hiv rt rnase h  (Worthington Biochemical)


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    Name:
    Deoxyribonuclease I Ribonuclease Protease Free
    Description:
    Molecular Biology Grade Chromatographically purified to remove RNase and protease Lyophilized in vials containing glycine and calcium as stabilizer
    Catalog Number:
    LS006331
    Price:
    41
    Source:
    Bovine Pancreas
    Size:
    2500 un
    Cas Number:
    9003.98.9
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    Structured Review

    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
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hiv rt rnase h - by Bioz Stars, 2021-04
    98/100 stars

    Images

    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

    Related Articles

    Isolation:

    Article Title: A murine model of Lyme disease demonstrates that Borrelia burgdorferi colonizes the dura mater and induces inflammation in the central nervous system
    Article Snippet: Images are composites generated using the extended focus z-stack function (0.2μM step size X 5 steps per image). .. Flow cytometrySingle cells were dissociated by incubating dura isolated from transcardially perfused mice with 500 units/ml type 4 collagenase (CAT # LS004188; Worthington Biochemicals, Lakewood, NJ) and 100 μg/ml DNaseI (Worthington; CAT # LS006333) in PBS (45 min, 37°C). .. Digestion reactions were quenched in PBS + 5% FBS, and the supernatant and remaining dura tissue were triturated on ice with a 200 μl pipette tip and strained through a 100 μm sieve.

    Mouse Assay:

    Article Title: A murine model of Lyme disease demonstrates that Borrelia burgdorferi colonizes the dura mater and induces inflammation in the central nervous system
    Article Snippet: Images are composites generated using the extended focus z-stack function (0.2μM step size X 5 steps per image). .. Flow cytometrySingle cells were dissociated by incubating dura isolated from transcardially perfused mice with 500 units/ml type 4 collagenase (CAT # LS004188; Worthington Biochemicals, Lakewood, NJ) and 100 μg/ml DNaseI (Worthington; CAT # LS006333) in PBS (45 min, 37°C). .. Digestion reactions were quenched in PBS + 5% FBS, and the supernatant and remaining dura tissue were triturated on ice with a 200 μl pipette tip and strained through a 100 μm sieve.

    Incubation:

    Article Title: Differential accumulation of U14 snoRNA and hsc70 mRNA in Chinese hamster cells after exposure to various stress conditions
    Article Snippet: Then 200 μL of the reaction buffer (10 mM Tris·Cl [pH 8.0], 5 mM MgCl2 , 0.3 M KCl, 5 mM dithiothreitol [DTT], and 1 mM of each nucleoside triphosphate [NTP] except uridine triphosphate [UTP]) plus 0.1 mCi of [α-32 P]UTP (760 Ci/mmol, 10 mCi/mL) (New England Nuclear, Boston, MA, USA (NEN)) was added to the nuclei, and the reaction mixture was incubated further at 30°C for 30 minutes. .. The reaction was stopped by the addition of 24 μg RNase-free DNase I (Worthington Biochemical, Lakewood, NJ, USA) and the incubation of the resulting mixture for 5 minutes at 37°C. ..

    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.

    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).

    Article Title: Transcriptional activation of HIV by Mycobacterium tuberculosis in human monocytes
    Article Snippet: .. Before infection, each aliquot of viral stock was depleted of contaminating pro-viral DNA by filtration through a 0.22-μm filter (Gelman Sciences, Ann Arbor, MI) and treatment with RNAse-free DNAse (1.8 μg/ml) (Worthington Biochemical Corp., Freehold, NJ). .. Briefly, monocytes were incubated with viral stock (at a TCID50 of 10–100) (a TCID50 of 1 corresponds to approximately 30 pg of HIV p24 antigen by ELISA) in the presence of polybrene (5 μg/ml) for 2 h at 37°C.

    Microarray:

    Article Title: GAGA factor, a positive regulator of global gene expression, modulates transcriptional pausing and organization of upstream nucleosomes
    Article Snippet: PCR primers are listed in Additional file : Tables S2, 3. .. Microarray analyses Total RNA was extracted from imaginal tissues of WT or mutant third-instar larvae by TRIzol (Invitrogen), treated with RNase-free DNase I (Worthington) and purified by RNeasy kit (Qiagen) according to the vendors’ protocols. .. Microarray analysis was conducted by our in-house Microarray Core Facility ( http://www.imb.sinica.edu.tw/mdarray/index.html ).

    Mutagenesis:

    Article Title: GAGA factor, a positive regulator of global gene expression, modulates transcriptional pausing and organization of upstream nucleosomes
    Article Snippet: PCR primers are listed in Additional file : Tables S2, 3. .. Microarray analyses Total RNA was extracted from imaginal tissues of WT or mutant third-instar larvae by TRIzol (Invitrogen), treated with RNase-free DNase I (Worthington) and purified by RNeasy kit (Qiagen) according to the vendors’ protocols. .. Microarray analysis was conducted by our in-house Microarray Core Facility ( http://www.imb.sinica.edu.tw/mdarray/index.html ).

    Purification:

    Article Title: GAGA factor, a positive regulator of global gene expression, modulates transcriptional pausing and organization of upstream nucleosomes
    Article Snippet: PCR primers are listed in Additional file : Tables S2, 3. .. Microarray analyses Total RNA was extracted from imaginal tissues of WT or mutant third-instar larvae by TRIzol (Invitrogen), treated with RNase-free DNase I (Worthington) and purified by RNeasy kit (Qiagen) according to the vendors’ protocols. .. Microarray analysis was conducted by our in-house Microarray Core Facility ( http://www.imb.sinica.edu.tw/mdarray/index.html ).

    other:

    Article Title: A rapid and robust assay for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine
    Article Snippet: Corp. cat. no: LS006331 ) in PBS with 5 mM MgSO4 .

    Filtration:

    Article Title: Transcriptional activation of HIV by Mycobacterium tuberculosis in human monocytes
    Article Snippet: .. Before infection, each aliquot of viral stock was depleted of contaminating pro-viral DNA by filtration through a 0.22-μm filter (Gelman Sciences, Ann Arbor, MI) and treatment with RNAse-free DNAse (1.8 μg/ml) (Worthington Biochemical Corp., Freehold, NJ). .. Briefly, monocytes were incubated with viral stock (at a TCID50 of 10–100) (a TCID50 of 1 corresponds to approximately 30 pg of HIV p24 antigen by ELISA) in the presence of polybrene (5 μg/ml) for 2 h at 37°C.

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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
    Hiv Rt Rnase H, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Worthington Biochemical 2015 worthington 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
    2015 Worthington H, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    Worthington Biochemical ayers 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
    Ayers H, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    Journal: BMC Infectious Diseases

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

    doi: 10.1186/s12879-016-1713-x

    Figure Lengend 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

    Article Snippet: For annealing, purified RNA2 transcripts were mixed with 50 nM of ODNs in hybridization buffer (50 mM NaCl, 10 mM MgCl2 , 1 mM DTT, 0.4 mM spermine hydrochloride, 25 mM Tris-acetate, pH 6.8), heat-treated for 3 min at 90 °C, cooled, and incubated at 37 °C for 30 min. After annealing, samples were incubated with 0.05 units/μl of HIV RT/RNase H (Worthington, USA) for a further 30 min at 37 °C.

    Techniques: 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

    Journal: BMC Infectious Diseases

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

    doi: 10.1186/s12879-016-1713-x

    Figure Lengend 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

    Article Snippet: For annealing, purified RNA2 transcripts were mixed with 50 nM of ODNs in hybridization buffer (50 mM NaCl, 10 mM MgCl2 , 1 mM DTT, 0.4 mM spermine hydrochloride, 25 mM Tris-acetate, pH 6.8), heat-treated for 3 min at 90 °C, cooled, and incubated at 37 °C for 30 min. After annealing, samples were incubated with 0.05 units/μl of HIV RT/RNase H (Worthington, USA) for a further 30 min at 37 °C.

    Techniques: 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

    Journal: BMC Infectious Diseases

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

    doi: 10.1186/s12879-016-1713-x

    Figure Lengend 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

    Article Snippet: For annealing, purified RNA2 transcripts were mixed with 50 nM of ODNs in hybridization buffer (50 mM NaCl, 10 mM MgCl2 , 1 mM DTT, 0.4 mM spermine hydrochloride, 25 mM Tris-acetate, pH 6.8), heat-treated for 3 min at 90 °C, cooled, and incubated at 37 °C for 30 min. After annealing, samples were incubated with 0.05 units/μl of HIV RT/RNase H (Worthington, USA) for a further 30 min at 37 °C.

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