Cleavage of specific sites of RNA by designed ribozymes

Two ribozymes were designed for site-specific cleavage of RNA. A UA site in an undecaribonucleotide was cleaved by a ribozyme consisting of two partially paired oligoribonucleotides with chain lengths of 19 and 15. The other ribozyme, which consists of a 19-mer and a 13-mer, recognized a UC sequence at positions 42 and 43 of 5 S rRNA.     

RNA catalysis: ribozymes, ribosomes, and riboswitches

The catalytic mechanisms employed by RNA are chemically more diverse than initially suspected. Divalent metal ions, nucleobases, ribosyl hydroxyl groups, and even functional groups on metabolic cofactors all contribute to the various strategies employed by RNA enzymes. This catalytic breadth raises intriguing evolutionary questions about how RNA lost its biological role in some cases, but not in others, and what catalytic roles RNA might still be playing in biology.     

New ligase-derived RNA polymerase ribozymes

The search is underway for a catalytic RNA molecule capable of self-replication. Finding such a ribozyme would lend crucial support to the RNA World hypothesis, which holds that very early life-forms relied on RNA for both replicating and storing genetic information. We previously reported an RNA polymerase isolated from a pool of variants of an existing RNA ligase ribozyme. Here we report eight additional ligase-derived polymerase ribozymes isolated from this pool. Because each of them is a new potential starting point for further in vitro evolution and engineering, together they substantially enrich the set of candidates from which an RNA replicase ribozyme might eventually emerge.     

A minihelix-loop RNA acts as a trans-aminoacylation catalyst.

We previously reported a bifunctional ribozyme that catalyzes self-aminoacylation and subsequent acyl-transfer to a tRNA. The ribozyme selectively recognizes a biotinyl-glutamine substrate, and charges the tRNA molecule in trans. Structurally, there are two catalytic domains, referred to as glutamine-recognition (QR) and acyl-transferase (ATRib). We report here the essential catalytic core of the QR domain as determined by extensive biochemical probing, mutation, and structural minimization. The minimal core of the QR domain is a 29-nt helix-loop RNA, which is also able to glutaminylate ATRib in trans. Its amino acid binding site is embedded in an 11-nt cluster that is adjacent to the loop that interacts with the ATRib domain. Our study shows that a minihelix-loop RNA can act as a trans-aminoacylation catalyst, which lends support for the critical role of minihelix-loops in the early evolution of the aminoacylation system.     

Natural montmorillonite clay as prebiotic catalyst

It is shown that complex intramolecular and intermolecular transformations of natural pinene terpenoids can proceed in the presence of natural montmorillonite clays with the preservation of optical activity. These facts support our assumption that natural clays could act as prebiotic catalysts and favor preservation of chirality in complex compounds formed from simple optically active molecules at early stages of life. The article is published in the original.     

RNA self-ligation: from oligonucleotides to full length ribozymes

The RNA-world-theory is one possible explanation of how life on earth has evolved. In this context it is of high interest to search for molecular systems, capable of self-organization into structures with increasing complexity. We have engineered a simple catalytic system in which two short RNA molecules can catalyze their own ligation to form a larger RNA construct. The system is based on the hairpin ribozyme using a 2',3'-cyclophosphate as activated species for ligation. 2',3'-cyclic phosphates can be easily formed and occur in many natural systems, thus being superior candidates for activated building blocks in RNA world scenarios.     

Activity and stability of hammerhead ribozymes containing 2'-C-methyluridine: a new RNA mimic

We propose 2'-C-methylnucleotides as a new class of 2'-modified RNA mimics. These analogues are expected to provide 2'-OH groups capable of reproducing the interactions observed in natural RNA and, due to the presence of the Me group, to possess increased stability towards nucleases. In this work, we investigate the catalytic activity and nuclease resistance of hammerhead ribozymes carrying 2'-C-methyluridines in positions 4 and 7 of the catalytic core. We describe the in vitro activity of these chimeric molecules and their stability in cell lysate, fetal calf serum, and cell culture medium. The data show that, when only position 4 is modified, activity decreases twofold; while, when both 4 and 7 positions are substituted, a sevenfold drop in activity is observed. Regarding biological stability, the main increase of the half-life time is observed when position 7 is modified. These results suggest that 2'-C-methylnucleotides may be useful in the design of chemically synthesized RNA mimics with biological activity.     

Turnover ability of a designed RNA acting as a template for chemical peptide ligation

A designed self-folding RNA possessing two peptide-recognition motifs served as a template for the chemical ligation of two RNA-binding peptides under stoichiometric conditions. In this study, we investigated the turnover ability of this template RNA in facilitation of peptide ligation and found that the RNA exhibited modest turnover ability under conditions in which its 3D structure was marginally stable.     

The origins of RNA catalysis in ribozymes

The discovery of RNA catalysis provided a paradigm shift in biology, insight into the evolution of life on the planet and a challenge to understand its mechanistic origins. RNA has limited catalytic resources that must be used to maximal effect. Consequently, RNA catalysis tends to be multifactorial, with several processes contributing to an overall significant enhancement of reaction rate. These include general acid-base catalysis, electrostatic effects, and substrate orientation and proximity. The main players are the RNA nucleobases and bound metal ions. Although most ribozymes carry out phosphoryl transfer, the same considerations appear to apply to peptidyl transfer in the ribosome.     

A short primer on RNAi: RNA-directed RNA polymerase acts as a key catalyst.

One of the many intriguing features of gene silencing by RNA interference is the apparent catalytic nature of the phenomenon. New biochemical and genetic evidence now shows that an RNA-directed RNA polymerase chain reaction, primed by siRNA, amplifies the interference caused by a small amount of "trigger" dsRNA.     

Complete RNA inverse folding: computational design of functional hammerhead ribozymes

Nanotechnology and synthetic biology currently constitute one of the most innovative, interdisciplinary fields of research, poised to radically transform society in the 21st century. This paper concerns the synthetic design of ribonucleic acid molecules, using our recent algorithm, RNAiFold, which can determine all RNA sequences whose minimum free energy secondary structure is a user-specified target structure. Using RNAiFold, we design ten cis-cleaving hammerhead ribozymes, all of which are shown to be functional by a cleavage assay. We additionally use RNAiFold to design a functional cis-cleaving hammerhead as a modular unit of a synthetic larger RNA. Analysis of kinetics on this small set of hammerheads suggests that cleavage rate of computationally designed ribozymes may be correlated with positional entropy, ensemble defect, structural flexibility/rigidity and related measures. Artificial ribozymes have been designed in the past either manually or by SELEX (Systematic Evolution of Ligands by Exponential Enrichment); however, this appears to be the first purely computational design and experimental validation of novel functional ribozymes. RNAiFold is available at http://bioinformatics.bc.edu/clotelab/RNAiFold/.     

A designed RNA selection: establishment of a stable complex between a target and selectant RNA via two coordinated interactions

In this paper, we describe a new method for selecting RNA aptamers that cooperatively bind to two specific sites within a target RNA. We designed a selection system in which two RNAs, a target RNA and a RNA pool, were assembled by employing a pre-organized GAAA tetraloop-11-nt receptor interaction. This allows us to select the binding sequence against a targeted internal loop as well as a linker region optimized for binding of the two binding sites. After the selection, the aptamers bound with dissociation constants in the nanomolar range, thereby forming a stable complex with the target RNA. Thus this method enables identification of aptamers for a specific binding site together with a linker for cooperative binding of the two RNAs. It appears that our new method can be applied generally to select RNAs that adhere tightly to a target RNA via two specific sites. The method can also be applicable for further engineering of both natural and artificial RNAs.     

Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.

Positions 2-6 of the substrate-binding internal guide sequence (IGS) of the L-21 Sca I form of the Tetrahymena thermophila intron were mutagenized to produce a GN5 IGS library. Ribozymes within the GN5 library capable of efficient cleavage of an 818-nt human immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were identified by ribozyme-catalyzed guanosine addition to the 3' cleavage product. Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the GN5 library that actively cleaved the long substrate were characterized kinetically and compared to the wild-type ribozyme (GGAGGG) and two control ribozymes (GGAGUC and GGAGAU). The two control ribozymes have specific sites within the long substrate, but were not identified during screening of the library. Under single-turnover conditions, ribozymes GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold faster than control ribozymes. Short cognate substrates, which should be structureless and therefore accessible to ribozyme binding, were cleaved at similar rates by all ribozymes except GGGGCU, which showed a fourfold rate enhancement. The rate of cleavage of long relative to short substrate under single-turnover conditions suggests that GGCUCC and GUGGCU were identified because of accessibility to their specific cleavage sites within the long substrate (substrate-specific effects), whereas GGGGCU was identified because of an enhanced rate of substrate binding despite a less accessible site in the long substrate. Even though screening was performed with 100-fold excess substrate (relative to total ribozyme), the rate of multiple-turnover catalysis did not contribute to identification of trans-cleaving ribozymes in the GN5 library.     

Quantitative studies of Mn(2+)-promoted specific and non-specific cleavages of a large RNA: Mn(2+)-GAAA ribozymes and the evolution of small ribozymes

Manganese (Mn²⁺) promotes specific cleavage at two major (I and III) and four minor (II, IV, V and VI) sites, in addition to slow non-specific cleavage, in a 659-nucleotide RNA containing the Cr.LSU group I intron. The specific cleavages occurred between G and AAA sequences and thus can be considered Mn²⁺-GAAA ribozymes. We have estimated rates of specific and non-specific cleavages under different conditions. Comparisons of the rates of major-specific and background cleavages gave a maximal specificity of approximately 900 for GAAA cleavage. Both specific and non-specific cleavages showed hyperbolic kinetics and there was no evidence of cooperativity with Mn²⁺ concentration. Interestingly, at site III, Mg²⁺ alone promoted weak, but the same specific cleavage as Mn²⁺. When added with Mn²⁺, Mg²⁺ had a synergistic effect on cleavage at site III, but inhibited cleavage at the other sites. Mn²⁺ cleavage at site III also exhibited lower values of K (Mn²⁺ requirement), pH-dependency and activation energy than did cleavage at the other sites. In contrast, the pH-dependency and activation energy for cleavage at site I was similar to non-specific cleavage. These results increase our understanding of the Mn²⁺-GAAA ribozyme. The implications for evolution of small ribozymes are also discussed.