Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recently, we described a new class of antisense oligonucleotides that can be used to direct the cleavage of mRNAs in Xenopus laevis embryos by RNase H (Dagle et al., Nucleic Acids Res. 18, 4751-4757). In this study, we have examined several factors that determine the activity of these derivatives. In embryos, oligodeoxyribonucleotides were found to be rapidly degraded by a 3' exonuclease. Modification of 3'-terminal phosphodiester linkages as phosphoramidates blocks this activity. The predominant sites of endonucleolytic cleavage within the embryo are localized close to the 5' termini demonstrating the necessity of multiply modifying phosphodiester linkages at each end of the molecule. A stretch of at least six consecutive phosphodiester linkages is required to form an effective substrate for Xenopus RNase H; mRNA degradation with an oligonucleotide containing fewer than six contiguous unmodified internucleoside linkages is greatly diminished. Injection of an anti-cyclin B oligonucleotide containing eight unmodified residues results in degradation of cyclin B mRNAs and subsequent inhibition of embryonic cell division. An oligonucleotide with the same sequence but containing four consecutive phosphodiesters has no observable effect on the cell cycle. This last observation suggests that, in Xenopus embryos, hybridization alone has a limited role, if any, in oligonucleotide-mediated inhibition of gene expression.
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PMID:Pathways of degradation and mechanism of action of antisense oligonucleotides in Xenopus laevis embryos. 166 7

During meiotic maturation and early embryonic cycles, the activity of maturation-promoting factor (MPF) cycles in exact correspondence with the mitotic cycles. For the appearance of MPF activity in starfish, protein synthesis is required except in the first meiotic cycle. In order to identify newly synthesized proteins involved in the regulation of MPF activity, we extracted poly(A)+ RNA from starfish eggs, and found that the egg poly(A)+ RNA induced germinal vesicle breakdown (GVBD) upon injection into immature oocytes of starfish and Xenopus. The molecular size of the poly(A)+ RNA responsible for GVBD was estimated to be approximately 22S by sucrose density gradient centrifugation. Since these characteristics of the starfish egg poly(A)+ RNA are similar to those of cyclin mRNAs from sea urchin and surf clam eggs, we synthesized a 50-mer antisense-cyclin oligonucleotide probe coding for a part of the sea urchin cyclin cDNA and used this to screen starfish RNA. The Northern blot analysis showed that the starfish egg RNA contained cyclin homologous transcripts. Incubation of the starfish egg poly(A)+ RNA and the antisense-cyclin oligonucleotide with RNase H completely destroyed its GVBD-inducing activity. These results indicated that starfish cyclin mRNA was the only poly(A)+ RNA responsible for GVBD. We constructed a starfish egg cDNA library to clone starfish cyclin cDNA. The longest cDNA clone containing 2190 base pairs was sequenced. The longest open reading frame consisted of 395 amino acid residues, and the predicted molecular size was 48 kDa. Comparison of the deduced amino acid sequences of starfish cyclin with known cyclins indicated that the starfish cyclin belongs to the B-type. Injection of synthetic mRNA of starfish cyclin caused GVBD in immature oocytes of starfish and Xenopus, while injection of synthetic mRNA of human CDC2 had no effect. The Northern blot analysis of starfish RNA extracted at various stages of the meiotic cycles suggested that the starfish cyclin transcript was stored in its polyadenylated form even in immature oocytes and was further polyadenylated at maturation.
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PMID:The starfish egg mRNA responsible for meiosis reinitiation encodes cyclin. 169 83

We have designed antisense oligodeoxyribonucleotides which are both highly resistant to nucleolytic degradation and also serve as substrates for ribonuclease H. Using these compounds we have targeted the specific degradation of several maternal mRNAs present in Xenopus laevis oocytes and early embryos. Several internucleoside linkages at both the 3' and 5' ends of the oligonucleotides were modified as phosphoramidates to provide complete protection against exonucleases, the predominant nucleolytic activity found in both oocytes and embryos. Eight Internal linkages were left unmodified to provide a substrate for RNase H. Degradation of specific embryonic mRNAs was accomplished using subtoxic amounts of the modified oligonucleotides. Specific depletion of An2, a localized mRNA encoding the alpha subunit of the mitochondrial ATPase, produced embryos that gastrulated later than control embryos and arrested in development prior to neurulation. A modified oligonucleotide targeting Xenopus cyclin B1 and cyclin B2 mRNA was also synthesized. Following the injection of one blastomere of a two-cell embryo with the anti-cyclin oligonucleotide, cell division in that half of the embryo was inhibited, demonstrating the in vivo importance of these cyclins in mitosis. The oligonucleotide analogs described here should be useful in studying developmentally significant proteins in Xenopus.
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PMID:Targeted degradation of mRNA in Xenopus oocytes and embryos directed by modified oligonucleotides: studies of An2 and cyclin in embryogenesis. 169 75

Transcripts representing mRNAs of three Xenopus cyclins, B1, B4 and B5, were hybridised to arrays of oligonucleotides scanning the first 120 nt of the coding region to assess the ability of the immobilised oligonucleotides to form heteroduplexes with their targets. Oligonucleotides that produced high heteroduplex yield and others that showed little annealing were assayed for their effect on translation of endogenous cyclin mRNAs in Xenopus egg extracts and their ability to promote cleavage of cyclin mRNAs in oocytes by RNase H. Excellent correlation was found between antisense potency and affinity of oligonucleotides for the cyclin transcripts as measured by the array, despite the complexity of the cellular environment.
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PMID:Antisense oligonucleotides selected by hybridisation to scanning arrays are effective reagents in vivo. 1135 73

We show that binding of an antisense oligonucleotide can lead to considerable changes in the target mRNA structure. The approaches described here are not only useful in the study of intra-molecular interactions in RNAs but can also be used to design oligonucleotides that facilitate binding of other antisense reagents. Such "cooperatively acting" antisense reagents have the potential to overcome several problems faced in their use, for example, low efficacy and non-specificity. To provide proof-of-principle, radiolabelled cyclin B5 transcript, a model mRNA, was hybridised with an antisense oligonucleotide array. An oligonucleotide sequence was selected from the array hybridisation data and was used in an RNase H/oligonucleotide library (dN12) assay to assess its ability to enhance cleavage of target RNA. This oligonucleotide ("facilitator") greatly enhanced cleavage of B5 RNA at a neighbouring site. The precise position and sequence of this "new" site was determined by further hybridisation of RNA-facilitator mixture to the B5 antisense array. Antisense oligonucleotides designed from the new region were used in combination with the facilitator in a cell-free system. The presence of the facilitator considerably enhanced cleavage of B5 RNA with these oligonucleotides. These approaches may be useful in designing antisense reagents against sequences of specific interest, such as, gene fusion sites, splice variants, mutant alleles and tightly structured RNA sites.
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PMID:Structural rearrangements in RNA on the binding of an antisense oligonucleotide: implications for the study of intra-molecular RNA interactions and the design of cooperatively acting antisense reagents with enhanced efficacy. 1584 55