hiv rt rnase h (Worthington Biochemical)

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Deoxyribonuclease I Ribonuclease Protease Free

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Molecular Biology Grade Chromatographically purified to remove RNase and protease Lyophilized in vials containing glycine and calcium as stabilizer

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ls006331

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Bovine Pancreas

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2500 un

Cas Number:

9003.98.9

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Worthington Biochemical hiv rt rnase h

Antiviral mechanism of ODN sequences. a Schematic representation of reverse transcription of <t>HIV</t> and mechanism of ODN A action. The HIV-1 extended polypurine tract (PPT) is indicated as black box with the RT/RNase H cleavage side depicted in white. ODN A interacts with the highly conserved PPT, mimicking the natural replication intermediate RNA-DNA hybrid, which results in premature activation of reverse transcriptase (RT)/RNase H hydroloysis of the viral RNA genome. b Sequences of the ODNs used. All ODNs form hairpin-like structures with an antisense ( lower ) and passenger ( upper ) strand linked by four thymidines. Watson-Crick bonds are shown as vertical bars . Phosphorothioate-modified nucleotides are shown in bold and marked by a star . The ODN A sequence is complementary to the extended PPT, and ODN Co targets an HIV-1 RNA region outside of the PPT. ODN G serves as a further control, with a similar passenger strand sequence compared to ODN A but a non-complementary HIV-1 PPT sequence. c Sequence of the extended polypurine tract of HIV-1 recognized by the viral RNase H, whose specific cleavage site is indicated by an arrow

Molecular Biology Grade Chromatographically purified to remove RNase and protease Lyophilized in vials containing glycine and calcium as stabilizer
https://www.bioz.com/result/hiv rt rnase h/product/Worthington Biochemical
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hiv rt rnase h - by Bioz Stars, 2021-03

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1) Product Images from "Abolishing HIV-1 infectivity using a polypurine tract-specific G-quadruplex-forming oligonucleotide"

Article Title: Abolishing HIV-1 infectivity using a polypurine tract-specific G-quadruplex-forming oligonucleotide

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-016-1713-x

Antiviral mechanism of ODN sequences. a Schematic representation of reverse transcription of HIV and mechanism of ODN A action. The HIV-1 extended polypurine tract (PPT) is indicated as black box with the RT/RNase H cleavage side depicted in white. ODN A interacts with the highly conserved PPT, mimicking the natural replication intermediate RNA-DNA hybrid, which results in premature activation of reverse transcriptase (RT)/RNase H hydroloysis of the viral RNA genome. b Sequences of the ODNs used. All ODNs form hairpin-like structures with an antisense ( lower ) and passenger ( upper ) strand linked by four thymidines. Watson-Crick bonds are shown as vertical bars . Phosphorothioate-modified nucleotides are shown in bold and marked by a star . The ODN A sequence is complementary to the extended PPT, and ODN Co targets an HIV-1 RNA region outside of the PPT. ODN G serves as a further control, with a similar passenger strand sequence compared to ODN A but a non-complementary HIV-1 PPT sequence. c Sequence of the extended polypurine tract of HIV-1 recognized by the viral RNase H, whose specific cleavage site is indicated by an arrow

Figure Legend Snippet: Antiviral mechanism of ODN sequences. a Schematic representation of reverse transcription of HIV and mechanism of ODN A action. The HIV-1 extended polypurine tract (PPT) is indicated as black box with the RT/RNase H cleavage side depicted in white. ODN A interacts with the highly conserved PPT, mimicking the natural replication intermediate RNA-DNA hybrid, which results in premature activation of reverse transcriptase (RT)/RNase H hydroloysis of the viral RNA genome. b Sequences of the ODNs used. All ODNs form hairpin-like structures with an antisense ( lower ) and passenger ( upper ) strand linked by four thymidines. Watson-Crick bonds are shown as vertical bars . Phosphorothioate-modified nucleotides are shown in bold and marked by a star . The ODN A sequence is complementary to the extended PPT, and ODN Co targets an HIV-1 RNA region outside of the PPT. ODN G serves as a further control, with a similar passenger strand sequence compared to ODN A but a non-complementary HIV-1 PPT sequence. c Sequence of the extended polypurine tract of HIV-1 recognized by the viral RNase H, whose specific cleavage site is indicated by an arrow

Techniques Used: Activation Assay, Modification, Sequencing

ODN A is active in vitro and in cell culture infection assays after long-term storage at 37 °C. a ODN A (8 μM) was stored for the indicated duration in PBS or ( b ) in H 2 O at 37 °C. Samples were analyzed by non-denaturing 10 % PAGE. c ODN A (50 nM), either freshly thawed or stored for the indicated time periods at 37 °C in PBS or H 2 O was hybridized to 50 nM in vitro transcribed γ-32-ATP 5′-labeled PPT-containing RNA in the presence of HIV-1 RT/RNase H. The cleavage products were analyzed by denaturing polyacrylamide/8 M urea gel electrophoresis and are presented schematically on the right . Cleavage sites are indicated by arrowheads and labeled products are shown in black . ODN Co, annealing to sequences downstream of the PPT, served as a control. d Following the experimental procedure shown at the top : Freshly thawed ODN A or ODN Co (250 nM), or ODN A stored for 102 days at 37 °C in PBS were incubated with replication-competent HIV-1 particles (1 × 10 9 ) at 37 °C for 6 h in cell culture medium. Jurkat 1G5 T cells were infected with the mixtures overnight and HIV-1 p24 antigen in the supernatant was detected at 3–14 days post infection. Two-way ANOVA followed by Bonferroni posttest was used for statistical evaluation. ODN A-mediated inhibition (as compared to buffer alone) was highly significant ( p

Figure Legend Snippet: ODN A is active in vitro and in cell culture infection assays after long-term storage at 37 °C. a ODN A (8 μM) was stored for the indicated duration in PBS or ( b ) in H 2 O at 37 °C. Samples were analyzed by non-denaturing 10 % PAGE. c ODN A (50 nM), either freshly thawed or stored for the indicated time periods at 37 °C in PBS or H 2 O was hybridized to 50 nM in vitro transcribed γ-32-ATP 5′-labeled PPT-containing RNA in the presence of HIV-1 RT/RNase H. The cleavage products were analyzed by denaturing polyacrylamide/8 M urea gel electrophoresis and are presented schematically on the right . Cleavage sites are indicated by arrowheads and labeled products are shown in black . ODN Co, annealing to sequences downstream of the PPT, served as a control. d Following the experimental procedure shown at the top : Freshly thawed ODN A or ODN Co (250 nM), or ODN A stored for 102 days at 37 °C in PBS were incubated with replication-competent HIV-1 particles (1 × 10 9 ) at 37 °C for 6 h in cell culture medium. Jurkat 1G5 T cells were infected with the mixtures overnight and HIV-1 p24 antigen in the supernatant was detected at 3–14 days post infection. Two-way ANOVA followed by Bonferroni posttest was used for statistical evaluation. ODN A-mediated inhibition (as compared to buffer alone) was highly significant ( p

Techniques Used: In Vitro, Cell Culture, Infection, Polyacrylamide Gel Electrophoresis, Labeling, Nucleic Acid Electrophoresis, Incubation, Inhibition

The lubricant CMC (K-Y Jelly) does not influence the stability and antiviral activity of ODN A. a ODN A (8 μM) was incubated for the indicated days in 25 % K-Y Jelly (in PBS) at 37 °C. The samples were analyzed by 10 % non-denaturing polyacrylamide gel electrophoresis. b Analysis of ODN A-triggered, RNase H-mediated cleavage of PPT RNA in vitro. Assays were performed as described in Fig. 2c . Cleavage sites are indicated by arrowheads and the labeled products are indicated in black . c Following the experimental procedure shown at the top : ODN A pre-incubated in 25 % K-Y Jelly/PBS for 57 days, freshly thawed ODN A, or ODN Co (both 250 nM) were incubated with replication-competent HIV-1 virions (1 × 10 9 ) at 37 °C for 6 h in cell culture medium. Jurkat 1G5 T cells were subsequently infected overnight. Virus replication was monitored by HIV-1 p24 antigen ELISA of culture supernatants at day 3–14 post infection. Two-way ANOVA followed by Bonferroni posttest was used for statistical evaluation. ODN A-mediated inhibition (as compared to buffer alone) was significant (day 7, p

Figure Legend Snippet: The lubricant CMC (K-Y Jelly) does not influence the stability and antiviral activity of ODN A. a ODN A (8 μM) was incubated for the indicated days in 25 % K-Y Jelly (in PBS) at 37 °C. The samples were analyzed by 10 % non-denaturing polyacrylamide gel electrophoresis. b Analysis of ODN A-triggered, RNase H-mediated cleavage of PPT RNA in vitro. Assays were performed as described in Fig. 2c . Cleavage sites are indicated by arrowheads and the labeled products are indicated in black . c Following the experimental procedure shown at the top : ODN A pre-incubated in 25 % K-Y Jelly/PBS for 57 days, freshly thawed ODN A, or ODN Co (both 250 nM) were incubated with replication-competent HIV-1 virions (1 × 10 9 ) at 37 °C for 6 h in cell culture medium. Jurkat 1G5 T cells were subsequently infected overnight. Virus replication was monitored by HIV-1 p24 antigen ELISA of culture supernatants at day 3–14 post infection. Two-way ANOVA followed by Bonferroni posttest was used for statistical evaluation. ODN A-mediated inhibition (as compared to buffer alone) was significant (day 7, p

Techniques Used: Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, In Vitro, Labeling, Cell Culture, Infection, Enzyme-linked Immunosorbent Assay, Inhibition

other:

Article Title: Human Immunodeficiency Virus Type 1 Infection and Replication in Normal Human Oral Keratinocytes
Article Snippet: Virus stocks were filtered and treated with RNase-free DNase I (1 μg/ml; Worthington Biochemical Corp., Lakewood, N.J.) for 30 min at room temperature in the presence of 0.01 M MgCl2 to degrade contaminating HIV-1 DNA.

Incubation:

Article Title: Oligomeric Coiled-Coil Adhesin YadA Is a Double-Edged Sword
Article Snippet: To determine killing, samples were taken at 120 min after infection, diluted in ddH2 O and plated. .. As control for NET-dependent killing, NETs were degraded by incubation with 50 U/ml of RNase-free and protease-free DNase-1 (Worthington) prior to the addition of bacteria (data not shown). .. To determine reference values, an equal number of bacteria was incubated without PMNs.

Article Title: Activation of the Myc oncoprotein leads to increased turnover of thrombospondin-1 mRNA
Article Snippet: .. The samples were incubated at 29°C for 30 min and then treated with 50 µl RNase-free DNase I (Worthington, Lakewood, NJ) at 37°C for 10 min. Then 36 µl 10× SET (100 mM Tris–Cl pH 7.5, 50 mM EDTA, 10% SDS) and 10 µl Proteinase K (10 mg/ml) were added and the reactions were incubated for 1 h at 50°C. .. RNAs were extracted with phenol–chloroform, precipitated with ethanol and resuspended in 100 µl H2 O. Unincorporated [32 P]UTP and short oligonucleotides were removed by passing the RNAs through Sephadex G-50 columns (Boehringer Mannheim).

Article Title: A Novel Intron Element Operates Posttranscriptionally To Regulate Human N-myc Expression
Article Snippet: Nascent transcripts were labeled in a reaction with 0.25 mM rATP, rCTP, and rGTP in 5 mM Tris (pH 8.0)–2.5 mM MgCl2 –25 mM KCl–2.5 mM dithiothreitol–200 μCi of [α-32 P]UTP (3,000 Ci/mmol; NEN) for 30 min at 37°C. .. One unit of RNase-free DNase I (Worthington) or 500 U of RNase-free DNase I (Gibco-BRL) was added per reaction, and the mixtures were incubated at 37°C for 10 min. .. Reactions were stopped by the addition of 10× stop buffer (10% Sarkosyl or 2.5% sodium dodecyl sulfate [SDS], 1 mg of proteinase K per ml, 100 mM EDTA, 100 mM Tris [pH 7.6]) and additional incubation at 42°C for 30 min. RNA was purified either by extraction with 3 equal volumes of phenol-chloroform-isoamyl alcohol (25:24:1) and precipitation with 0.3 M sodium acetate and 100% ethanol using yeast tRNA as the carrier, or by purification through 3-ml Sephadex G-50 columns.

Infection:

Article Title: An Inducible Human Immunodeficiency Virus Type 1 (HIV-1) Vector Which Effectively Suppresses HIV-1 Replication
Article Snippet: .. Before infection, the NL-r-HSAS virus was treated with RNase-free DNase (20 μg/ml; Worthington) for 30 min at 37°C in the presence of 0.01 M MgCl2 . .. Mock-transduced and vector-transduced CEMx174 cells or SupT1 cells (7 × 105 ) were infected with replication-competent NL-r-HSAS virus for 2 h at 37°C in the presence of Polybrene (8 μg/ml) at different MOI (MOI of 0.01, 0.1, and 1 for CEMx174 and 0.01 and 0.1 for SupT1 cell experiments).

Purification:

Article Title: Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells
Article Snippet: Using hybridization ensured specificity of detection and allowed us to use PCR with a low number of cycles, thereby avoiding the problem of non-linear amplification rates at later cycles. .. First, total RNA (2.5 μg) was treated with 0.5 U of RNase-free DNAse (Worthington, Lakewood, NJ, USA) to remove contaminating genomic DNA in 100 μl of 10 mM Tris–HCl (pH 7.5), 2.5 mM MgCl2 and 0.1 mM CaCl2 for 7 min at 37°C, purified by phenol–chloroform extraction and precipitated with isopropanol. ..

Activity Assay:

Article Title: Patch Cramming Reveals the Mechanism of Long-Term Suppression of Cyclic Nucleotides in Intact Neurons
Article Snippet: Alkaline phosphatase, as well as peptide inhibitors of protein kinases, was prepared in distilled water at 10× the final cytoplasmic concentration and was pressure injected into recipient cells with a Picospritzer (General Valve, Fairfield, NJ). .. In some experiments we used Ultrapure alkaline phosphatase, free of detectable DNase, RNase, or protease activity (Worthington Biochemical, Freehold, NJ). .. Protein kinase C inhibitor peptide 19-31 (PKC-IP; ) and autocamtide-2 related inhibitory peptide (AIP) ( ) were obtained from Calbiochem.

Irradiation:

Article Title: A shared RNA-binding site in the Pet54 protein is required for translational activation and group I intron splicing in yeast mitochondria
Article Snippet: UV cross-linking For the cross-linking experiments, internally 32 P-labeled 4-thiouridine containing transcript (20 nM) was incubated in the absence or presence of Pet54p (∼250 nM) in 50 mM Tris–HCl, pH 7.5, 100 mM NaCl and 7 mM MgCl2 (TNM buffer) for 10 min. For competition experiments, unlabeled competitor RNAs were included at a final concentrations of 50 to 750 nM (see and legends). .. The reaction mixture was then placed in a 96-well plate on ice and irradiated at 312 nm in a UV Stratalinker 1800 (Stratagene, La Jolla, CA, USA) for 20 min. A ribonuclease cocktail of RNaseA and T1 was added (Worthington Biochemical Corp., Lakewood, NJ, USA) and the RNA was digested for 60 min at 42°C. ..