Application of computational technologies to ribozyme biotechnology products (original) (raw)
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Complete RNA inverse folding: computational design of functional hammerhead ribozymes
Nucleic Acids Research, 2014
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 userspecified 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.
Automated Design and Empirical Validation of Hammerhead Ribozymes
2015
Ribozymes are catalytic RNA molecules. Hammerhead ribozymes are one of a set of ribozymes capable of cleaving RNA molecules, in cis and in trans, without the help of other molecules, such as proteins. A trans-acting hammerhead ribozyme has a few related forms, which can be customized to target specific sites within RNA strands, including the transcripts of genes. As such, hammerhead ribozymes can be used to down-regulate or even silence any gene with valid cut-sites. All, except very short transcripts, will have multiple valid cut-sites. However, the efficiency of silencing by a hammerhead ribozyme depends on multiple often conflicting factors. Also, the efficiency of cleavage of any one ribozyme is normally low. Hence, it is useful to automate the process of design of hammerhead ribozymes to efficiently and without error explore the large space of possible designs. Computers are simply better than humans in doing a large amount of repetitive work without error. This thesis describe...
A structural analysis of in vitro catalytic activities of hammerhead ribozymes
BMC Bioinformatics, 2007
Background Ribozymes are small catalytic RNAs that possess the dual functions of sequence-specific RNA recognition and site-specific cleavage. Trans-cleaving ribozymes can inhibit translation of genes at the messenger RNA (mRNA) level in both eukaryotic and prokaryotic systems and are thus useful tools for studies of gene function. However, identification of target sites for efficient cleavage poses a challenge. Here, we have considered a number of structural and thermodynamic parameters that can affect the efficiency of target cleavage, in an attempt to identify rules for the selection of functional ribozymes. Results We employed the Sfold program for RNA secondary structure prediction, to account for the likely population of target structures that co-exist in dynamic equilibrium for a specific mRNA molecule. We designed and prepared 15 hammerhead ribozymes to target GUC cleavage sites in the mRNA of the breast cancer resistance protein (BCRP). These ribozymes were tested, and thei...
The effect of structure in a long target RNA on ribozyme cleavage efficiency
Nucleic Acids Research, 1997
Inhibition of gene expression by catalytic RNA (ribozymes) requires that ribozymes efficiently cleave specific sites within large target RNAs. However, the cleavage of long target RNAs by ribozymes is much less efficient than cleavage of short oligonucleotide substrates because of higher order structure in the long target RNA. To further study the effects of long target RNA structure on ribozyme cleavage efficiency, we determined the accessibility of seven hammerhead ribozyme cleavage sites in a target RNA that contained human immunodeficiency virus type 1 (HIV-1) vif-vpr. The base pairing-availability of individual nucleotides at each cleavage site was then assessed by chemical modification mapping. The ability of hammerhead ribozymes to cleave the long target RNA was most strongly correlated with the availability of nucleotides near the cleavage site for base pairing with the ribozyme. Moreover, the accessibility of the seven hammerhead ribozyme cleavage sites in the long target RNA varied by up to 400-fold but was directly determined by the availability of cleavage sites for base pairing with the ribozyme. It is therefore unlikely that steric interference affected hammerhead ribozyme cleavage. Chemical modification mapping of cleavage site structure may therefore provide a means to identify efficient hammerhead ribozyme cleavage sites in long target RNAs.
F1000 - Post-publication peer review of the biomedical literature, 2008
We have obtained precatalytic (enzyme-substrate complex) and postcatalytic (enzyme-product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pK a that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme-substrate and enzyme-product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures.
Biochemistry, 2007
The aim of this work was to shed some more light on factors influencing the effectiveness of delta ribozyme cleavage of structured RNA molecules. An oligoribonucleotide that corresponds to the 3′-terminal region X of HCV RNA and yeast tRNA Phe were used as representative RNA targets. Only a few sites susceptible to ribozyme cleavage were identified in these targets using a combinatorial library of ribozyme variants, in which the region responsible for ribozyme-target interaction was randomized. On the other hand, the targets were fairly accessible for binding of complementary oligonucleotides, as was shown by 6-mer DNA libraries and RNase H approach. Moreover, the specifically acting ribozymes cleaved the targets precisely but with unexpectedly modest efficacy. To explain these observations, six model RNA molecules were designed, in which the same seven nucleotide long sequence recognized by the delta ribozyme was always single stranded but was embedded into different RNA structural context. These molecules were cleaved with differentiated rates, and the corresponding k 2 values were in the range of 0.91-0.021 min-1 ; thus they differed almost 50-fold. This clearly shows that cleavage of structured RNAs might be much slower than cleavage of a short unstructured oligoribonucleotide, despite full accessibility of the targeted regions for hybridization. Restricted possibilities of conformational transitions, which are necessary to occur on the cleavage reaction trajectory, seem to be responsible for these differences. Their magnitude, which was evaluated in this work, should be taken into account while considering the use of delta ribozymes for practical applications.
Chemical models for ribozyme action
Current Opinion in Chemical Biology, 2005
Mechanistic studies of the action of catalytic ribonucleic acids, ribozymes, are highly challenging, because even a slight structural change can dramatically affect the chain folding. This, in turn, alters the binding properties of the catalytic core, making identification of the real origin of the observed influence on rate difficult. Unambiguous structure-reactivity correlations based on studies with structurally simplified chemical models may help to distinguish between alternative mechanistic interpretations. The results of such model studies are reviewed. The topics include intramolecular cleavage of RNA phosphodiester bonds by solvent-derived species, general acids/bases and metal ions, effect of molecular environment on their hydrolytic stability and trinucleoside monophosphates as models for large ribozymes.
Ribozymes: recent advances in the development of RNA tools
FEMS Microbiology Reviews, 2003
The discovery 20 years ago that some RNA molecules, called ribozymes, are able to catalyze chemical reactions was a breakthrough in biology. Over the last two decades numerous natural RNA motifs endowed with catalytic activity have been described. They all fit within a few well-defined types that respond to a specific RNA structure. The prototype catalytic domain of each one has been engineered to generate trans-acting ribozymes that catalyze the site-specific cleavage of other RNA molecules. On the 20th anniversary of ribozyme discovery we briefly summarize the main features of the different natural catalytic RNAs. We also describe progress towards developing strategies to ensure an efficient ribozyme-based technology, dedicating special attention to the ones aimed to achieve a new generation of therapeutic agents.
Substrate specificity and reaction kinetics of an X-motif ribozyme
RNA, 2003
The X-motif is an in vitro-selected ribozyme that catalyzes RNA cleavage by an internal phosphoester transfer reaction. This ribozyme class is distinguished by the fact that it emerged as the dominant clone among at least 12 different classes of ribozymes when in vitro selection was conducted to favor the isolation of high-speed catalysts. We have examined the structural and kinetic properties of the X-motif in order to provide a framework for its application as an RNA-cleaving agent and to explore how this ribozyme catalyzes phosphoester transfer with a predicted rate constant that is similar to those exhibited by the four natural self-cleaving ribozymes. The secondary structure of the X-motif includes four stem elements that form a central unpaired junction. In a bimolecular format, two of these base-paired arms define the substrate specificity of the ribozyme and can be changed to target different RNAs for cleavage. The requirements for nucleotide identity at the cleavage site are GD, where D = G, A, or U and cleavage occurs between the two nucleotides. The ribozyme has an absolute requirement for a divalent cation cofactor and exhibits kinetic behavior that is consistent with the obligate binding of at least two metal ions.
Capturing Hammerhead Ribozyme Structures in Action by Modulating General Base Catalysis
PLoS Biology, 2008
We have obtained precatalytic (enzyme-substrate complex) and postcatalytic (enzyme-product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pK a that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme-substrate and enzyme-product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation enzyme-product complex structure reveals RNA and metal ion interactions potentially relevant to transition-state stabilization that are absent in precatalytic structures.