e coli rnase h Thermo Fisher Search Results


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  • 90
    Thermo Fisher e coli rnase h
    <t>RNase</t> H cleavage analysis of truncated DNA substrates. (A) The substrates utilized are illustrated and are labeled A through F. The RNA portions are indicated in bold, and an asterisk indicates the radiolabel. The substrates were prepared as described in Materials and Methods. (B) Substrates B to F assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow. (C) Substrates B to F assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow.
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    87
    Thermo Fisher ambion rnase h
    <t>RNase</t> H cleavage analysis of truncated DNA substrates. (A) The substrates utilized are illustrated and are labeled A through F. The RNA portions are indicated in bold, and an asterisk indicates the radiolabel. The substrates were prepared as described in Materials and Methods. (B) Substrates B to F assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow. (C) Substrates B to F assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow.
    Ambion Rnase H, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher u e coli rnase h1
    R-loop formation at the studied genomic and mitochondrial regions in untreated control N-TERA-2 cells. DNA enrichment of each sample is subtracted of the enrichment value of the same sample treated with <t>RNase</t> H1 before DRIVE precipitation. Then the enrichment value is normalized against the 2-min CPT sample (see Fig 2 ) of the RPL13A amplicon of the same experiment. Values are means ±SEM of two to four independent experiments. The data show a higher SEM than commonly published as we report median values of several experiments and not a single representative one. (A) DRIVE assay was performed to determine R-loop levels downstream TSS (white bars) and upstream TSS (black bars). Three negative loci for R-loop formation are also reported (SNRPN, a-SAT, TNIK). (B) Mitochondrial DNA was analyzed with DRIVE assay. Three regions of interest were selected: red for the r-loop forming region (RB31-R3), green for the D-loop region (1A-1B) and blue for the non-D-loop region (4A-4B). Map on the right of the panel shows the heavy (H) and the light (L) strands of mitochondrial DNA, with the three Conserved Sequence Blocks (CSB) and the studied regions (in red, green and blue respectively). (C) Antisense transcription after CPT treatment in N-TERA-2 cells. Promoter-associated antisense transcripts were evaluated by rtqPCR after 4 hours CPT treatment at 10 μM. PCR determinations were normalized to cytochrome b mRNA and to untreated cells (dotted line). Values are means +/− SEM of two determinations from at least two independent experiments.
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    90
    Thermo Fisher superscript ii rnase hi reverse transcriptase
    R-loop formation at the studied genomic and mitochondrial regions in untreated control N-TERA-2 cells. DNA enrichment of each sample is subtracted of the enrichment value of the same sample treated with <t>RNase</t> H1 before DRIVE precipitation. Then the enrichment value is normalized against the 2-min CPT sample (see Fig 2 ) of the RPL13A amplicon of the same experiment. Values are means ±SEM of two to four independent experiments. The data show a higher SEM than commonly published as we report median values of several experiments and not a single representative one. (A) DRIVE assay was performed to determine R-loop levels downstream TSS (white bars) and upstream TSS (black bars). Three negative loci for R-loop formation are also reported (SNRPN, a-SAT, TNIK). (B) Mitochondrial DNA was analyzed with DRIVE assay. Three regions of interest were selected: red for the r-loop forming region (RB31-R3), green for the D-loop region (1A-1B) and blue for the non-D-loop region (4A-4B). Map on the right of the panel shows the heavy (H) and the light (L) strands of mitochondrial DNA, with the three Conserved Sequence Blocks (CSB) and the studied regions (in red, green and blue respectively). (C) Antisense transcription after CPT treatment in N-TERA-2 cells. Promoter-associated antisense transcripts were evaluated by rtqPCR after 4 hours CPT treatment at 10 μM. PCR determinations were normalized to cytochrome b mRNA and to untreated cells (dotted line). Values are means +/− SEM of two determinations from at least two independent experiments.
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    99
    Thermo Fisher rnaseh
    R-loop formation at the studied genomic and mitochondrial regions in untreated control N-TERA-2 cells. DNA enrichment of each sample is subtracted of the enrichment value of the same sample treated with <t>RNase</t> H1 before DRIVE precipitation. Then the enrichment value is normalized against the 2-min CPT sample (see Fig 2 ) of the RPL13A amplicon of the same experiment. Values are means ±SEM of two to four independent experiments. The data show a higher SEM than commonly published as we report median values of several experiments and not a single representative one. (A) DRIVE assay was performed to determine R-loop levels downstream TSS (white bars) and upstream TSS (black bars). Three negative loci for R-loop formation are also reported (SNRPN, a-SAT, TNIK). (B) Mitochondrial DNA was analyzed with DRIVE assay. Three regions of interest were selected: red for the r-loop forming region (RB31-R3), green for the D-loop region (1A-1B) and blue for the non-D-loop region (4A-4B). Map on the right of the panel shows the heavy (H) and the light (L) strands of mitochondrial DNA, with the three Conserved Sequence Blocks (CSB) and the studied regions (in red, green and blue respectively). (C) Antisense transcription after CPT treatment in N-TERA-2 cells. Promoter-associated antisense transcripts were evaluated by rtqPCR after 4 hours CPT treatment at 10 μM. PCR determinations were normalized to cytochrome b mRNA and to untreated cells (dotted line). Values are means +/− SEM of two determinations from at least two independent experiments.
    Rnaseh, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1371 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher dntp mix
    R-loop formation at the studied genomic and mitochondrial regions in untreated control N-TERA-2 cells. DNA enrichment of each sample is subtracted of the enrichment value of the same sample treated with <t>RNase</t> H1 before DRIVE precipitation. Then the enrichment value is normalized against the 2-min CPT sample (see Fig 2 ) of the RPL13A amplicon of the same experiment. Values are means ±SEM of two to four independent experiments. The data show a higher SEM than commonly published as we report median values of several experiments and not a single representative one. (A) DRIVE assay was performed to determine R-loop levels downstream TSS (white bars) and upstream TSS (black bars). Three negative loci for R-loop formation are also reported (SNRPN, a-SAT, TNIK). (B) Mitochondrial DNA was analyzed with DRIVE assay. Three regions of interest were selected: red for the r-loop forming region (RB31-R3), green for the D-loop region (1A-1B) and blue for the non-D-loop region (4A-4B). Map on the right of the panel shows the heavy (H) and the light (L) strands of mitochondrial DNA, with the three Conserved Sequence Blocks (CSB) and the studied regions (in red, green and blue respectively). (C) Antisense transcription after CPT treatment in N-TERA-2 cells. Promoter-associated antisense transcripts were evaluated by rtqPCR after 4 hours CPT treatment at 10 μM. PCR determinations were normalized to cytochrome b mRNA and to untreated cells (dotted line). Values are means +/− SEM of two determinations from at least two independent experiments.
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    Thermo Fisher rnase hi buffer
    R-loop formation at the studied genomic and mitochondrial regions in untreated control N-TERA-2 cells. DNA enrichment of each sample is subtracted of the enrichment value of the same sample treated with <t>RNase</t> H1 before DRIVE precipitation. Then the enrichment value is normalized against the 2-min CPT sample (see Fig 2 ) of the RPL13A amplicon of the same experiment. Values are means ±SEM of two to four independent experiments. The data show a higher SEM than commonly published as we report median values of several experiments and not a single representative one. (A) DRIVE assay was performed to determine R-loop levels downstream TSS (white bars) and upstream TSS (black bars). Three negative loci for R-loop formation are also reported (SNRPN, a-SAT, TNIK). (B) Mitochondrial DNA was analyzed with DRIVE assay. Three regions of interest were selected: red for the r-loop forming region (RB31-R3), green for the D-loop region (1A-1B) and blue for the non-D-loop region (4A-4B). Map on the right of the panel shows the heavy (H) and the light (L) strands of mitochondrial DNA, with the three Conserved Sequence Blocks (CSB) and the studied regions (in red, green and blue respectively). (C) Antisense transcription after CPT treatment in N-TERA-2 cells. Promoter-associated antisense transcripts were evaluated by rtqPCR after 4 hours CPT treatment at 10 μM. PCR determinations were normalized to cytochrome b mRNA and to untreated cells (dotted line). Values are means +/− SEM of two determinations from at least two independent experiments.
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    80
    Thermo Fisher flag tagged human rnase h1
    R-loop formation at the studied genomic and mitochondrial regions in untreated control N-TERA-2 cells. DNA enrichment of each sample is subtracted of the enrichment value of the same sample treated with <t>RNase</t> H1 before DRIVE precipitation. Then the enrichment value is normalized against the 2-min CPT sample (see Fig 2 ) of the RPL13A amplicon of the same experiment. Values are means ±SEM of two to four independent experiments. The data show a higher SEM than commonly published as we report median values of several experiments and not a single representative one. (A) DRIVE assay was performed to determine R-loop levels downstream TSS (white bars) and upstream TSS (black bars). Three negative loci for R-loop formation are also reported (SNRPN, a-SAT, TNIK). (B) Mitochondrial DNA was analyzed with DRIVE assay. Three regions of interest were selected: red for the r-loop forming region (RB31-R3), green for the D-loop region (1A-1B) and blue for the non-D-loop region (4A-4B). Map on the right of the panel shows the heavy (H) and the light (L) strands of mitochondrial DNA, with the three Conserved Sequence Blocks (CSB) and the studied regions (in red, green and blue respectively). (C) Antisense transcription after CPT treatment in N-TERA-2 cells. Promoter-associated antisense transcripts were evaluated by rtqPCR after 4 hours CPT treatment at 10 μM. PCR determinations were normalized to cytochrome b mRNA and to untreated cells (dotted line). Values are means +/− SEM of two determinations from at least two independent experiments.
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    Image Search Results


    RNase H cleavage analysis of truncated DNA substrates. (A) The substrates utilized are illustrated and are labeled A through F. The RNA portions are indicated in bold, and an asterisk indicates the radiolabel. The substrates were prepared as described in Materials and Methods. (B) Substrates B to F assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow. (C) Substrates B to F assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow.

    Journal: Journal of Virology

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: RNase H cleavage analysis of truncated DNA substrates. (A) The substrates utilized are illustrated and are labeled A through F. The RNA portions are indicated in bold, and an asterisk indicates the radiolabel. The substrates were prepared as described in Materials and Methods. (B) Substrates B to F assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow. (C) Substrates B to F assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: Labeling

    Second-strand transfer assay with model substrates. This illustrates the model strand transfer assay with the truncated substrate possessing only 12 (Δ6), 9 (Δ9), and 6 (Δ12) positions of the RNA sequence. Step 1 illustrates the input substrate for each truncated substrate, along with their respective input RNA-DNA sizes. Step 2 illustrates the polymerization reaction which can occur in the presence of RT and dNTPs and the size of the polymerization product for each substrate, 52-mer (Δ6), 49-mer (Δ9), and 46-mer (Δ12). DNA polymerization creates the RNA-DNA hybrid, which is a substrate for the RNase H domain (step 3). Once the RNA has been removed between the terminal ribo-A and ribo-C, the acceptor molecule can enter and produce a strand transfer product, 70-mer (step 4). In each step, the RNA portion is indicated in bold and the 5′ radiolabel is indicated by an asterisk. The size of the strand transfer product (70-mer) would be the same for each truncated substrate.

    Journal: Journal of Virology

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: Second-strand transfer assay with model substrates. This illustrates the model strand transfer assay with the truncated substrate possessing only 12 (Δ6), 9 (Δ9), and 6 (Δ12) positions of the RNA sequence. Step 1 illustrates the input substrate for each truncated substrate, along with their respective input RNA-DNA sizes. Step 2 illustrates the polymerization reaction which can occur in the presence of RT and dNTPs and the size of the polymerization product for each substrate, 52-mer (Δ6), 49-mer (Δ9), and 46-mer (Δ12). DNA polymerization creates the RNA-DNA hybrid, which is a substrate for the RNase H domain (step 3). Once the RNA has been removed between the terminal ribo-A and ribo-C, the acceptor molecule can enter and produce a strand transfer product, 70-mer (step 4). In each step, the RNA portion is indicated in bold and the 5′ radiolabel is indicated by an asterisk. The size of the strand transfer product (70-mer) would be the same for each truncated substrate.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: Sequencing

    Truncated substrates assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Lanes 1 to 6, 7 to 12, and 13 to 18 represent HIV-1 RT incubated with the Δ6, Δ9, and Δ12 constructs, respectively. Time points are indicated above each lane in minutes. Strand transfer products (70-mer), DNA primer (26-mer), and RNase H products are indicated by arrows. Input substrates for the Δ6, Δ9, and Δ12 constructs are 44-mer, 41-mer, and 38-mer, respectively. Initial RNase H cleavage products for the Δ6, Δ9, and Δ12 constructs are 11-mer, 8-mer, and 5-mer, respectively.

    Journal: Journal of Virology

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: Truncated substrates assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Lanes 1 to 6, 7 to 12, and 13 to 18 represent HIV-1 RT incubated with the Δ6, Δ9, and Δ12 constructs, respectively. Time points are indicated above each lane in minutes. Strand transfer products (70-mer), DNA primer (26-mer), and RNase H products are indicated by arrows. Input substrates for the Δ6, Δ9, and Δ12 constructs are 44-mer, 41-mer, and 38-mer, respectively. Initial RNase H cleavage products for the Δ6, Δ9, and Δ12 constructs are 11-mer, 8-mer, and 5-mer, respectively.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: Incubation, Construct

    Truncated substrates assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Lanes 1 to 6, 7 to 12, and 13 to 18 represent E478Q RT assayed with the Δ6, Δ9, and Δ12 constructs, respectively. Time points are indicated above each lane in minutes. Strand transfer products (70-mer), DNA primer (26-mer), and RNase H products are indicated by arrows. Input substrates for the Δ6, Δ9, and Δ12 constructs are 44-mer, 41-mer, and 38-mer, respectively. Initial RNase H cleavage products for the Δ6, Δ9, and Δ12 constructs are 11-mer, 8-mer, and 5-mer, respectively.

    Journal: Journal of Virology

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: Truncated substrates assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Lanes 1 to 6, 7 to 12, and 13 to 18 represent E478Q RT assayed with the Δ6, Δ9, and Δ12 constructs, respectively. Time points are indicated above each lane in minutes. Strand transfer products (70-mer), DNA primer (26-mer), and RNase H products are indicated by arrows. Input substrates for the Δ6, Δ9, and Δ12 constructs are 44-mer, 41-mer, and 38-mer, respectively. Initial RNase H cleavage products for the Δ6, Δ9, and Δ12 constructs are 11-mer, 8-mer, and 5-mer, respectively.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques: Construct

    (A) Complementation of HIV-1 RT with E. coli RNase H. Reactions were performed as described in Materials and Methods. Input RNA-DNA, DNA primer, and RNase H cleavage products are indicated by arrows. Reactions were allowed to proceed for 12 min in the presence of Mg 2+ , and then of E. coli RNase H was added (indicated by the vertical arrows). Time points are indicated above each lane in minutes. (B) Complementation of E478Q RT with E. coli RNase H. Reactions were performed as described for panel A. Time points are indicated above each lane in minutes. Input RNA-DNA, DNA primer, and RNase H cleavage products are indicated by arrows.

    Journal: Journal of Virology

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: (A) Complementation of HIV-1 RT with E. coli RNase H. Reactions were performed as described in Materials and Methods. Input RNA-DNA, DNA primer, and RNase H cleavage products are indicated by arrows. Reactions were allowed to proceed for 12 min in the presence of Mg 2+ , and then of E. coli RNase H was added (indicated by the vertical arrows). Time points are indicated above each lane in minutes. (B) Complementation of E478Q RT with E. coli RNase H. Reactions were performed as described for panel A. Time points are indicated above each lane in minutes. Input RNA-DNA, DNA primer, and RNase H cleavage products are indicated by arrows.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques:

    ) (1rtd), and substrates are shown as stick models. Positively charged amino acids are shown in blue, and negatively charged amino acids are shown in red. In the right-hand panel, the substrate found in the 1rtd structure has been truncated to include only 12 bp of template-primer extending from the RNase H active site. This truncated substrate makes very limited interactions with the thumb. T, thumb; F, fingers; RH, RNase H active site; Pol, polymerase active site.

    Journal: Journal of Virology

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    doi:

    Figure Lengend Snippet: ) (1rtd), and substrates are shown as stick models. Positively charged amino acids are shown in blue, and negatively charged amino acids are shown in red. In the right-hand panel, the substrate found in the 1rtd structure has been truncated to include only 12 bp of template-primer extending from the RNase H active site. This truncated substrate makes very limited interactions with the thumb. T, thumb; F, fingers; RH, RNase H active site; Pol, polymerase active site.

    Article Snippet: E. coli RNase H was purchased from Gibco BRL.

    Techniques:

    Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.

    Article Snippet: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    Techniques: Sequencing, Construct

    Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.

    Article Snippet: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    Techniques: Functional Assay, Preserving, Sequencing, Binding Assay

    RNase H Sequence Preferences correlate with gapmer efficiency. ( A ) Correlation between the log 2 fold changes of different hexamers observed for the R4b construct in the experiment and the corresponding log 2 fold changes as predicted by a single nucleotide model prepared from the data obtained in the R4a experiment. ( B ) As in (A), but with prediction with a dinucleotide model. ( C ) Prediction of RNase H1 mediated downregulation of the different 11-mers present in RNA sequence used for the RNase H RNA–DNA heteroduplex crystal structure [PDB: 2QK9]. Each bar corresponds to the cleavage site of a potential binding mode of RNase H1. The filled bar corresponds to the RNase H1 binding mode observed in the crystal structure and is also indicated in the drawing below the plot. ( D ) Correlation between the change of target RNA level for MAPT ( 30 ) after treatment with 1518 different gapmers and the corresponding downregulation predicted by the dinucleotide model for the different binding modes of RNase H1 on each gapmer target duplex. The error bars show the 99% confidence intervals. The drawing below the plot indicates the RNase H1 binding mode associated with the best-observed correlation. ( E ) The same analysis as in (D), but with 1581 different gapmers targeted against ANGPTL3 ( 31 ).

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: RNase H Sequence Preferences correlate with gapmer efficiency. ( A ) Correlation between the log 2 fold changes of different hexamers observed for the R4b construct in the experiment and the corresponding log 2 fold changes as predicted by a single nucleotide model prepared from the data obtained in the R4a experiment. ( B ) As in (A), but with prediction with a dinucleotide model. ( C ) Prediction of RNase H1 mediated downregulation of the different 11-mers present in RNA sequence used for the RNase H RNA–DNA heteroduplex crystal structure [PDB: 2QK9]. Each bar corresponds to the cleavage site of a potential binding mode of RNase H1. The filled bar corresponds to the RNase H1 binding mode observed in the crystal structure and is also indicated in the drawing below the plot. ( D ) Correlation between the change of target RNA level for MAPT ( 30 ) after treatment with 1518 different gapmers and the corresponding downregulation predicted by the dinucleotide model for the different binding modes of RNase H1 on each gapmer target duplex. The error bars show the 99% confidence intervals. The drawing below the plot indicates the RNase H1 binding mode associated with the best-observed correlation. ( E ) The same analysis as in (D), but with 1581 different gapmers targeted against ANGPTL3 ( 31 ).

    Article Snippet: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    Techniques: Sequencing, Construct, Binding Assay

    Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.

    Article Snippet: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    Techniques: Refining, Sequencing

    R-loop formation at the studied genomic and mitochondrial regions in untreated control N-TERA-2 cells. DNA enrichment of each sample is subtracted of the enrichment value of the same sample treated with RNase H1 before DRIVE precipitation. Then the enrichment value is normalized against the 2-min CPT sample (see Fig 2 ) of the RPL13A amplicon of the same experiment. Values are means ±SEM of two to four independent experiments. The data show a higher SEM than commonly published as we report median values of several experiments and not a single representative one. (A) DRIVE assay was performed to determine R-loop levels downstream TSS (white bars) and upstream TSS (black bars). Three negative loci for R-loop formation are also reported (SNRPN, a-SAT, TNIK). (B) Mitochondrial DNA was analyzed with DRIVE assay. Three regions of interest were selected: red for the r-loop forming region (RB31-R3), green for the D-loop region (1A-1B) and blue for the non-D-loop region (4A-4B). Map on the right of the panel shows the heavy (H) and the light (L) strands of mitochondrial DNA, with the three Conserved Sequence Blocks (CSB) and the studied regions (in red, green and blue respectively). (C) Antisense transcription after CPT treatment in N-TERA-2 cells. Promoter-associated antisense transcripts were evaluated by rtqPCR after 4 hours CPT treatment at 10 μM. PCR determinations were normalized to cytochrome b mRNA and to untreated cells (dotted line). Values are means +/− SEM of two determinations from at least two independent experiments.

    Journal: PLoS ONE

    Article Title: Dynamic Effects of Topoisomerase I Inhibition on R-Loops and Short Transcripts at Active Promoters

    doi: 10.1371/journal.pone.0147053

    Figure Lengend Snippet: R-loop formation at the studied genomic and mitochondrial regions in untreated control N-TERA-2 cells. DNA enrichment of each sample is subtracted of the enrichment value of the same sample treated with RNase H1 before DRIVE precipitation. Then the enrichment value is normalized against the 2-min CPT sample (see Fig 2 ) of the RPL13A amplicon of the same experiment. Values are means ±SEM of two to four independent experiments. The data show a higher SEM than commonly published as we report median values of several experiments and not a single representative one. (A) DRIVE assay was performed to determine R-loop levels downstream TSS (white bars) and upstream TSS (black bars). Three negative loci for R-loop formation are also reported (SNRPN, a-SAT, TNIK). (B) Mitochondrial DNA was analyzed with DRIVE assay. Three regions of interest were selected: red for the r-loop forming region (RB31-R3), green for the D-loop region (1A-1B) and blue for the non-D-loop region (4A-4B). Map on the right of the panel shows the heavy (H) and the light (L) strands of mitochondrial DNA, with the three Conserved Sequence Blocks (CSB) and the studied regions (in red, green and blue respectively). (C) Antisense transcription after CPT treatment in N-TERA-2 cells. Promoter-associated antisense transcripts were evaluated by rtqPCR after 4 hours CPT treatment at 10 μM. PCR determinations were normalized to cytochrome b mRNA and to untreated cells (dotted line). Values are means +/− SEM of two determinations from at least two independent experiments.

    Article Snippet: Each sample was then splitted in three: 1,5 μg were used as input, 1,5 μg were incubated for 2 hours at 37°C with 10U of RNase H1 and 1,5 μg were incubated for 2 hours at 37°C without RNase H1 (Life Technologies).

    Techniques: Cycling Probe Technology, Amplification, Sequencing, Polymerase Chain Reaction

    Chromosome fragmentation analysis by RNase HI, RNase HII and RNase A treatment in vitro A. A scheme of various hypothetical R-lesions (R-tract, two types of R-gaps) with positions of cleavage by RNase HI, HII and A (in low salt (LS) and high salt (HS) conditions) shown with arrows of the corresponding color. Small blue “d” letters, dNs; small orange “r” letters, rNs. The strand polarity in a duplex is identified on the left. B. A representative pulsed-field gel detecting chromosomal fragmentation after RNase HII treatment. The lanes are marked either with “b” (buffer treatment control) or “H2” (RNase HII treatment). Strains: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB , L-417. C. Quantification of the RNase treatment-induced fragmentation. The plotted values are means ± SEM from 3-6 independent measurements from gels like in “B”. For RNase A treatment, both low salt (LS) and high salt (HS) conditions are plotted. Since individual fragmentation values are differences between the enzyme and the buffer treatments, some values are negative.

    Journal: Journal of molecular biology

    Article Title: RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation

    doi: 10.1016/j.jmb.2017.08.004

    Figure Lengend Snippet: Chromosome fragmentation analysis by RNase HI, RNase HII and RNase A treatment in vitro A. A scheme of various hypothetical R-lesions (R-tract, two types of R-gaps) with positions of cleavage by RNase HI, HII and A (in low salt (LS) and high salt (HS) conditions) shown with arrows of the corresponding color. Small blue “d” letters, dNs; small orange “r” letters, rNs. The strand polarity in a duplex is identified on the left. B. A representative pulsed-field gel detecting chromosomal fragmentation after RNase HII treatment. The lanes are marked either with “b” (buffer treatment control) or “H2” (RNase HII treatment). Strains: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB , L-417. C. Quantification of the RNase treatment-induced fragmentation. The plotted values are means ± SEM from 3-6 independent measurements from gels like in “B”. For RNase A treatment, both low salt (LS) and high salt (HS) conditions are plotted. Since individual fragmentation values are differences between the enzyme and the buffer treatments, some values are negative.

    Article Snippet: Then half of the plug was treated in 50 μl of fresh 1× buffer and with 25 units RNase HI (Ambion) or 12.5 units RNase HII (NEB) or heat-treated 100 μg/ml RNase A (Boehringer) for 4 hours at 37°C.

    Techniques: In Vitro, Pulsed-Field Gel

    Growth, morphology and viability of the double rnhAB mutants A. A scheme of in vivo substrates of the two RNase H enzymes. The common substrate, framed in bright green, is the RNA-run with at least four contiguous rNs, which we call “R-tract”. HI and H1, HII and H2 refer to RNase H enzymes of prokaryotes and eukaryotes accordingly. B. Colony size on LB agar, 37°C, 24 hours. Strains: WT, AB1157; Δ rnhA , L-413; Δ rnhB , L-415; Δ rnhAB , L-416. C. Images of rnh and wild type strains stained with DAPI and observed by Hiraga's fluo-phase combined method. Cells were grown at 37°C in LB. The strains are like in “B”. D. Viability of the strains, determined as the ratio of the colony forming units (CFUs) to the microscopic counts in the same volume of the culture. Overnight cultures grown at 30°C were diluted and grown at the temperature (indicated by the first number) to OD 0.2-0.3 (about 2 hours), then cultures were serially diluted and plated on LB plates developed for 16 hours at the temperature indicated by the second number in pairs. Average viability (± SEM) of the eight WT measurements and six measurements for the rnhAB mutant cells is shown (the low titers of the two MG1655 Δ rnhAB cultures at 42°C were not used in the calculation). Strains: AB1157, L-416, MG1655, L-419. E. An enlarged image of the rnhAB mutant cells (processed as in panel C), to show nucleoids of both filamenting and normal-looking cells in some detail. F. Anaerobic growth inhibition of the rnhA and anaerobic lethality of rnhAB strains. Dilution-spotting of strains (like in “B”) was done in an anaerobic chamber on LB plates. Plates were incubated at room temperature in the chamber for 24 hours, then shifted to 28°C aerobic conditions for another 48 hours. G. The uvrA defect further reduces the colony size of the rnhAB double mutant. Strains: rnhAB , L-416; uvrA rnhA , L-414; uvrA rnhAB , L-417.

    Journal: Journal of molecular biology

    Article Title: RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation

    doi: 10.1016/j.jmb.2017.08.004

    Figure Lengend Snippet: Growth, morphology and viability of the double rnhAB mutants A. A scheme of in vivo substrates of the two RNase H enzymes. The common substrate, framed in bright green, is the RNA-run with at least four contiguous rNs, which we call “R-tract”. HI and H1, HII and H2 refer to RNase H enzymes of prokaryotes and eukaryotes accordingly. B. Colony size on LB agar, 37°C, 24 hours. Strains: WT, AB1157; Δ rnhA , L-413; Δ rnhB , L-415; Δ rnhAB , L-416. C. Images of rnh and wild type strains stained with DAPI and observed by Hiraga's fluo-phase combined method. Cells were grown at 37°C in LB. The strains are like in “B”. D. Viability of the strains, determined as the ratio of the colony forming units (CFUs) to the microscopic counts in the same volume of the culture. Overnight cultures grown at 30°C were diluted and grown at the temperature (indicated by the first number) to OD 0.2-0.3 (about 2 hours), then cultures were serially diluted and plated on LB plates developed for 16 hours at the temperature indicated by the second number in pairs. Average viability (± SEM) of the eight WT measurements and six measurements for the rnhAB mutant cells is shown (the low titers of the two MG1655 Δ rnhAB cultures at 42°C were not used in the calculation). Strains: AB1157, L-416, MG1655, L-419. E. An enlarged image of the rnhAB mutant cells (processed as in panel C), to show nucleoids of both filamenting and normal-looking cells in some detail. F. Anaerobic growth inhibition of the rnhA and anaerobic lethality of rnhAB strains. Dilution-spotting of strains (like in “B”) was done in an anaerobic chamber on LB plates. Plates were incubated at room temperature in the chamber for 24 hours, then shifted to 28°C aerobic conditions for another 48 hours. G. The uvrA defect further reduces the colony size of the rnhAB double mutant. Strains: rnhAB , L-416; uvrA rnhA , L-414; uvrA rnhAB , L-417.

    Article Snippet: Then half of the plug was treated in 50 μl of fresh 1× buffer and with 25 units RNase HI (Ambion) or 12.5 units RNase HII (NEB) or heat-treated 100 μg/ml RNase A (Boehringer) for 4 hours at 37°C.

    Techniques: In Vivo, Staining, Mutagenesis, Inhibition, Incubation

    Verification of RNase HI and RNase HII rN-DNA substrate specificity in vitro and the rN-density in DNA of the RNase H + cells and rnh mutants A. A scheme of the two double stranded oligo substrates: 38R1 (single rN) and 34R5 (five consecutive rN). The 32 P label at the 5′ end is shown as a red asterisk. DNA nucleotides are shown as blue lower case “d”, ribonucleotides are orange uppercase “R”. B. Products of the rN-DNA substrate hydrolysis by E. coli RNase HI and RNase HII enzymes. The radiolabelled rN-containing dsDNA oligos (shown in A) were incubated with the RNase HI or RNase HII enzymes. “0.1 M NaOH” and “Na Carb. pH 9.3” refer to alkali conditions in which rN hydrolysis produces reference size products. Numbers “1” or “5” refer to 38R1 or 34R5 oligos (A); ss/ds refers to whether the substrate used in the reaction was single-stranded or double-stranded. RNase H1 and RNase H2 were the E. coli enzymes RNase HI and RNase HII. RNase H1-1 and RNase H1-2 were RNase HI enzymes from different producers. The numbers on the side of the gel represent the sizes of the substrate and cleavage products. The reaction products were analyzed in 18% urea-PAGE gel. C. Only 34R5 oligo was used as either ss or ds substrate. All designations are like in “B”. D. Treatment with RNase HII of the plasmid isolated by alkaline lysis protocol. SCM, supercoiled monomer; b, buffer; H2, RNase HII. Plasmid: pEAK86, plasmid isolation was done at 0°C. Strains for results shown in panels D-I were: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB L-417. Product of the reactions were run in 1.1% agarose gel; autoradiogram of the representative Southern blot with the radiolabelled pEAK86 DNA as a probe is shown here and also in E and G. E. Treatment with either RNase HI or RNase HII enzymes of the plasmid isolated by the total genomic DNA protocol. SC, supercoiled plasmid; relaxed, relaxed plasmid; chrom., chromosomal DNA. Plasmid: pEAK86. Analysis of plasmid species was carried out as in D. F. Summary of quantification of the RNaseHII-revealed density of rNs in plasmid DNA isolated by various methods from the rnhAB double mutant. The density calculations are described in Methods. “Form.”, formamide. G. Alkali treatment analysis of rN-density. The plasmid DNA isolated by alkaline lysis at 0°C, was linearized and treated with NaOH. Treatment: “—”, no treatment; 0°, 0.2 M NaOH, 20 mM EDTA treatment on ice for 20 min; 45°, 0.3 M NaOH, 20 mM EDTA treatment at 45°C for 90 minutes. ds, linearized plasmid DNA, ss -single stranded plasmid. The samples were run in 1.1% agarose in TAE buffer, at 4°C. H. Summary of quantification of the rN-density determined by either RNase HII or by alkali treatments (from gels like in “G”). Various mutant comparison data are shown, pEAK86 was purified by alkaline lysis only, values are means of three independent measurements ± SEM. The star identifies the value already reported in panel “F”. I. R-loop removal by RNase HI or by RNase A. pAM34 isolated from rnhA (strain L-413) by the total genomic DNA protocol.

    Journal: Journal of molecular biology

    Article Title: RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation

    doi: 10.1016/j.jmb.2017.08.004

    Figure Lengend Snippet: Verification of RNase HI and RNase HII rN-DNA substrate specificity in vitro and the rN-density in DNA of the RNase H + cells and rnh mutants A. A scheme of the two double stranded oligo substrates: 38R1 (single rN) and 34R5 (five consecutive rN). The 32 P label at the 5′ end is shown as a red asterisk. DNA nucleotides are shown as blue lower case “d”, ribonucleotides are orange uppercase “R”. B. Products of the rN-DNA substrate hydrolysis by E. coli RNase HI and RNase HII enzymes. The radiolabelled rN-containing dsDNA oligos (shown in A) were incubated with the RNase HI or RNase HII enzymes. “0.1 M NaOH” and “Na Carb. pH 9.3” refer to alkali conditions in which rN hydrolysis produces reference size products. Numbers “1” or “5” refer to 38R1 or 34R5 oligos (A); ss/ds refers to whether the substrate used in the reaction was single-stranded or double-stranded. RNase H1 and RNase H2 were the E. coli enzymes RNase HI and RNase HII. RNase H1-1 and RNase H1-2 were RNase HI enzymes from different producers. The numbers on the side of the gel represent the sizes of the substrate and cleavage products. The reaction products were analyzed in 18% urea-PAGE gel. C. Only 34R5 oligo was used as either ss or ds substrate. All designations are like in “B”. D. Treatment with RNase HII of the plasmid isolated by alkaline lysis protocol. SCM, supercoiled monomer; b, buffer; H2, RNase HII. Plasmid: pEAK86, plasmid isolation was done at 0°C. Strains for results shown in panels D-I were: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB L-417. Product of the reactions were run in 1.1% agarose gel; autoradiogram of the representative Southern blot with the radiolabelled pEAK86 DNA as a probe is shown here and also in E and G. E. Treatment with either RNase HI or RNase HII enzymes of the plasmid isolated by the total genomic DNA protocol. SC, supercoiled plasmid; relaxed, relaxed plasmid; chrom., chromosomal DNA. Plasmid: pEAK86. Analysis of plasmid species was carried out as in D. F. Summary of quantification of the RNaseHII-revealed density of rNs in plasmid DNA isolated by various methods from the rnhAB double mutant. The density calculations are described in Methods. “Form.”, formamide. G. Alkali treatment analysis of rN-density. The plasmid DNA isolated by alkaline lysis at 0°C, was linearized and treated with NaOH. Treatment: “—”, no treatment; 0°, 0.2 M NaOH, 20 mM EDTA treatment on ice for 20 min; 45°, 0.3 M NaOH, 20 mM EDTA treatment at 45°C for 90 minutes. ds, linearized plasmid DNA, ss -single stranded plasmid. The samples were run in 1.1% agarose in TAE buffer, at 4°C. H. Summary of quantification of the rN-density determined by either RNase HII or by alkali treatments (from gels like in “G”). Various mutant comparison data are shown, pEAK86 was purified by alkaline lysis only, values are means of three independent measurements ± SEM. The star identifies the value already reported in panel “F”. I. R-loop removal by RNase HI or by RNase A. pAM34 isolated from rnhA (strain L-413) by the total genomic DNA protocol.

    Article Snippet: Then half of the plug was treated in 50 μl of fresh 1× buffer and with 25 units RNase HI (Ambion) or 12.5 units RNase HII (NEB) or heat-treated 100 μg/ml RNase A (Boehringer) for 4 hours at 37°C.

    Techniques: In Vitro, Incubation, Polyacrylamide Gel Electrophoresis, Plasmid Preparation, Isolation, Alkaline Lysis, Agarose Gel Electrophoresis, Southern Blot, Mutagenesis, Purification