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)

Recent interest in the use of adriamycin-DNA complex as an approach to improve the therapeutic effectiveness and to reduce toxicity of adriamycin for cancer chemotherapy requires an in-depth understanding of the physicochemical and biochemical properties of such complexes. The interactions of adriamycin with single-strand polydeoxyribonucleotides, double-strand DNA, and double-strand ribodeoxyribopolynucleotide hybrids were therfore investigated. Association constants (Kapp) of adriamycin and polynucleotides were obtained. These data showed that the inherent variable in such complex lies in the composition of the polynucleotides. Alternate deoxyguanylate (dG)-deoxycytidylate (dC) sequence binds 7-fold better than alternate deoxyadenylate (dA)-deoxythymidylate (dT) sequence. Comparative studies of the hydrolysis of DNA duplexes by deoxyribonucleases I and II with and without adriamycin were also carried out. The rate of hydrolysis decreased in the order poly(dA-dT) greater than calf thymus DNA greater than poly(dG-dC) greater than poly(dA)-poly(dT) greater than poly(dG)-poly(dC) for DNase I and poly(dA)-dT) greater than calf thymus DNA greater than poly(dG-dC) greater than poly(dA)-poly(dT) greater than poly(dG)-poly(dC) for DNase II. Intercalation of adriamycin to deoxyribopolynucleotide duplex resulted in inhibition of DNase II two to three times more than tat of DNase I. On the other hand, intercalation of adriamycin to homodeoxypolynucleotide duplex poly(dA)-poly(dT) and poly(dG)-poly(dC) enhanced the DNase I hydrolysis. If DNase I activity could be related to serum DNase and DNase II related to tumor lyososomal DNase as in the endocytosis mechanism proposed by Trouet et al. (Cancer Chemotherapy Rept., 59: 260, 1975), the best adriamycin carrier suggested by this investigation could be poly(dA)-poly(dT) and poly(dG-dC). It is also suggested in this study that adriamycin-RNA-DNA hybrid could be of interest as an antiviral agent by a similar release mechanism via RNase H, an enzyme associated with viral reverse transcriptase.
Cancer Res 1976 Sep
PMID:Effect of deoxyribonuclease on adriamycin-polynucleotide complexes. 97 96

Inhibition of polypeptide chain elongation with the mRNA-complementary (antisense) oligonucleotide has been realized through a RNase H independent mechanism. Nuclease resistant complementary non-natural alpha-17-mer oligonucleotide did not inhibit cell-free protein biosynthesis of beta-globin in the wheat germ system because it did not elicit RNase H activity. Linkage of alkylating group [4-(N-2-chloroethyl-N-methyl)-aminobenzyl]-methylamine to the 5'-terminus of the alpha-oligomer led to the formation of its covalent adduct with mRNA which could not be translated in vitro. Linkage of hydrophobic residues to the terminal phosphates of natural oligonucleotides increased their stability against nucleases and uptake by human cancer cells. A porphyrin, substituted in the meso-position by aromatic groups, gave a rise to an approximately six-fold increase of uptake and cholesterol a 30-100-fold increase. Eighty percent of bound derivatives were found in cytoplasmic cellular fractions.
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PMID:Effect of the terminal phosphate derivatization of beta- and alpha-oligodeoxynucleotides on their antisense activity in protein biosynthesis, stability and uptake by eucaryotic cells. 132 80

Phosphodiester oligonucleotides (ODNs) and their analogs are presently being investigated as potential antisense therapeutics in the treatment of viral infections and various forms of cancer. here, we would like to report results from an investigation of activity for a ribonuclease H (RNase H) mediated RNA digestion assay in the duplexes formed by an ODN or the ODN analog, N3'-->P5' phosphoramidate (3'-phosphoramidate), and complimentary RNA strands. Capillary gel electrophoresis (CGE) proved to be an effective method for determining RNA hydrolysis in the presence of RNase H. RNA and an ODN or RNA and a 3'-phosphoramidate were hybridized in a Tris-HCl, MgCl2 buffer at room temperature (RT) and incubated with RNase H. Digestions were carried out at RT or at 37 degrees C. Control samples were unhybridized RNA with RNase H, RNA without RNase H, and duplexes (RNA-ODN or 3'-phosphoramidate) without RNase H. All controls were incubated in Tris-HCl, MgCl2 buffer, and sample aliquots were analyzed at various time intervals. A homodecamer, (dT)10, was used as an internal standard to determine the relative migration time of the RNA strand. The final digestion products for the duplexes and the various controls were monitored by CGE. In addition, polyacrylamide gel electrophoresis (PAGE) was used in conjunction with Stains-All (staining) and a densitometric analysis to verify CGE results.
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PMID:Analysis of a ribonuclease H digestion of N3'-->P5' phosphoramidate-RNA duplexes by capillary gel electrophoresis. 758 76

Antisense oligonucleotides (ODNs) and peptide nucleic acids (PNAs) are potential therapeutics for eradication of malignancies, viral infections, and other pathologies. However, ODNs and PNAs in general are unable to cross cellular membranes and blood-tissue barriers, such as the blood-brain barrier (BBB), which is only permeable to lipophilic molecules of molecular weight <600 Da. Cellular delivery systems based on conjugates of streptavidin (SA) and the OX26 monoclonal antibody directed to the transferrin receptor may be employed as a universal carrier for the transport of mono-biotinylated peptides, ODNs, or PNAs. 3'-Biotinylation of phosphodiester (PO)-ODN produces complete protection of ODN against serum and cellular 3'-exonucleases, facilitating the conjugation to avidin-based delivery systems and maintaining the activation of RNase H. These delivery systems markedly increased the cellular uptake and antisense efficacy of 3'-biotinylated ODNs in models of Alzheimer's disease and HIV-AIDS. In vivo brain delivery studies demonstrated that 3'-protected PO-ODNs and PO-phosphorothioate(PS)-ODN hybrids containing a single PO linkage are subjected to endonuclease degradation in vivo. On the contrary PS-ODNs, which were also protected at 3'-terminus by biotinylation, are metabolically stable in vivo and resistant to exo/endonuclease degradation. However, because of the strong binding of these oligomers to plasma protein, PS-ODNs are poorly transported into the brain through the BBB by the OX26-SA delivery vector following intravenous administration. PNAs are also resistant to exo/endonuclease and protease degradation, and these molecules biotinylated at the amino terminal group were transported into the brain by the OX26-SA delivery system with brain uptake levels comparable to that of morphine. Using the rev gene of HIV as a model target, RNase protection assays and cell-free translation arrest showed that the PNA-OX26-SA conjugate maintained active recognition and inactivation of target mRNA, respectively. The overall experimental evidence suggests that PNA-OX26-SA conjugates represent optimal antisense molecules for drug delivery to the brain.
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PMID:Drug delivery of antisense molecules to the brain for treatment of Alzheimer's disease and cerebral AIDS. 981 82

This study was aimed at creating a more effective tumor cell vaccine by suppressing Ii protein in the presence of MHC class II molecules within a cancer cell. Absence of the Ii protein, which normally blocks the antigenic-peptide-binding site of MHC class II molecules at synthesis in the endoplasmic reticulum, presumably increases the range of cancer-related epitopes presented to CD4+ helper T cells. Effective suppression of Ii protein was achieved with an antisense, phosphorothioate oligonucleotide, which was selected on the basis of (1) the RNase H activation assay, (2) an assay for Ii protein suppression, and (3) a test for potency with respect to the extent of base sequence ("sequence walking"). The SaI murine sarcoma, which is MHC-class-I+ and MHC-class-II-, Ii-protein-, upon transfection with genes for either interferon gamma or the MHC class II transactivator, came to express MHC class II molecules and Ii protein. In each line of transfected tumor cells, the antisense oligonucleotide profoundly suppressed Ii protein in 35%-55% cells, without affecting expression of MHC class II molecules. Inoculation of mice with such Ii-protein-suppressed tumor vaccine cells, after either formaldehyde fixation or X-irradiation, led to much greater protection against challenge with the parental SaI sarcoma than did inoculation with untreated cells. This approach to cancer cell vaccination can be applied in a wide range of human tumors.
Cancer Immunol Immunother 1999 Dec
PMID:Cancer immunotherapy by antisense suppression of Ii protein in MHC-class-II-positive tumor cells. 1060 87

We have designed and studied antisense oligodeoxynucleotides (oligonucleotides; oligos) which we call 'pseudo-cyclic oligonucleotides' (PCOs). PCOs contain two oligonucleotide segments attached through their 3'-3'- or 5'-5'-ends. One of the segments of the PCO is an antisense oligo complementary to a target mRNA, and the other is a short protective oligo that is 5-8 nucleotides long and complementary to the 3'- or 5'-end of the antisense oligo. As a result of complementarity between the antisense and protective oligo segments, PCOs form intramolecular pseudo-cyclic structures in the absence of the target RNA. The antisense oligo segment of PCOs used for the studies described here is complementary to an 18-nucleotide-long site on the mRNA of the protein kinase A regulatory subunit RIalpha (PKA-RIalpha). Thermal melting studies of PCOs in the absence and presence of the complementary RNA suggest that the pseudo-cyclic structures formed in the absence of the target RNA dissociate, bind to the target RNA, and form heteroduplexes. The results of RNase H cleavage assays suggest that PCOs bind to complementary RNA and activate RNase H in a manner similar to that of an 18-mer conventional antisense PS-oligo. In snake venom (a 3'-exonuclease) or spleen (a 5'-exonuclease) phosphodiesterase digestion studies, PCOs are more stable than conventional antisense oligos because of the presence of 3'-3'- or 5'-5'-linkages and the formation of intramolecular pseudo-cyclic structures. PCOs with a phosphorothioate antisense oligo segment inhibited cell growth of MDA-MB-468 and GEO cancer cell lines similar to that of the conventional antisense PS-oligo, suggesting efficient cellular uptake and target binding. The nuclease stability studies in mice suggest that PCOs have higher in vivo stability than antisense PS-oligos. The studies in mice showed similar pharmacokinetic and tissue distribution profiles for PCOs to those of antisense PS-oligos in general, but rapid elimination from selected tissues.
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PMID:Pseudo-cyclic oligonucleotides: in vitro and in vivo properties. 1065 77

Stereo-enriched [Rp] and [Sp]-phosphorothioate oligodeoxynucleotides are synthesized using oxazaphospholidine derivatized monomers. Three different designs of phosphorothioate oligodeoxynucleotides (PS-oligos), (i) stereo-enriched all-[Rp] or all-[Sp] PS-linkages, (ii) stereo-random mixture of PS-linkages, and (iii) segments containing certain number of stereo-enriched [Rp] and [Sp] PS-linkages ([Sp-Rp-Sp] or [Rp-Sp-Rp]), have been studied. Thermal melting studies of these PS-oligos with RNA complementary strands showed that the binding affinities are in the order [Rp] > [Sp-Rp-Sp]-[Rp-Sp-Rp] > stereo-random > [Sp]. Circular dichroism (CD) studies suggest that the stereochemistry of the PS-oligo does not affect the global conformation of the duplex. The in vitro nuclease stability of these PS-oligos is in the order [Sp] > [Sp-Rp-Sp] > stereo-random > [Rp]. The RNase H activation is in the order [Rp] > stereo-random > [Rp-Sp-Rp] > [Sp] > [Sp-Rp-Sp]. Studies in a cancer cell line of PS-oligos targeted to MDM2 mRNA showed that all oligos had similar biological activity under the experimental conditions employed. Protein- and enzyme-binding studies showed insignificant stereo-dependent binding to proteins. The [Sp] and [Sp-Rp-Sp] chimeric and stereo-random PS-oligos that contained a CpG motif showed higher cell proliferation than [Rp] PS-oligo of the same sequence.
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PMID:Stereo-enriched phosphorothioate oligodeoxynucleotides: synthesis, biophysical and biological properties. 1096 87

Kirsten-ras is frequently mutated in colorectal cancers and may be an important therapeutic target, particularly because we have previously shown that acquisition of a mutation is associated with a poorer outcome. Understanding the role of Kirsten-ras and the consequences of inhibiting its activity or expression will contribute to our comprehension of colorectal cancer biology and may help to rationalize the choice of molecular targets suitable for therapeutic manipulation. Therefore we undertook a simple screen, incubating a library of oligonucleotides with Kirsten-ras mRNA and RNase H to identify an antisense oligonucleotide that effectively inhibited Kirsten-ras expression. We show for the first time in a human colon cancer cell line that inhibition of Kirsten-ras expression inhibits constitutive phosphorylation of Erk1/2, but not c-Akt, suggesting that in these cells constitutive phosphorylation of Erk 1/2 is dependent upon Kirsten-ras. Successful inhibition of Kirsten-ras had little effect on cell number or cell death and there was no evidence for accumulation of cells in any particular phase of the cell cycle. Kirsten-ras inhibition significantly reduced secretion of VEGF-A165 into the culture medium. Gene expression profiling by microarray detected altered expression of a number of genes. Of particular interest for future studies was the altered expression of genes encoding products involved in protein trafficking and the potential effects of these changes on cell adhesion. Our results suggest that, at least in this model, Kirsten-ras may contribute to malignancy predominantly through effects on angiogenesis, invasion, and metastasis, and that therapies directed at Kirsten-ras, including antisense approaches, may have particular utility through these mechanisms.
Mol Cancer Ther 2001 Nov
PMID:Inhibition of Kirsten-ras expression in human colorectal cancer using rationally selected Kirsten-ras antisense oligonucleotides. 1246 36

Chemotherapy resistance is a significant obstacle in lung cancer therapy, and has been found to frequently correlate with amplification and overexpression of the c-myc oncogene. Earlier studies have shown that c-Myc inhibition alone is not always effective in cancer models. The purpose of this study was to test different dosing regimen, which included commonly used chemotherapeutic drugs in combination with c-Myc inhibition in a Lewis lung syngeneic drug-resistant murine tumor model. Inhibition of c-myc was specifically achieved by using phosphorodiamidate Morpholino oligomer (PMOs), a novel, non-toxic antisense DNA chemistry for inhibition of gene expression by an RNase H-independent mechanism. When administration of cisplatin overlapped with c-myc PMO (AVI-4126) treatment there was no additional effect on tumor growth inhibition compared to cisplatin alone. In contrast, using a dosing regimen in which cisplatin or taxol treatment preceded AVI-4126, a dramatic decrease in tumor growth rate was observed with tumor areas less then 0.5 cm2 in 60% of the animals at the end of the study. This effect was specific to c-Myc inhibition as other antisense PMOs against p21 or Rad51 showed no such effect in combination with chemotherapy. Immunoblot and HPLC-based analysis of tumor lysates at the end of the study confirmed c-Myc inhibition and detection of intact AVI-4126, respectively. In conclusion, AVI-4126 potentiates the efficacy of chemotherapeutic drugs in a manner that is schedule dependent.
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PMID:Resistance to chemotherapeutic drugs overcome by c-Myc inhibition in a Lewis lung carcinoma murine model. 1254 57

The antisense method is one of the most promising anti-cancer methods, however, the design of antisense oligonucleotides is difficult because many factors affecting their activitiy and stability must be considered. Recently, the oligonucleotide stabilities related to the antisense effects were quantitatively investigated based on nearest-neighbor parameters. We demonstrated that DeltaG(o) (37, hyb), a free energy change for the hybridization of antisense oligodeoxynucleotides (ODNs) with target RNAs is related to the RNase H cleavage of TAg (SV40 large T antigen) mRNA, the expression of a rabbit globin mRNA, and the protein function encoded by hMDR1 (human multidrug resistance-1) mRNA, while DeltaG(o) (37, hp), a free-energy change for hairpin formations of the antisense ODNs significantly affected the arrest efficiency of the DHFR (dihydrofolate reductase) mRNA transcription, the expression of the proalpha1(I) chain of human, and the hybridization extent for HIV-1 alpha-1. For ras RNA (Ha-ras mRNA), DeltaG(o) (37, sc), a free energy change for the conformational change of the mRNA required for antisense ODN binding showed the best correlation with the equilibrium constants for the hybridization with their target RNA. On the other hand, the antisense effects ifor the HSV-1 IE5 (herpes simplex virus type 1 immediate early pre-mRNA5) showed less of a relationship to the hybridization stability of the antisense ODNs with the target pre-mRNA, because the antisense ODNs targeting the pre-mRNA must collapse its secondary structure around the splicing site to cancel out the expected antisense effects. Based on these results, we illustrate a new concept for the design of antisense ODNs based on DeltaG(o) (37, hyb), DeltaG(o) (37, hp), and DeltaG(o) (37, sc).
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PMID:A new concept for the design of antisense oligonucleotides based on nucleic acid thermostability. 1267 72


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