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)

Ribonuclease H (RNase H) is an endonuclease that cleaves only the RNA strand of an RNA-DNA hybrid to produce 5'-phosphate and 3'-hydroxy termini and lacks useful sequence specific recognition properties. A mutant form of the E. coli enzyme has been prepared that is suited for selective chemical modification at a site proximal to the substrate binding region. The chemical derivatization involves the formation of a disulfide linkage to a modified octadeoxyribonucleotide. The conjugate retains only 0.3% of the normal sequence independent RNase H activity demonstrating that substrate recognition can be modulated by a covalent appendage. A beta-globin RNA transcript containing a sequence complementary to that of the octadeoxyribonucleotide was cleaved in a catalytic fashion to two products upon treatment with the conjugate. The selectivity in the phosphodiester bond cleavage mediated by the conjugate was found to be different than that displayed by the nonderivatized enzyme. These results demonstrate the potential of semi-synthetic RNase H conjugates for mechanistic studies and their application as RNA targeted diagnostic or therapeutic agents.
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PMID:Sequence specific cleavage of messenger RNA by a modified ribonuclease H. 752 7

Ribonuclease H (RNase H) which recognizes and cleaves the RNA strand of mismatched RNA-DNA heteroduplexes can induce non-specific effects of antisense oligonucleotides. In a previous paper [Larrouy et al. (1992), Gene, 121, 189-194], we demonstrated that ODN1, a phosphodiester 15mer targeted to the AUG initiation region of alpha-globin mRNA, inhibited non-specifically beta-globin synthesis in wheat germ extract due to RNase H-mediated cleavage of beta-globin mRNA. Specificity was restored by using MP-ODN2, a methylphosphonate-phosphodiester sandwich analogue of ODN1, which limited RNase H activity on non-perfect hybrids. We report here that 2'-O-alkyl RNA-phosphodiester DNA sandwich analogues of ODN1, with the same phosphodiester window as MP-ODN2, are non-specific inhibitors of globin synthesis in wheat germ extract, whatever the substituent (methyl, allyl or butyl) on the 2'-OH. These sandwich oligomers induced the cleavage of non-target beta-globin RNA sites, similarly to the unmodified parent oligomer ODN1. This is likely due to the increased affinity of 2'-O-alkyl-ODN2 chimeric oligomers for both fully and partly complementary RNA, compared to MP-ODN2. In contrast, the fully modified 2'-O-methyl analogue of ODN1 was a very effective and highly specific antisense sequence. This was ascribed to its inability (i) to induce RNA cleavage by RNase H and (ii) to physically prevent the elongation of the polypeptide chain.
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PMID:RNase H is responsible for the non-specific inhibition of in vitro translation by 2'-O-alkyl chimeric oligonucleotides: high affinity or selectivity, a dilemma to design antisense oligomers. 756 53

To assess the usefulness of computer-assisted modeling of mRNA as an aid in design of antisense DNA, the efficiency of inhibition of translation of rabbit beta-globin mRNA by various antisense sequences was compared with calculated structures of the mRNA. The model obtained by consideration of 30 lowest-energy computer-simulated structures is consistent with the high accessibility of the AUG initiation codon region known from digestion with nucleases and with previous antisense inhibition studies reported in the literature. Additional antisense inhibition data were obtained with 20-mer phosphorothioate oligonucleotides, targeted to regions of beta-globin mRNA differing moderately in their degree of participation in intramolecular folding. The efficiency of translation arrest by the oligonucleotides in cell-free expression systems (wheat germ extract and rabbit reticulocyte lysate) was obtained by measuring incorporation of [35S]methionine into total protein, and corrected for sequence-nonspecific inhibition using brome mosaic virus mRNA. In the presence of RNase H (wheat germ system), the inhibitory activity of the oligonucleotides showed correlation with the calculated secondary structure of mRNA, in particular at low oligonucleotide-to-mRNA ratios (correlation coefficient, 0.95). No correlation was observed in the reticulocyte lysate system, in which the inhibition is mediated by translational arrest.
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PMID:Targeting of antisense DNA: comparison of activity of anti-rabbit beta-globin oligodeoxyribonucleoside phosphorothioates with computer predictions of mRNA folding. 815 75

Efforts have been made to reduce the disadvantages associated with the natural oligonucleotides (all-PO) for antisense application by introducing phosphorothioate (PS) linkages into the molecule. A series of such oligodeoxynucleotide copolymers (17-mers) complementary to the coding region of the rabbit beta-globin mRNA, and containing different proportions and arrangements of PO and PS bonds, were synthesized and tested for their protein-binding properties, nuclease stability in vitro, hybridizing ability with the complementary DNA (cDNA), ability to form RNase H-sensitive substrates and antisense activity in cell-free systems. The melting temperatures (Tm) of the co-polymers were reduced by up to 6 degrees C relative to the all-PO oligo, compared to 11 degrees C for the all-PS compound, indicating intermediate hybridizing abilities of the co-polymers. The protein-binding studies with human serum albumin exhibited a linear correlation with the percentage of PS linkage present in the molecule. Nuclease susceptibilities of the co-polymers were also improved, but the number and position of the PS linkages played a significant role in such improvement. Translation inhibition by these oligonucleotides was only found in wheat germ agglutinin (WGA) extract, but not in rabbit reticulocyte lysate (RRL) cell-free system, suggesting the involvement of RNase H in their antisense activities. Provided they have > or = 50% PS linkages, the co-polymers produced almost the same increased inhibition in the WGA system as that of the all-PS oligo. The translation arrest in WGA extract is in good agreement with the in vitro cleavage found for rabbit globin mRNA in the oligo:mRNA duplex by RNase H alone. It is concluded that a copolymer of PO and PS might be preferable to either all-PO or all-PS for antisense applications.
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PMID:Phosphorothioate-phosphodiester oligonucleotide co-polymers: assessment for antisense application. 838 13

An array of 1,938 oligodeoxynucleotides (ONs) ranging in length from monomers to 17-mers was fabricated on the surface of a glass plate and used to measure the potential of oligonucleotide for heteroduplex formation with rabbit beta-globin mRNA. The oligonucleotides were complementary to the first 122 bases of mRNA comprising the 5' UTR and bases 1 to 69 of the first exon. Surprisingly few oligonucleotides gave significant heteroduplex yield. Antisense activity, measured in a RNase H assay and by in vitro translation, correlated well with yield of heteroduplex on the array. These results help to explain the variable success that is commonly experienced in the choice of antisense oligonucleotides. For the optimal ON, the concentration required to inhibit translation by 50% was found to be five times less than for any other ON. We find no obvious features in the mRNA sequence or the predicted secondary structure that can explain the variation in heteroduplex yield. However, the arrays provide a simple empirical method of selecting effective antisense oligonucleotides for any RNA target of known sequence.
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PMID:Selecting effective antisense reagents on combinatorial oligonucleotide arrays. 918 66

A rapid and simple method for determining accessible sites in RNA that is independent of the length of target RNA and does not require RNA labeling is described. In this method, target RNA is allowed to hybridize with sequence-randomized libraries of DNA oligonucleotides linked to a common tag sequence at their 5'-end. Annealed oligonucleotides are extended with reverse transcriptase and the extended products are then amplified by using PCR with a primer corresponding to the tag sequence and a second primer specific to the target RNA sequence. We used the combination of both the lengths of the RT-PCR products and the location of the binding site of the RNA-specific primer to determine which regions of the RNA molecules were RNA extendible sites, that is, sites available for oligonucleotide binding and extension. We then employed this reverse transcription with the random oligonucleotide libraries (RT-ROL) method to determine the accessible sites on four mRNA targets, human activated ras (ha-ras), human intercellular adhesion molecule-1 (ICAM-1), rabbit beta-globin, and human interferon-gamma (IFN-gamma). Our results were concordant with those of other researchers who had used RNase H cleavage or hybridization with arrays of oligonucleotides to identify accessible sites on some of these targets. Further, we found good correlation between sites when we compared the location of extendible sites identified by RT-ROL with hybridization sites of effective antisense oligonucleotides on ICAM-1 mRNA in antisense inhibition studies. Finally, we discuss the relationship between RNA extendible sites and RNA accessibility.
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PMID:Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase. 1123 88

Antisense oligonucleotides act as exogenous inhibitors of gene expression by binding to a complementary sequence on the target mRNA, preventing translation into protein. Antisense technology is being applied successfully as a research tool and as a molecular therapeutic. However, a quantitative understanding of binding energetics between short oligonucleotides and longer mRNA targets is lacking, and selecting a high-affinity antisense oligonucleotide sequence from the many possibilities complementary to a particular RNA is a critical step in designing an effective antisense inhibitor. Here, we report measurements of the thermodynamics and kinetics of hybridization for a number of oligodeoxynucleotides (ODNs) complementary to the rabbit beta-globin (RBG) mRNA using a binding assay that facilitates rapid separation of bound from free species in solution. A wide range of equilibrium dissociation constants were observed, and association rate constants within the measurable range correlated strongly with binding affinity. In addition, a significant correlation was observed of measured binding affinities with binding affinity values predicted using a thermodynamic model involving DNA and RNA unfolding, ODN hybridization, and RNA restructuring to a final free energy minimum. In contrast to the behavior observed for hybridization of short strands, the association rate constant increased with temperature, suggesting that the kinetics of association are related to disrupting the native structure of the target RNA. The rate of cleavage of the RBG mRNA in the presence of ribonuclease H and ODNs of varying association kinetics displayed apparent first-order kinetics, with the rate constant exhibiting binding-limited behavior at low association rates and reaction-limited behavior at higher rates. Implications for the rational design of effective antisense reagents are discussed.
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PMID:Thermodynamic and kinetic characterization of antisense oligodeoxynucleotide binding to a structured mRNA. 1175 23

Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides up to 17 nt in length, and evaluated their binding efficiency and selectivity towards complementary RNA, their biological stability in serum, their RNase H inducing potential and their antisense activity in a cellular assay. Relative to RNA or 2'-O-Me-phosphorothioate (PS)-RNA, fully modified tc-oligodeoxynucleotides, 10-17 nt in length, show enhanced selectivity and enhanced thermal stability by approximately 1 degrees C/modification in binding to RNA targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37 degree C. Moreover, tc-DNA-RNA duplexes are not substrates for RNase H. To test for antisense effects in vivo, we used HeLa cell lines stably expressing the human beta-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in beta-globin mRNA. Lipofectamine-mediated delivery of a 17mer tc-oligodeoxynucleotide complementary to the 3'-cryptic splice site results in correction of aberrant splicing already at nanomolar concentrations with up to 100-fold enhanced efficiency relative to a 2'-O-Me-PS-RNA oligonucleotide of the same length and sequence. In contrast to 2'-O-Me-PS-RNA, tc-DNA shows antisense activity even in the absence of lipofectamine, albeit only at much higher oligonucleotide concentrations.
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PMID:Antisense properties of tricyclo-DNA. 1208 57

AU-rich elements (AREs) in 3'-untranslated regions of mRNAs confer instability. They target mRNAs for rapid deadenylation and degradation and may enhance decapping. The p38 MAPK pathway stabilizes many otherwise unstable ARE-containing mRNAs encoding proteins involved in inflammation; however, the mRNA decay step(s) regulated by the signaling pathway are unknown. To investigate whether it regulates deadenylation or the decay of the mRNA body, we used a tetracycline-regulated beta-globin mRNA reporter system to transcribe pulses of mRNA of uniform length. We measured on Northern gels the migration of reporter mRNAs isolated from cells transfected only with reporter plasmid or co-transfected with an active mutant of MAPK kinase-6, and treated either with or without the p38 MAPK inhibitor SB 203580. Differences in migration were shown by RNase H mapping with oligo(dT) to be due to poly(A) shortening. Insertion of an ARE into the beta-globin reporter mRNA promoted rapid deadenylation and decay of hypo-adenylated reporter mRNA. p38 MAPK activation inhibited the deadenylation of reporter mRNAs containing either the cyclooxygenase-2 or tumor necrosis factor AREs. The regulation of deadenylation by p38 MAPK was found to be specific because deadenylation of the beta-globin reporter mRNA either lacking an ARE or containing the c-Myc 3'-untranslated region (which is not p38 MAPK-responsive) was unaffected by p38 MAPK. It was concluded that the p38 MAPK pathway predominantly regulates deadenylation, rather than decay of the mRNA body, and this provides an explanation for why p38 MAPK regulates mRNA stability in some situations and translation in others.
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PMID:p38 Mitogen-activated protein kinase stabilizes mRNAs that contain cyclooxygenase-2 and tumor necrosis factor AU-rich elements by inhibiting deadenylation. 1288 63

Phosphoenolpyruvate carboxykinase (PEPCK) is regulated solely by alterations in gene expression that involve changes in rates of PEPCK mRNA transcription and degradation. A tetracycline-responsive promoter system was used to quantify the half-life of various chimeric beta-globin-PEPCK (betaG-PCK) mRNAs in LLC-PK -F(+) cells. The control betaG mRNA was extremely stable (t(1/2) = 5 days). However, betaG-PCK-1 mRNA, which contains the entire 3'-UTR of the PEPCK mRNA, was degraded with a half-life of 1.2 h. RNase H treatment indicated that rapid deadenylation occurred concomitant with degradation of the betaG-PCK-1 mRNA. Previous studies indicate that PCK-7, a 50-nucleotide segment at the 3'-end of the 3'-UTR, binds an unidentified protein that may contribute to the rapid decay of the PEPCK mRNA. However, the chimeric betaG-PCK-7 mRNA has a half-life of 17 h. Inclusion of the adjacent PCK-6 segment, a 23-bp AU-rich region, produced the betaG-PCK-6/7 mRNA, which has a half-life of 3.6 h. The betaG-PCK-3 mRNA that contains the 3'-half of 3'-UTR was degraded with the same half-life. Surprisingly, the betaG-PCK-2 mRNA, containing the 5'-end of the 3'-UTR, was also degraded rapidly (t((1/2)) = 5.4 h). RNA gel shift analyses established that AUF1 (hnRNP D) binds to the PCK-7, PCK-6, and PCK-2 segments with high affinity and specificity. Mutational analysis indicated that AUF1 binds to a UUAUUUUAU sequence within PCK-6 and the stem-loop structure and adjacent CU-region of PCK-7. Thus, AUF1 binds to multiple destabilizing elements within the 3'-UTR that participate in the rapid turnover of the PEPCK mRNA.
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PMID:3'-Untranslated region of phosphoenolpyruvate carboxykinase mRNA contains multiple instability elements that bind AUF1. 1595 44


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