Complete RNA inverse folding: computational design of functional hammerhead ribozymes (original) (raw)
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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...
Application of computational technologies to ribozyme biotechnology products
Journal of Molecular Structure, 1994
Ribozymes are RNA molecules that act enzymatically to cleave other RNA molecules. The cleavage reaction requires the binding of ribozyme to specific sites on the target RNA through (mostly) Watson-Crick base-pairing interactions. Association ofribozyme with target completes a three-dimensional ribozymejtarget complex which results in cleavage of the target RNA. We are employing both computational and experimental approaches to identify sites on target RNA molecules that are open to ribozyme attack and to determine which ribozymes are most active against those sites. Two types of computational technologies are available for aiding in the identification of target sites and design of active ribozymes. First, DNA/RNA sequence analysis software is employed to identify sequence motifs necessary for ribozyme cleavage and to look for sequence conservation between different sources of the target organism so that ribozymes with the broadest possible target range can be designed. Second, RNA folding algorithms are employed to predict the secondary structure of both ribozyme and target RNA in an attempt to identify combinations of ribozyme and target site that will successfully associate prior to ribozyme cleavage. The RNA folding algorithms utilize a set of thermodynamic parameters obtained from measurements on short RNA duplexes; while these rules give reasonable predictions of secondary structure for a small set of highly structured RNAs, they remain largely untested for predicting the structure of messenger RNAs. This paper outlines the current status of designing ribozymes that fold correctly and of locating target sites that are sufficiently unfolded to allow ribozyme cleavage.
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.
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...
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.
RNAiFold 2.0: a web server and software to design custom and Rfam-based RNA molecules
Nucleic Acids Research, 2015
Several algorithms for RNA inverse folding have been used to design synthetic riboswitches, ribozymes and thermoswitches, whose activity has been experimentally validated. The RNAiFold software is unique among approaches for inverse folding in that (exhaustive) constraint programming is used instead of heuristic methods. For that reason, RNAiFold can generate all sequences that fold into the target structure or determine that there is no solution. RNAiFold 2.0 is a complete overhaul of RNAiFold 1.0, rewritten from the now defunct COMET language to C++. The new code properly extends the capabilities of its predecessor by providing a user-friendly pipeline to design synthetic constructs having the functionality of given Rfam families. In addition, the new software supports amino acid constraints, even for proteins translated in different reading frames from overlapping coding sequences; moreover, structure compatibility/incompatibility constraints have been expanded. With these features, RNAiFold 2.0 allows the user to design single RNA molecules as well as hybridization complexes of two RNA molecules. Availability: the web server, source code and linux binaries are publicly accessible at http: //bioinformatics.bc.edu/clotelab/RNAiFold2.0.
Hammerhead ribozyme engineering
Current Opinion in Structural Biology, 1996
Of all the catalytic RNAs the hammerhead ribozyme is the most chemically modified and structurally studied. Such studies have resulted in improvements in the nuclease resistance of ribozymes, reductions in their size, and improvements in their catalytic efficiency. These improvements have facilitated the use of ribozymes for therapeutic applications, and have allowed us to study how the three-dimensional structure of the enzyme and its array of functional groups interact to create a catalytic site where a phosphodiester bond is cleaved.
A prototypic hammerhead ribozyme has three helices that surround an asymmetrical central core loop. We have mutagenlzed a hammerhead type ribozyme. In agreement with previous studies, progressive removal of stem-loop II from a three stemmed ribozyme showed that this region Is not absolutely critical for catalysis. However, complete elimination of stern II and its loop did reduce, but did not eliminate, function. In a stem-loop 11-deleted ribozyme, activity was best pre served when a purine, preferably a G, was present at position 1 0.1. This G contributed to catalysis irregard less of its role as either one part of a canonical pair with a C residue at 11.1 or a lone nucleotide with C (11.1) deleted. Computational methods using lattices gener ated 87 million three-dimensional chain fonns for a stem-loop 11-deleted RNA complex that preserved one potential G•C base pair at positions 10.1 and 11.1. This exhaustive set of chain forms Included one major class of structures with G(1 0.1) being spatially proximal to the GUCX cleavage site of the substrate strand. Strong coiTelatlons were observed between collnear arrange ment of stems I and Ill, constraints of base-pairing In the central core loop, and one particular placement of G(1 0.1) relative to the cleavage site. Our calculations of a stem-loop 11-deleted rlbozyme Indicate that without needing to Invoke any other constraints, the Inherent asymmetry In the lengths of the two loop strands (3 nt in one and 7 nt In the other) that compose the core and flank G10.1-C11.1 stipulated strongly this particular G placement. This suggests that the hammerhead rlbo zyme maintains an asymmetry In its internal loop for a necessary structureffunctlon reason.
Modulating RNA structure and catalysis: lessons from small cleaving ribozymes
Cellular and Molecular Life Sciences, 2009
RNA is a key molecule in life, and comprehending its structure/function relationships is a crucial step towards a more complete understanding of molecular biology. Even though most of the information required for their correct folding is contained in their primary sequences, we are as yet unable to accurately predict both the folding pathways and active tertiary structures of RNA species. Ribozymes are interesting molecules to study when addressing these questions because any modifications in their structures are often reflected in their catalytic properties. The recent progress in the study of the structures, the folding pathways and the modulation of the small ribozymes derived from natural, self-cleaving, RNA motifs have significantly contributed to today's knowledge in the field.
Structure—function studies of the hammerhead ribozyme
Current Opinion in Chemical Biology, 1997
Elucidation of the catalytic mechanism and structure-function relationship studies of the hammerhead ribozyme continue to be an area of intensive research. A combination of diverse approaches, such as X ray crystallography, spectral studies, chemical modifications, sequence variations and kinetic analyses, have provided valuable insight into the cleavage mechanism of this ribozyme. The hammerhead ribozyme crystal structures have provided valuable insight into conformational deformations needed to attain the catalytically active structure. Similarly, determination of ribozyme solution structure by spectroscopic analyses and the effect of divalent metal ions on RNA folding has further aided in the construction of a model for hammermead catalysis.