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

Baertschi et al. (Antiviral Chem. Chemother. 1997, 8, 353-362) clarified the nature of a polymeric degradation product formed from the cephalosporin ceftazidime. Interest in the polymeric material arises from its ability to inhibit the RNase H and polymerase activities of HIV-1 reverse transcriptase (RT). To shed light on the structure of the polymeric material like that which forms from degradation of third-generation cephalosporins, we apply molecular modeling and other computational chemistry techniques. Aminothiazole methoxime (2-amino-4-thiazolyl-methoxyimino; ATMO) is the parent structure related to the isolated degradation product of ceftazidime. The MMFF94 force field and Monte Carlo multiple minimum method as implemented in MacroModel are used to generate low-energy conformers. We built up oligomeric models starting from the trimer to the 16-mer and performed distribution analyses on the dihedral angles from the Monte Carlo runs to analyze the three-dimensional shapes of the oligomers. Although the larger oligomers are too long for a complete search of conformational space, the low-energy conformers examined do not show secondary structure or repetitive conformations. Polymeric ATMO material may, therefore, exhibit only random coil conformations. Topological similarity of ATMO structures to other reported RT inhibitors is also examined.
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PMID:Exploration of the conformational space of a polymeric material that inhibits human immunodeficiency virus. 1671 51

A blunt-ended 19-mer short interfering hybrid (siHybrid) (H) comprised of sense-DNA/antisense-RNA targeting HER-2 mRNA was encapsulated in a liposomal nanoplex with anti-transferrin receptor single-chain antibody fragment (TfRscFv) as the targeting moiety for clinically relevant tumor-specific delivery. In vitro delivery to a human pancreatic cell line (PANC-1) was shown to exhibit sequence-specific inhibition of 48-h cell growth with an IC50 value of 37 nM. The inhibitory potency of this siHybrid was increased (IC50 value of 7.8 nM) using a homologous chemically modified siHybrid (mH) in which the 19-mer sense strand had the following pattern of 2 '-deoxyinosine (dI) and 2 '-O-methylribonucleotide (2 '-OMe) residues: 5'-d(TITIT)-2'OMe(GCGGUGGUU)-d(GICIT). These modifications were intended to favor antisense strand-mediated RNAi while mitigating possible sense strand-mediated off-target effects and RNase H-mediated cleavage of the antisense RNA strand. The presently reported immunoliposomal delivery system was successfully used in vivo to inhibit HER-2 expression, and thus induce apoptosis in human breast carcinoma tumors (MDA-MB-435) in mice upon repeated i.v. treatment at a dose of 3 mg/kg of H or mH. The in vivo potency of modified siHybrid mH appeared to be qualitatively greater than that of H, as was the case in vitro.
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PMID:Chemically modified short interfering hybrids (siHYBRIDS): nanoimmunoliposome delivery in vitro and in vivo for RNAi of HER-2. 1690 21

The 2'-deoxy-2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T) has been synthesized using a key cyclization step involving 2'-ara-trifluoromethylsufonyl-4'-cyanomethylene 11 to give a pair of 3',5'-bis-OBn-protected diastereomerically pure aza-ENA-Ts (12a and 12b) with the fused piperidino skeleton in the chair conformation, whereas the pentofuranosyl moiety is locked in the North-type conformation (7 degrees < P < 27 degrees, 44 degrees < phi m < 52 degrees). The origin of the chirality of two diastereomerically pure aza-ENA-Ts was found to be due to the endocyclic chiral 2'-nitrogen, which has axial N-H in 12b and equatorial N-H in 12a. The latter is thermodynamically preferred, while the former is kinetically preferred with Ea = 25.4 kcal mol-1, which is thus far the highest observed inversion barrier at pyramidal N-H in the bicyclic amines. The 5'-O-DMTr-aza-ENA-T-3'-phosphoramidite was employed for solid-phase synthesis to give four different singly modified 15-mer antisense oligonucleotides (AONs). Their AON/RNA duplexes showed a Tm increase of 2.5-4 degrees C per modification, depending upon the modification site in the AON. The relative rates of the RNase H1 cleavage of the aza-ENA-T-modified AON/RNA heteroduplexes were very comparable to that of the native counterpart, but the RNA cleavage sites of the modified AON/RNA were found to be very different. The aza-ENA-T modifications also made the AONs very resistant to 3' degradation (stable over 48 h) in the blood serum compared to the unmodified AON (fully degraded in 4 h). Thus, the aza-ENA-T modification in the AON fulfilled three important antisense criteria, compared to the native: (i) improved RNA target affinity, (ii) comparable RNase H cleavage rate, and (iii) higher blood serum stability.
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PMID:Conformationally constrained 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies of aza-ENA-T-modified oligonucleotides. 1711 69

RNase H binds RNA-DNA hybrid and double-stranded RNA (dsRNA) duplexes with similar affinity, but only cleaves the RNA in the former. To potentially gain insight into the conformational origins of substrate recognition by the enzyme from Escherichia coli, cocrystallization experiments were carried out with RNase HI-dsRNA (enzyme-inhibitor) complexes. Crystals were obtained of two complexes containing 9-mer and 10-mer RNA duplexes that diffracted X-rays to 3.5 and 4 A resolution, respectively.
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PMID:Crystallization and preliminary X-ray analysis of Escherichia coli RNase HI-dsRNA complexes. 1727 45

The synthesis of oligonucleotides containing 2'-deoxy-2'-fluoro-4'-thioarabinonucleotides is described. 2'-Deoxy-2'-fluoro-5-methyl-4'-thioarabinouridine (4'S-FMAU) was incorporated into 18-mer antisense oligonucleotides (AONs). 4'S-FMAU adopts a predominantly northern sugar conformation. Oligonucleotides containing 4'S-FMAU, unlike those containing FMAU, were unable to elicit E. coli or human RNase H activity, thus corroborating the hypothesis that RNase H prefers duplexes containing oligonucleotides that can adopt eastern conformations in the antisense strand. The duplex structure and stability of these oligonucleotides was also investigated via circular dichroism (CD)- and UV- binding studies. Replacement of the 4'-oxygen by a sulfur atom resulted in a marked decrease in melting temperature of AON:RNA as well as AON:DNA duplexes. 2'-deoxy-2'-fluoro-4'-thioarabinouridine (4'S-FAU) was incorporated into 21-mer small interfering RNA (siRNA) and the resulting siRNA molecules were able to trigger RNA interference with good efficiency. Positional effects were explored, and synergy with 2'F-ANA, which has been previously established as a functional siRNA modification, was demonstrated.
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PMID:2'-fluoro-4'-thioarabino-modified oligonucleotides: conformational switches linked to siRNA activity. 1728 57

The aim of this work was to shed some more light on factors influencing the effectiveness of delta ribozyme cleavage of structured RNA molecules. An oligoribonucleotide that corresponds to the 3'-terminal region X of HCV RNA and yeast tRNAPhe were used as representative RNA targets. Only a few sites susceptible to ribozyme cleavage were identified in these targets using a combinatorial library of ribozyme variants, in which the region responsible for ribozyme-target interaction was randomized. On the other hand, the targets were fairly accessible for binding of complementary oligonucleotides, as was shown by 6-mer DNA libraries and RNase H approach. Moreover, the specifically acting ribozymes cleaved the targets precisely but with unexpectedly modest efficacy. To explain these observations, six model RNA molecules were designed, in which the same seven nucleotide long sequence recognized by the delta ribozyme was always single stranded but was embedded into different RNA structural context. These molecules were cleaved with differentiated rates, and the corresponding k2 values were in the range of 0.91-0.021 min-1; thus they differed almost 50-fold. This clearly shows that cleavage of structured RNAs might be much slower than cleavage of a short unstructured oligoribonucleotide, despite full accessibility of the targeted regions for hybridization. Restricted possibilities of conformational transitions, which are necessary to occur on the cleavage reaction trajectory, seem to be responsible for these differences. Their magnitude, which was evaluated in this work, should be taken into account while considering the use of delta ribozymes for practical applications.
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PMID:Structural features of target RNA molecules greatly modulate the cleavage efficiency of trans-acting delta ribozymes. 1742 88

Light-activated antisense oligodeoxynucleotides (asODNs) were developed to control the degradation of target mRNA in living cells by RNase H. A 20-mer asODN previously shown to target c-myb, a hematopoietic transcription factor, was covalently attached via a photocleavable linker (PL) to partially complementary 20-mer sense strands (sODNs). In the 'caged' state, the sODN blocked hybridization of the asODN to c-myb mRNA. Six asODN-PL-sODN conjugates, C1-C6, were synthesized. C5, with twelve complementary bases, gave the largest decrease in melting temperature (T(m)) upon UV irradiation (DeltaT(m) = -29 degrees C). The most thermally stable conjugate, C6 (T(m) = 84 degrees C), gave the lowest background RNase H activity, with just 8.6% degradation of an RNA 40-mer after 1 h incubation. In biochemical assays with C6, RNA digestion increased 10-fold 10 min after UV irradiation. Finally, phosphorothioated analogs S-C5 and S-C6 were synthesized to test activity in cultured K562 (human leukemia) cells. No knockdown of c-myb mRNA or protein was observed with intact S-C5 or S-C6, whereas more than half of c-myb mRNA was degraded 24 h after photoactivation. Two-fold photomodulation of c-MYB protein levels was also observed with S-C5. However, no photomodulation of c-MYB protein levels was observed with S-C6, perhaps due to the greater stability of this duplex.
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PMID:Regulating gene expression in human leukemia cells using light-activated oligodeoxynucleotides. 1805 83

In this review, we report DNA duplex-based fluorescence probes/sensors using pyrene monomer-excimer switching. The review mainly comprises two topics: 1) excimer-monomer switching molecular beacons (EMS-MB) and 2) monomer-excimer switching sensors based on the structural motif of antibodies. The EMS-MBs have two pyrene fluorophores connected both at 3' and 5' ends of a single-stranded oligonucleotide. Emission switching occurs from excimer to monomer accompanying isoemissive points when the probes hybridized with target DNAs. The isoemissive points indicate the presence of only two fluorescent species, nonhybridized and hybridized probes in the mixtures, and thereby unambiguous detection of the targets is available. The probes can detect target 19-mer DNAs and can discriminate the targets from their single-nucleotide mismatches at 1 nM concentration. Furthermore, the EMS-MBs have been recently applied to kinetic study for RNase H activity by Tan et al. The structures and emission-switching properties of the EMS-MBs encourage us to develop a new class of fluorescent sensors based on the structural motif of antibodies. The sensors consist of three functional regions, benzo-15-crown-5 ether (or per-O-methylated beta-cyclodextrin), DNA, and pyrene as guest-binding, dimerizing, and sensing sites, respectively. The crown- and CD-modified sensors can detect potassium cation and porphyrin derivatives, respectively, by monomer-excimer emission switching.
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PMID:[DNA duplex-based fluorescence probes/sensors using monomer-excimer switching]. 1898 96

The Herpes simplex virus (HSV) is known as an infectious agent and widespread in the human population. The symptoms of HSV infections can range from mild to life threatening, especially in immune-compromised individuals. HSV infections are commonly treated with the guanosine analogue Aciclovir, but reports of resistance are increasing. Efforts are made to establish single-stranded antisense oligodeoxynucleotides (as) and small interfering ribonucleic acids (siRNAs) for antiviral treatment. Recently, another class of short interfering nucleic acids, partially double-stranded hairpin loop-structured 54 mer oligodeoxynucleotides (ODNs), was shown to allow hydrolysis of HIV RNA by binding to the viral RNA. This leads to a substrate for the viral RNase H. To assess the potential of such ODNs for inhibition of HSV-1 replication, five partially double-stranded ODNs were designed based on the sequences of known siRNAs against HSV-1 with antiviral activity. Three of them are directed against early and two against leaky late genes. Primary human lung fibroblasts, MRC-5, and African green monkey kidney cells, Vero, were transfected with ODNs and subsequently infected. The effect on HSV-1 replication was determined by analyzing the virus titer in cell culture supernatants by quantitative PCR and plaque assays. An inhibitory effect was observed with all five selected ODNs, with two cases showing statistical significance in both cell types. The observed effect was sequence-specific and dose dependent. In one case the ODN was more efficient than a previously described siRNA directed against the same target site in the mRNA of UL5, a component of the helicase/primase complex. HSV-1 virions and ODNs can be applied simultaneously without transfection reagent, but at a 50-fold higher concentration to Vero cells with similar efficiencies. The results underline the potential of partially double-stranded hairpin loop-structured ODNs as antiviral agents.
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PMID:Short hairpin-loop-structured oligodeoxynucleotides reduce HSV-1 replication. 1939 93

The optimal antisense accessible sites (AAS) of uroplakin II (UP II) mRNA, a specific gene expressed in bladder urothelium, were selected in order to provide a novel method for targeted therapy of transitional cell carcinoma (TCC) of bladder. The 20 mer random oligonucleotide library was synthesized, hybridized with in vitro transcripted total UP II cRNA, then digested by RNase H. After primer extension and autoradiography, the AAS of UP II were selected. The RNADraw software was used to analyze and choose the AAS with obvious stem-loop structures, according to which the complementary antisense oligonucleotides (AS-ODN) were synthesized. With the AS-ODN(0) designed only by RNADraw as a control, the AS-ODNs were transferred into UP II highly-expressing cell line RT(4). The cellular expression of UP II mRNA was detected by RT-PCR and Western blot. Twelve AAS of UP II mRNA were selected in vitro. Four AAS with stem-loop structures were chosen, locating at 558-577, 552-571, 217-236 and 97-116 bp of UP II mRNA respectively. After transfection with the corresponding AS-ODN (AS-ODN(1), AS-ODN(2), AS-ODN(3) and AS-ODN(4)) for 18 h, the UP II mRNA levels in RT(4) cells were reduced by 29.3%, 82.7%, 71.3% and 70.9%, while UP II protein levels were decreased by 20.2%, 78.5%, 65.2% and 64.4% respectively, which were significantly higher than those of AS-ODN(0) (14.3%, 12.1% respectively) (P<0.01). The AAS of UP II mRNA was effectively selected in vitro by random oligonucletide library/RNase H cleavage method in combination with computer software analysis, which had important reference values for further studying biological functions of UP II gene and targeted therapeutic strategy for TCC of bladder.
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PMID:Selection of optimal antisense accessible sites of uroplakin II mRNA for bladder urothelium. 1951 19


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