Journal: Nucleic Acids Research

Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

doi: 10.1093/nar/gkx1073

Figure Lengend Snippet: Overview and validation of the H-SPA method. ( A ) Sequences of the cleavable randomized gaps used in this study. ( B ) Schematic representation of the experimental strategy. ( C ) Denaturing gel electrophoresis showing the limited digestion of double stranded substrates. The uncleaved duplexes are indicated with the asterisk and the cleaved fragment with the tilde. The reaction conditions are indicated: w: 37°C; c: 16°C and EDTA: 0.1 mM. ( D ) Correlation of heptamer counts for the construct R4b (positions 9–15) between two untreated controls (top left), two human RNase H1 treated samples (top right) and a control and a treated sample (bottom).

Article Snippet: Hydrolysis reactions with recombinant human RNase H1 (Creative BioMart cat. RNASEH1-433H) were performed at 37°C in a buffer RNH1 (20 mM Tris–HCl pH 7.5, 20 mM KCl, 20 mM 2-mercaptoethanol, 2 mM MgCl 2 and 0.1 mM EDTA (modified from Lima et al. , 2001 ( )).

Techniques: Biomarker Discovery, Nucleic Acid Electrophoresis, Construct, Control

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: Hydrolysis reactions with recombinant human RNase H1 (Creative BioMart cat. RNASEH1-433H) were performed at 37°C in a buffer RNH1 (20 mM Tris–HCl pH 7.5, 20 mM KCl, 20 mM 2-mercaptoethanol, 2 mM MgCl 2 and 0.1 mM EDTA (modified from Lima et al. , 2001 ( )).

Techniques: Sequencing, Construct

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 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 .

Article Snippet: Hydrolysis reactions with recombinant human RNase H1 (Creative BioMart cat. RNASEH1-433H) were performed at 37°C in a buffer RNH1 (20 mM Tris–HCl pH 7.5, 20 mM KCl, 20 mM 2-mercaptoethanol, 2 mM MgCl 2 and 0.1 mM EDTA (modified from Lima et al. , 2001 ( )).

Techniques: Sequencing, Construct, Binding Assay

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: Hydrolysis reactions with recombinant human RNase H1 (Creative BioMart cat. RNASEH1-433H) were performed at 37°C in a buffer RNH1 (20 mM Tris–HCl pH 7.5, 20 mM KCl, 20 mM 2-mercaptoethanol, 2 mM MgCl 2 and 0.1 mM EDTA (modified from Lima et al. , 2001 ( )).

Techniques: Refining, Sequencing

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: Hydrolysis reactions with recombinant human RNase H1 (Creative BioMart cat. RNASEH1-433H) were performed at 37°C in a buffer RNH1 (20 mM Tris–HCl pH 7.5, 20 mM KCl, 20 mM 2-mercaptoethanol, 2 mM MgCl 2 and 0.1 mM EDTA (modified from Lima et al. , 2001 ( )).

Techniques: Functional Assay, Reverse Transcription, Comparison, Preserving, Sequencing, Binding Assay

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 1. Knockout of Rnaseh2a gene of HEK293 cells with the CRISPR/Cas9 system.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques: Knock-Out, CRISPR

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 2. Screening of Rnaseh2a-/- homozygote HEK293 cells.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques:

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 7. Knockout of Rnaseh2a gene of HEK293

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques: Knock-Out

Journal: bioRxiv

Article Title: Cytoplasmic circular dsDNA is a key constituent of stress granules

doi: 10.64898/2026.03.12.711345

Figure Lengend Snippet: (A) Abundance of histones (brown) and other representative DNA-binding proteins (gray) in the proteomes of yeast stress granule cores (strain JD1370 grown in synthetic defined (SD) media), normalized to that of the stress granule marker eIF4A [peptide spectrum matches (PSMs); means from n = 2 biological replicates ± s.d.; n.s., no significance; one-tailed Student’s t -test]. (B) Abundance of histones and other representative DNA-binding proteins in the proteomes of HEK293T cell stress granule cores, normalized to that of the stress granule marker G3BP1 (PSMs; means from n = 3 biological replicates ± s.d.; *, p < 0.05; one-tailed Student’s t -test. Stress granule markers, histones and non-histone DNA-binding proteins in green, yellow, and gray, respectively. (C) Negative-stain immuno-electron micrograph of stress granule cores from yeast strain YAG1021 carrying a chromosomally encoded FLAG-tagged histone H3. Anti-FLAG antibodies were conjugated to NanoGold beads (arrows; Methods). Magnification (× 23,000). (D) Native agarose gel electrophoresis (SYBR Gold stain) of yeast stress granule cores treated with DNase I. BP, bromophenol blue. (E) DNase I, psDNase and RNase H treatment (the first at two different concentrations) of total nucleic acids extracted from yeast stress granule cores analyzed as in (D). Bottom graph, integrated signal for the dashed box. (F) Comparison of nuclease susceptibility of total nucleic acids extracted from yeast and mammalian stress granule cores, analyzed as in (D). (G) Alkaline hydrolysis of total nucleic acids extracted from yeast stress granule cores (15, 10 and 5 ng) and rRNA (15 ng), analyzed by non-denaturing agarose gel electrophoresis (SYTOX Green stain). Samples marked (+) were incubated with 50 mM KOH for 15 minutes at 95 °C. Bottom graph, integrated signal for the dashed box.

Article Snippet: Next, 5 units of thermostable RNase H (New England Biolabs, Cat# M0523) was added and the reaction was incubated for 30 min at 86 °C in the RNase H reaction buffer.

Techniques: DNA Binding Assay, Marker, One-tailed Test, Staining, Agarose Gel Electrophoresis, Comparison, Incubation