Acyl-CoAs from coenzyme ribozymes.

We describe in vitro selection of two novel ribozymes that mediate coenzyme reactions. The first is a trans-capping ribozyme that attaches coenzyme A (CoA) at the 5' end of any RNA with the proper short terminal sequence, including RNAs with randomized internal sequences. From such a trans-capped CoA-RNA pool, we derive ribozymes that attack biotinyl-AMP using the SH group of CoA. These ribozymes, selected to acylate CoA with the valeryl side chain of biotin, also produce the crucial metabolic intermediates acetyl-CoA and butyryl-CoA with substantial velocities. Thus, we argue that RNAs might have used the chemical functionality offered by coenzymes to support an RNA world metabolism. In particular, we can combine our results with those of other labs to argue that simple chemistry and RNA catalysis suffice to proceed from simple chemicals to catalysis with acyl-CoAs. The trans-capping method can be generalized for production of varied coenzyme ribozymes using a single catalytic RNA subunit. Finally, the long-suggested RNA origin for CoA itself appears to be chemically feasible.     

Computational design and experimental validation of oligonucleotide-sensing allosteric ribozymes

Allosteric RNAs operate as molecular switches that alter folding and function in response to ligand binding. A common type of natural allosteric RNAs is the riboswitch; designer RNAs with similar properties can be created by RNA engineering. We describe a computational approach for designing allosteric ribozymes triggered by binding oligonucleotides. Four universal types of RNA switches possessing AND, OR, YES and NOT Boolean logic functions were created in modular form, which allows ligand specificity to be changed without altering the catalytic core of the ribozyme. All computationally designed allosteric ribozymes were synthesized and experimentally tested in vitro. Engineered ribozymes exhibit >1,000-fold activation, demonstrate precise ligand specificity and function in molecular circuits in which the self-cleavage product of one RNA triggers the action of a second. This engineering approach provides a rapid and inexpensive way to create allosteric RNAs for constructing complex molecular circuits, nucleic acid detection systems and gene control elements.     

RNA with polynucleotide kinase properties, isolated by in vitro selection

Data on isolation from a large pool of RNAs of a fragment characterized by high-affinity ATP binding are reviewed. This ATP-binding domain flanking the regions of randomly sequenced nucleotide residues was used for preparation of an RNA pool, from which ribozymes displaying a polynucleotide kinase activity were isolated. The isolated ribozymes catalyzed the transfer of gamma-thiophosphate from ATP-gamma S to the 5' hydroxyl or to internal 2'-hydroxyls of their own chains. ATP was also used as a donor of phosphate; however, in this case the reaction rate was 55-300 times lower. Similarly to a true enzyme, one of these ribozymes shortened by 40 nucleotide residues at the 5' end repeatedly catalyzed the transfer of thiophosphate or phosphate to the 5' hydroxyl of an exogenous oligonucleotide.     

The emergence of molecular machines as a prerequisite of the ancient RNA world evolution

The problem of the start of biological evolution in the ancient RNA world is considered. It is postulated that the appearance of catalytic RNAs — ribozymes — via spontaneous cis- and trans-rearrangements of polyribonucleotides in primordial Darwin ponds should not have been sufficient for the start of evolution, until a new class of functional RNA, namely energy-dependent molecular machines, arose. The proposed hypothesis is that the simplest and primary type of molecular machines could be nucleoside triphosphate-dependent RNA-based helicases, which were capable of unwinding the stable double-helical RNAs inevitably formed during RNA syntheses on complementary templates. Thereupon, unwinding RNA polymerases could appear as a result of association or fusion of helicases and polyribonucleotide-polymerizing ribozymes. The latter event provided the mechanism of RNA replication using the double-helical RNAs as a communal genofond (gene pool) of a Darwin pond, and thus initiated the fast evolution of the ancient RNA world.     

Anchoring hairpin ribozymes to long target RNAs by loop-loop RNA interactions

Efficient ribozyme-mediated gene silencing requires the effective binding of a ribozyme to its specific target sequence. Stable stem-loop domains are key elements for efficiency of natural antisense RNAs. This work tests the possibility of using such naturally existing structural motifs for anchoring hairpin ribozymes when targeting long RNAs. Assays were performed with four catalytic antisense RNAs, based on the hairpin ribozyme (HP), that carried a stable stem-loop motif at their 3' end. Extensions consisted of one of the following motifs: the stem-loop II of the natural antisense RNA-CopA, its natural target in CopT, the TAR-RNA motif, or its complementary sequence alphaTAR. Interestingly, the presence of any of these antisense motifs resulted in an enhancement of catalytic performance against the ribozyme's 14-nucleotide-long target RNA (Swt). A series of artificial, long RNA substrates containing the Swt sequence and the natural TAR-RNA stem-loop were constructed and challenged with a catalytic antisense RNA carrying the TAR-complementary stem-loop. This cleaves each of these substrates significantly more efficiently than HP. The deletion of the TAR domain in the substrate, or its substitution by its complementary counterpart alphaTAR, abolishes the positive effect. These results suggest that the enhancement is owed to the interaction of both complementary stem-loop domains. Moreover, they demonstrate that the TAR domain can be used as an anchoring site to facilitate the access of hairpin ribozymes to their specific target sequences within TAR-containing RNAs.     

Ribozymes: the characteristics and properties of catalytic RNAs

Ribozymes, or catalytic RNAs, were discovered a little more than 15 years ago. They are found in the organelles of plants and lower eukaryotes, in amphibians, in prokaryotes, in bacteriophages, and in viroids and satellite viruses that infect plants. An example is also known of a ribozyme in hepatitis delta virus, a serious human pathogen. Additional ribozymes are bound to be found in the future, and it is tempting to regard the RNA component(s) of various ribonucleoprotein complexes as the catalytic engine, while the proteins serve as mere scaffolding--an unheard-of notion 15 years ago! In nature, ribozymes are involved in the processing of RNA precursors. However, all the characterized ribozymes have been converted, with some clever engineering, into RNA enzymes that can cleave or modify targeted RNAs (or even DNAs) without becoming altered themselves. While their success in vitro is unquestioned, ribozymes are increasingly used in vivo as valuable tools for studying and regulating gene expression. This review is intended as a brief introduction to the characteristics of the different identified ribozymes and their properties.     

Efficient substrate cleavage catalyzed by hammerhead ribozymes derivatized with selenium for X-ray crystallography

Because oxygen and selenium are in the same group (Family VI) in the periodic table, the site-specific mutagenesis at the atomic level by replacing RNA oxygen with selenium can provide insights on the structure and function of catalytic RNAs. We report here the first Se-derivatized ribozymes transcribed with all nucleoside 5'-(alpha-P-seleno)triphosphates (NTPalphaSe, including A, C, G, and U). We found that T7 RNA polymerase recognizes NTPalphaSe Sp diastereomers as well as the natural NTPs, whereas NTPalphaSe Rp diastereomers are neither substrates nor inhibitors. We also demonstrated the catalytic activity of these Se-derivatized hammerhead ribozymes by cleaving the RNA substrate, and we found that these phosphoroselenoate ribozymes can be as active as the native one. These hammerhead ribozymes site-specifically mutagenized by selenium reveal the close relationship between the catalytic activities and the replaced oxygen atoms, which provides insight on the participation of oxygen in catalysis or intramolecular interaction. This demonstrates a convenient strategy for the mechanistic study of functional RNAs. In addition, the active ribozymes site-specifically derivatized by selenium will allow for convenient MAD phasing in X-ray crystal structure studies.     

Gaining target access for deoxyribozymes

Antisense oligonucleotides and ribozymes have been used widely to regulate gene expression by targeting mRNAs in a sequence-specific manner. Long RNAs, however, are highly structured molecules. Thus, up to 90% of putative cleavage sites have been shown to be inaccessible to classical RNA based ribozymes or DNAzymes. Here, we report the use of modified nucleotides to overcome barriers raised by internal structures of the target RNA. In our attempt to cleave a broad range of picornavirus RNAs, we generated a DNAzyme against a highly conserved sequence in the 5' untranslated region (5' UTR). While this DNAzyme was highly efficient against the 5' UTR of the human rhinovirus 14, it failed to cleave the identical target sequence within the RNA of the related coxsackievirus A21 (CAV-21). After introduction of 2'-O-methyl RNA or locked nucleic acid (LNA) monomers into the substrate recognition arms, the DNAzyme degraded the previously inaccessible virus RNA at a high catalytic rate even to completion, indicating that nucleotides with high target affinity were able to compete successfully with internal structures. We then adopted this strategy to two DNAzymes that we had found to be inactive in our earlier experiments. The modified DNAzymes proved to be highly effective against their respective target structures. Our approach may be useful for other ribozyme strategies struggling with accessibility problems, especially when being restricted to unique target sites.     

Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes

Allosteric ribozymes are engineered RNAs that operate as molecular switches whose rates of catalytic activity are modulated by the binding of specific effector molecules. New RNA molecular switches can be created by using "allosteric selection," a molecular engineering process that combines modular rational design and in vitro evolution strategies. In this report, we describe the characterization of 3',5'-cyclic nucleotide monophosphate (cNMP)-dependent hammerhead ribozymes that were created using allosteric selection (Koizumi et al., Nat Struct Biol, 1999, 6:1062-1071). Artificial phylogeny data generated by random mutagenesis and reselection of existing cGMP-, cCMP-, and cAMP-dependent ribozymes indicate that each is comprised of distinct effector-binding and catalytic domains. In addition, patterns of nucleotide covariation and direct mutational analysis both support distinct secondary-structure organizations for the effector-binding domains. Guided by these structural models, we were able to disintegrate each allosteric ribozyme into separate ligand-binding and catalytic modules. Examinations of the independent effector-binding domains reveal that each retains its corresponding cNMP-binding function. These results validate the use of allosteric selection and modular engineering as a means of simultaneously generating new nucleic acid structures that selectively bind ligands. Furthermore, we demonstrate that the binding affinity of an allosteric ribozyme can be improved through random mutagenesis and allosteric selection under conditions that favor tighter binding. This "affinity maturation" effect is expected to be a valuable attribute of allosteric selection as future endeavors seek to apply engineered allosteric ribozymes as biosensor components and as controllable genetic switches.     

Energetics of RNA cleavage: implications for the mechanism of action of ribozymes.

A new class of ribozymes produce 2',3'-cyclic phosphate upon self-catalyzed cleavage of RNA molecules, similar to those observed during enzymatic (RNase-catalyzed) as well as non-enzymatic hydrolyses of RNAs. This product suggests that the reaction intermediate/transition state is a pentacoordinated oxyphosphorane. In order to elucidate the energetics of these RNA cleaving reactions, the reaction coordinate has been simulated and a pentacoordinated intermediate has been characterized via ab initio molecular orbital calculations utilizing the dianionic hydrolysis-intermediate of methyl ethylene phosphate as a model compound. The calculated reaction coordinate indicates that the transition state for the P-O(2') bond cleavage is lower in energy than that for the P-O(5') bond cleavage under uncatalyzed conditions. Thus, the dianionic pentacoordinated phosphorus intermediate tends to revert back to the starting RNA by cleaving the P-O(2') bond rather than productively cleaving the P-O(5') bond. In order for ribozymes to effectively cleave RNA molecules, it is therefore mandatory to stabilize the leaving 5'-oxygen, e.g. by means of a divalent magnesium ion.     

Nonreplicative RNA Recombination in Poliovirus

Current models of recombination between viral RNAs are based on replicative template-switch mechanisms. The existence of nonreplicative RNA recombination in poliovirus is demonstrated in the present study by the rescue of viable viruses after cotransfections with different pairs of genomic RNA fragments with suppressed translatable and replicating capacities. Approximately 100 distinct recombinant genomes have been identified. The majority of crossovers occurred between nonhomologous segments of the partners and might have resulted from transesterification reactions, not necessarily involving an enzymatic activity. Some of the crossover loci are clustered. The origin of some of these "hot spots" could be explained by invoking structures similar to known ribozymes. A significant proportion of recombinant RNAs contained the entire 5' partner, if its 3' end was oxidized or phosphorylated prior to being mixed with the 3' partner. All of these observations are consistent with a mechanism that involves intermediary formation of the 2',3'-cyclic phosphate and 5'-hydroxyl termini. It is proposed that nonreplicative RNA recombination may contribute to evolutionarily significant RNA rearrangements.     

Selective cleavage of bcr-abl chimeric RNAs by a ribozyme targeted to non-contiguous sequences.

Conventionally designed ribozymes may be unable to cleave RNA at sites which are inaccessible due to secondary structure. In addition, it may also be difficult to specifically target a conventionally designed ribozyme to some chimeric RNA molecules. Novel approaches for ribozyme targeting were developed by using the L6 bcr-abl fusion RNA as a model. Using one approach, we successfully directed ribozyme nucleation to a site on the bcr-abl RNA that is distant from the GUA cleavage site. These ribozymes bound to the L6 substrate RNA via an anchor sequence that was complementary to bcr sequences. The anchor was necessary for efficient cleavage as the anchor minus ribozyme, a conventionally designed ribozyme, was inefficient at catalyzing cleavage at this same site. The effect of anchor sequences on catalytic rates was determined for two of these ribozymes. Ribozymes generated by a second approach were designed to cleave at a CUU site in proximity to the bcr-abl junction. Both approaches have led to the development of a series of ribozymes specific for both the L6 and K28 bcr-abl chimeric RNAs, but not normal abl or bcr RNAs. The specificity of the ribozyme correlated in part with the ability of the ribozyme to bind substrate as demonstrated by gel shift analyses. Secondary structure predictions for the RNA substrate support the experimental results and may prove useful as a theoretical basis for the design of ribozymes.     

Ribozymes, riboswitches and beyond: regulation of gene expression without proteins

Although various functions of RNA are carried out in conjunction with proteins, some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that cells use to regulate gene expression in response to internal and external changes. Structural studies have shown how these RNAs can carry out a range of functions. In addition, the contribution of ribozymes and riboswitches to gene expression is being revealed as far more widespread than was previously appreciated. These findings have implications for understanding how cellular functions might have evolved from RNA-based origins.     

DNA polymerization catalysed by a group II intron RNA in vitro.

The excised group II intron bI1 from Saccharomyces cerevisiae can act as a ribozyme catalysing various chemical reactions with different substrate RNAs in vitro . Recently, we have described an editing-like RNA polymerization reaction catalysed by the bI1 intron lariat that proceeds in the 3'-->5'direction. Here we show that the bI1 lariat RNA can also catalyse successive deoxyribonucleotide polymerization reactions on exogenous substrate molecules. The basic mechanism of the reaction involved interacting cycles between an alternative version of partial reverse splicing (lariat charging) and canonical forward splicing (lariat discharging by exon ligation). With an overall chain growth in the 3'-->5' direction, the 5' exon RNAs (IBS1dN) were elongated by successive insertion of deoxyribonucleotides derived from single deoxyribonucleotide substitutions (dA, dG, dC or dT). All four deoxyribonucleotides were used as substrates, although with different efficiencies. Our findings extend the catalytic repertoire of group II intron RNAs not only by a novel DNA polymerization activity, but also by a DNA-DNA ligation capacity, supporting the idea that ribozymes might have been part of the first primordial polymerization machinery for both RNA and DNA.     

Serial passage of tobacco rattle virus under different selection conditions results in deletion of structural and nonstructural genes in RNA 2.

The RNA genome of tobacco rattle virus (TRV) is bipartite. RNA 2 of the nematode-transmissible TRV isolate PPK20 encodes the viral coat protein (cp) and proteins with molecular weights of 29,400 and 32,800 (29.4K and 32.8K proteins). When this isolate was serially passaged in tobacco by using phenol-extracted RNA as the inoculum in each transfer, defective interfering (DI) RNAs rapidly accumulated. A number of these DI RNAs were cloned. Six DI RNAs had single internal deletions in RNA 2 that removed most of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. The borders of the deletions in these DI RNAs were found to be flanked in the genomic RNA 2 by short nucleotide repeats or sequences resembling the 5' end of TRV genomic and subgenomic RNAs. Two DI RNAs were found to be recombinants containing a 5' sequence derived from RNA 2 and a 3' sequence derived from RNA 1. When serial passage of TRV isolate PPK20 was carried out by using leaf homogenates as inocula in each transfer, accumulation of a DI RNA (designated D7) with a functional cp gene was observed. The deletion in D7 covered the 3' end of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. An infectious cDNA clone of D7 RNA was made. In mixed infections, D7 RNA rapidly outcompeted RNA 2 but did not compete with RNA 1. The deletion in D7 RNA abolished the nematode transmissibility of the PPK20 isolate. These results may explain the observation that many laboratory isolates of tobraviruses have lost their nematode transmissibility and contain RNA 2 molecules of widely different lengths.