Targeting RNA structures by antisense oligonucleotides

The presence of folded regions in RNA competes with the binding of a complementary oligonucleotide, resulting in a weak antisense effect. Due to the key role played by a number of RNA structures in the natural regulation of gene expression it might be of interest to design antisense sequences able to selectively interact with such motifs in order to interfere with the biological processes they mediate. Different possibilities have been explored. A high affinity oligomer will disrupt the structure; if the target structure is solved one can take advantage of unpaired bases (bulges, loops) to minimize the thermodynamic cost of the binding. Alternatively, the folded structure can be accommodated within the complex via the formation of a local triple helix. Oligomers able to adapt to the RNA structure (aptamers) can be extracted by in vitro selection from randomly synthesized libraries comprising several billions of sequences.     

Selective inhibition of cell-free translation by oligonucleotides targeted to a mRNA hairpin structure

Using an in vitro selection approach we have previously isolated oligodeoxy aptamers that can bind to a DNA hairpin structure without disrupting the double-stranded stem. We report here that these oligomers can bind to the RNA version of this hairpin, mostly through pairing with a designed 6 nt anchor. The part of the aptamer selected against the DNA hairpin did not increase stability of the RNA-aptamer complex. However, it contributed to the binding site for Escherichia coli RNase H, leading to very efficient cleavage of the target RNA. In addition, a 2'- O -methyloligoribonucleotide analogue of one selected sequence selectively blocked in vitro translation of luciferase in wheat germ extract by binding to the hairpin region inserted upstream of the initiation codon of the reporter gene. Therefore, non-complementary oligomers can exhibit antisense properties following hybridization with the target RNA. Our study also suggests that in vitro selection might provide a means to extend the repertoire of sequences that can be targetted by antisense oligonucleotides to structured RNA motifs of biological importance.     

Structure-based discovery of ligands targeted to the RNA double helix

Ligands capable of specific recognition of RNA structures are of interest in terms of the principles of molecular recognition as well as potential chemotherapeutic applications. We have approached the problem of identifying small molecules with binding specificity for the RNA double helix through application of the DOCK program [Kuntz, I. D., Meng, E. C., and Shoichet, B. K. (1994) Acc. Chem. Res. 27, 117-123], a structure-based method for drug discovery. A series of lead compounds was generated through a database search for ligands with shape complementarity to the RNA deep major groove. Compounds were then evaluated with regard to their fit into the minor groove of B DNA. Those compounds predicted to have an optimal fit to the RNA groove and strong discrimination against DNA were examined experimentally. Of the 11 compounds tested, 3, all aminoglycosides, exhibited pronounced stabilization of RNA duplexes against thermal denaturation with only marginal effects on DNA duplexes. One compound, lividomycin, was examined further, and shown to facilitate the ethanol-induced B to A transition in calf thymus DNA. Fluorine NMR solvent isotope shift measurements on RNA duplexes containing 5-fluorouracil provided evidence that lividomycin binds in the RNA major groove. Taken together, these results indicate that lividomycin recognizes the general features of the A conformation of nucleic acids through deep groove binding, confirming the predictions of our DOCK analysis. This approach may be of general utility for identifying ligands possessing specificity for additional RNA structures as well as other nucleic acid structural motifs.     

Deconvolution of combinatorial libraries for Drug discovery: theoretical comparison of pooling strategies

Synthesis and testing of mixtures of compounds in a combinatorial library allow much greater throughput than synthesis and testing of individual compounds. When mixtures of compounds are screened, however, the possibility exists that the most active compound will not be identified. The specific strategies employed for pooling and deconvolution will affect the likelihood of success. We have used a nucleic acid hybridization example to develop a theoretical model of library deconvolution for a library of more than 250,000 compounds. This model was used to compare various strategies for pooling and deconvolution. Simulations were performed in the absence and presence of experimental error. We found iterative deconvolution to be most reliable when active molecules were assigned to the same subset in early rounds. Reliability was reduced only slightly when active molecules were assigned randomly to all subsets. Iterative deconvolution with as many as 65,536 compounds per subset did not drastically reduce the reliability compared to one-at-a-time testing. Pooling strategies compared using this theoretical model are compared experimentally in an accompanying paper.     

In vitro selection of RNA lectins: using combinatorial chemistry to interpret ribozyme evolution

BACKGROUND: It has been hypothesized that the fact that both ribosomal RNA and the group I intron can bind to aminoglycoside antibiotics implies that these RNAs are evolutionarily related. This hypothesis requires the assumption that there are relatively few ways for RNA molecules to form aminoglycoside-binding sites. RESULTS: We have used in vitro selection to determine the diversity of aminoglycoside-binding sites that can be formed by RNA molecules. We have generated RNA 'lectins' that can bind aminoglycosides tightly and specifically. Sequence analysis indicates that there are many different ways to form tight and specific aminoglycoside binding sites. These artificially selected binding sites are functionally similar to those that have arisen from natural selection. CONCLUSIONS: Our results suggest that the presence of aminoglycoside-binding sites on RNA molecules may not be a useful trait for determining evolutionary relatedness. Instead, the fact that RNA molecules can bind these 'low molecular-weight effectors' may indicate that natural products such as aminoglycosides have evolved to exploit sequence- and structure-specific recognition of nucleic acids, in much the same way that lexitropsins have been designed by chemists to recognise specific nucleic acid sequences.     

Synthesis and anti-influenza virus-A activity of circular dumbbell RNA DNA chimeric oligonucleotides

We have designed a new type of antisense oligonucleotide, containing two hairpin loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA)) in the double helical stem (nicked and circular dumbbell DNA/RNA chimeric oligonucleotides). The reaction of the nicked and circular dumbbell DNA/RNA chimeric oligonucleotides with RNase H gave the corresponding anti-DNA together with the sense RNA cleavage products. These oligonucleotides were more resistant to exonuclease attack. We also describe the anti-Fluv activities of circular dumbbell DNA/RNA chimeric oligonucleotides.     

An in vitro messenger RNA binding assay as a tool for identifying hybridization-competent antisense oligonucleotides

Targeting, use of appliances, and standards of outcome for General Dental Service orthodontic cases collected between 1990 and 1991 were compared with a sample of cases from an earlier study, collected between 1987 and 1988, using the PAR index and IOTN. Comparisons are made generally and in relation to the changes in prior approval regulations for cases started since October 1987. More lower-need cases were treated, but there were no more "unnecessary' treatments and there has been a limited improvement in outcomes, as assessed by the indices, associated with increased use of fixed appliances since 1987. Prior approval appeared to give no tangible benefits in terms of levels of unnecessary treatment or quality of outcome.     

A genetic algorithm based molecular modeling technique for RNA stem-loop structures

A new modeling technique for arriving at the three dimensional (3-D) structure of an RNA stem-loop has been developed based on a conformational search by a genetic algorithm and the following refinement by energy minimization. The genetic algorithm simultaneously optimizes a population of conformations in the predefined conformational space and generates 3-D models of RNA. The fitness function to be optimized by the algorithm has been defined to reflect the satisfaction of known conformational constraints. In addition to a term for distance constraints, the fitness function contains a term to constrain each local conformation near to a prepared template conformation. The technique has been applied to the two loops of tRNA, the anticodon loop and the T-loop, and has found good models with small root mean square deviations from the crystal structure. Slightly different models have also been found for the anticodon loop. The analysis of a collection of alternative models obtained has revealed statistical features of local variations at each base position.     

Physicochemical approaches to designing NiAl-based alloys for high-temperature operation

The structure and properties of new-type materials based on light refractory nickel monoaluminide NiAl as a structural material are analytically reviewed. The choice of various alloying systems and structural-phase states of NiAl-based structural materials, including structural materials, is analyzed, and the choice of the processes of production of the materials is grounded, as applied to their composition.     

如何应用SSA与SSD方法设计开发MIS

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