5' exon mimic d(ATGACT) binds nearly 32 000 times more tightly to the P-8/4x ribozyme than to r(GGUCAU). Therefore, oligonucleotides without 2' OH groups can exploit tertiary stabilization to bind dramatically more tightly and with more specificity than possible from base pairing. These results suggest a new paradigm for antisense targeting: targeting the tertiary interactions of structural RNAs with short antisense oligonucleotides.

The hammerhead ribozyme has an invariant nucleotide sequence in the core region. In order to search for alternative sequences which can support the cleavage after the triplet GUC, the core region of 10 nucleotides was randomized and subjected to in vitro selection by repeated cycles of transcription, reverse transcription, and PCR. Active sequences were isolated after each transcription by denaturing PAGE, and after nine cycles of selection, two sequences dominated the pool. Both sequences conformed broadly to the consensus core region except that in one sequence a single A9U mutation was observed while in the other two mutations at A9U and U7A were seen. The catalytic efficiencies of these ribozymes were 6.4 and 14.1 microM(-1) min(-1), respectively, as compared to 163 microM(-1) min(-1) for the consensus sequence. Interestingly, the consensus was not found in any of the selected sequences. This discrimination against the consensus sequence was attributed to the specificity of the enzymes used in the selection procedure.

A variant of the Tetrahymena ribozyme that efficiently cleaves single-stranded DNA under simulated physiological conditions [Tsang, J., & Joyce, G. F. (1994) Biochemistry 33, 5966-5973] was evaluated as a potential therapeutic agent on the basis of its ability to cleave synthetic oligonucleotide substrates corresponding to conserved target sites within HIV-I cDNA. In order to increase the sequence selectivity of the ribozyme, its substrate recognition domain was extended from 6 to 12 nucleotides, allowing base pairing with substrate nucleotides that lie both upstream and downstream of the cleavage site. The sequence of the extended recognition domain could be changed to allow cleavage of a variety of different DNA targets. The ribozyme exhibited a high degree of sequence specificity, discriminating by a factor of 10(2) to more than 10(4) against substrates that form a single-base mismatch with the ribozyme's recognition domain. Mismatches that occurred close to the cleavage site led to a greater decrease in activity compared to those that occurred farther away.

Five modified hammerhead ribozyme/substrate complexes have been prepared in which individual adenosine N3-nitrogens have been excised and replaced with carbon. The modified complexes were chemically synthesized with the substitution of a single 3-deazzaadenosine (c3A) base analogue for residues A6, A9, A13, A14, or A15.1. Steady-state kinetic analyses indicate that the cleavage efficiencies, as measured by kcat/K(M), for the c3A6, c3A9, and c3A14 complexes were only marginally reduced (< or = 5-fold) relative to the native complex. By comparison, the cleavage efficiencies for the c3A13 and c315.1 complexes were reduced by 9-fold and 55-fold, respectively. these reductions in cleavage efficiency are primarily a result of lower kcat values. Profiles of pH and cleavage rate suggest that the chemical cleavage step is the rate-limiting reaction for these complexes. These results suggest that the N3-nitrogen of the A13 residue and particularly the A15.1 residue in the hammerhead ribozyme/substrate complex are critical for transition state stabilization and efficient cleavage activity. We have additionally compared the locations of these critical functional groups, as well as those identified from other studies, with recent crystallographic analyses. In some cases, the critical functional groups are clustered around proposed metal binding sites and may reflect functional groups critical for binding the metal cofactor. In other cases, clusters of functional groups may form a network of hydrogen bonds necessary for transition state stabilization.

In this study we investigated the effects of the relocation of the wild type termini on the folding of a cis-cleaving RNA Rz 1 that was modified from the autolytic domains of hepatitis delta virus (HDV) RNA. Ten circularly permuted (CP) isomers of this ribozyme were synthesized. The structure homogeneity of RNA molecules, the molar ratio of the active species that undergoes cis-cleavage, and the rate of cis-cleavage were examined for each construct. CP isomers with new termini in H1 or at the junction of H2-H3, H1-J1/4, J1/4-H4, or H4-J4/2 of the proposed pseudoknot-like structure were inactive. The single breaks of phosphodiester bond in H2, J1/2, Lp4, and at the 3'-end of Lp3 decreased but did not abolish autolytic activity. The structural heterogeneity of RNA molecules may account for the limited cis-cleavage of the latter three isomers. The findings of circular permutation analysis were used as the basis for designing an active trans-cleaving ribozyme by dividing the cis-cleaving ribozyme into two subdomains at J1/2 and Lp4. The ribozyme subdomain catalyzed the site-specific cleavage of the circularly permuted composite substrate RNA in trans. Thus, the structure of HDV autolytic domain could be re-formed after two subdomains were associated through the base-pairing interactions of H1, H2, and H4.

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.

In the cyanobacterium Anabaena PCC7120, the precursor to tRNA(Leu) contains a 249-nucleotide group I intron that undergoes efficient self-splicing in vitro. By deleting the 5' and 3' splice sites, this intron has now been converted to an RNA enzyme that uses a guanosine nucleophile to cleave substrate RNAs (S) with multiple turnover. This Anabaena ribozyme has a second-order rate constant for RNA cleavage (kcat/Km)S that is 250-500-fold smaller than that of the Tetrahymena ribozyme, and a multiple-turnover rate constant at saturating S [kcat(mt)] that is approximately 400-fold larger. Several lines of evidence, including kinetic analysis of cleavage of phosphorothioate- and deoxynucleotide-substituted substrates and pH dependence, support the conclusion that both (kcat/Km)S and kcat(mt) are limited by the actual chemical cleavage step. In contrast, for the Tetrahymena ribozyme, it has been shown that neither of these rate constants reflects the chemical step. These kinetic differences are expected from the shorter guide sequence-substrate pairing of the Anabaena ribozyme; for example, weaker binding of RNA speeds product release during multiple turnover and thereby overcomes the rate-limiting product release observed for the Tetrahymena ribozyme. Thus, the large kinetic differences represent superficial rather than fundamental differences between these ribozymes. Furthermore, the strength of the guanosine-binding interaction, the stereospecificity for Rp-phosphorothioate at the cleavage site, and the 10(3)-fold slower cleavage with a deoxyribonucleoside leaving group are properties conserved between the Anabaena and Tetrahymena ribozymes. Finally, log(kcat/Km)S increases linearly with pH in the acid range where chemistry is rate-limiting and becomes pH-independent above pH 7, perhaps because a conformational step becomes rate-limiting; again, these are characteristics shared with the Tetrahymena ribozyme. We conclude that two group I ribozymes, although differing in the identity of many of their active site nucleotides, nevertheless provide functionally similar active sites for sequence-specific RNA cleavage.

We have identified an essential UV-sensitive tertiary structure domain within the hairpin ribozyme. Irradiation at 254 nm produces two cross-linked RNA species that are resolved from the unmodified structure by denaturing gel electrophoresis. One cross-link forms at high efficiency and maps between nucleotides G21 and/or A22 and U41, all essential bases located within an internal loop joining helices 3 and 4. A second cross-link forms between nucleotides A20 and U42 as a result of ribozyme dimerization at concentrations greater than 0.5 microM. Both cross-linked species retain cleavage activity and so presumably reflect catalytically proficient structures of the ribozyme. Formation of the intramolecular cross-link is independent of Mg2+ and substrate and is blocked by base substitutions within the reactive domain that inhibit catalysis. A 36-nt RNA fragment containing the photoreactive domain but lacking the substrate binding domain also cross-links with high efficiency and maps between G21 and U41, as observed with the intact molecule. The sequence and cross-linking sites of the UV-sensitive internal loop are strikingly similar to those found in several other RNA molecules, including loop E of 5S rRNA. These results suggest that the loop E-like structure may be a common RNA folding domain that is utilized in a variety of functionally important RNA molecules.

The Tetrahymena thermophila pre-rRNA contains a 413-nucleotide self-splicing group I intron. This intron has been converted into a sequence-specific endonuclease or ribozyme. A 160-nucleotide portion of the ribozyme consisting of both highly conserved sequence elements (P4 and P6) and nonconserved peripheral extensions (P5abc and P6ab) was synthesized as a separate molecule. Solvent-based Fe(II)-EDTA, a probe that monitors higher-order RNA structure, revealed a protection pattern that was a large subset of that observed in the whole ribozyme. Data from dimethyl sulfate modification and partial digestion with nucleases were also consistent with maintenance of the proper secondary and tertiary structure in the shortened RNA molecule. Thus, this 160-nucleotide molecule (P4-P6 RNA) is an independently folding domain of RNA tertiary structure. A series of mutations and deletions were made within the P4-P6 domain to further dissect its tertiary structure. Fe(II)-EDTA and dimethyl sulfate analysis of these mutants revealed that the domain consists of two substructures, a localized subdomain involving the characteristic adenosine-rich bulge in P5a, and a subdomain-stabilized structure involving long-range interactions. Therefore, like some proteins, the intron RNA is modular, containing a separable domain and subdomain of tertiary structure.

The hammerhead ribozyme is a small RNA motif that catalyzes the cleavage and ligation of RNA. The well-studied minimal hammerhead motif is inactive under physiological conditions and requires high Mg(2+) concentrations for efficient cleavage. In contrast, natural hammerheads are active under physiological conditions and contain motifs outside the catalytic core that lower the requirement for Mg(2+). Single-turnover kinetics were used here to characterize the Mg(2+) and pH dependence for cleavage of a trans-cleaving construct of the Schistosoma mansoni natural hammerhead ribozyme. Compared to the minimal hammerhead motif, the natural Schistosoma ribozyme requires 100-fold less Mg(2+) to achieve a cleavage rate of 1 min(-1). The improved catalysis results from tertiary interactions between loops in stems I and II and likely arises from increasing the population of the active conformation. Under optimum pH and Mg(2+) conditions this ribozyme cleaves at over 870 min(-1) at 25 degrees C, further demonstrating the impressive catalytic power of this ribozyme.

In a novel approach to gene therapy, researchers at Duke University Medical Center have shown that ribozymes can "edit" genetic information encoded in messenger RNA molecules inside mammalian cells. If further development is successful, the technique could, in effect, bypass a defective gene and instead correct the RNA blueprint the gene produces to direct protein synthesis. Ribozymes are RNA segments that catalyze reactions within cells. Because they selectively bind and react with other RNA segments, they have drawn attention as potential tools that could protect against viral infection by cleaving and destroying an invading virus's RNA. Clinical trials are now testing the potential of several modified ribozymes to protect against HIV infection. But the Duke team's approach is quite different. Assistant professor of surgery and genetics Bruce A. Sullenger, research assistant Joshua T. Jones, and postdoctoral fellow Seong-Wook Lee modified a ribozyme from a different class than those that cleave viral RN...