e coli rnase h1  (Thermo Fisher)

Journal: PLoS ONE

Article Title: Antisense Oligonucleotides Capable of Promoting Specific Target mRNA Reduction via Competing RNase H1-Dependent and Independent Mechanisms

doi: 10.1371/journal.pone.0108625

Figure Lengend Snippet: Proposed mechanism for non-RNase H activity with a DNA-like ASO. A) In the absence of ASO treatment hnRNPs bind the pre-mRNA, and it is efficiently spliced. B) A fully modified 2′MOE ASO binds the hnRNP site preventing hnRNP interaction with the pre-mRNA, leading to accumulation of pre-mRNA in the nucleus and reduction of spliced mRNA is the cytoplasm. C) An RNase H-activating ASO also competes with hnRNP for the binding site on the pre-mRNA; however, RNase H cleavage is prevented when HSPA8 binds the RNA/ASO heteroduplex.

Article Snippet: For RNase H reduction experiments, 107 cells were seeded in 10-cm plates and treated for 4 h with 25 nM siRNA (Ambion Silencer siRNA, # s48358) in OptiMem media with 6 µg/ml Lipofectamine RNAiMax.

Techniques: Activity Assay, Allele-specific Oligonucleotide, Modification, Binding Assay

Journal: PLoS ONE

Article Title: Antisense Oligonucleotides Capable of Promoting Specific Target mRNA Reduction via Competing RNase H1-Dependent and Independent Mechanisms

doi: 10.1371/journal.pone.0108625

Figure Lengend Snippet: Effect of RNase H1 reduction on activity of SOD1 minigene ASOs. A) SOD/TO cells were treated with an siRNA targeting human RNase H1. After 48 hours, RNase H-deficient and control cells were transfected with SOD1 ASOs at 50 nM. Following TET induction, expression of minigene-derived spliced mRNA was assessed by qRT/PCR. Expression is shown relative to mock-treated control. Solid bars, SOD/TO cells; striped bars, SOD/TO treated with siRNA targeting gene encoding RNase H1. B) Northern analysis of levels of SOD/TO minigene transcript. Cells were treated as in panel A.

Article Snippet: For RNase H reduction experiments, 107 cells were seeded in 10-cm plates and treated for 4 h with 25 nM siRNA (Ambion Silencer siRNA, # s48358) in OptiMem media with 6 µg/ml Lipofectamine RNAiMax.

Techniques: Activity Assay, Transfection, Expressing, Derivative Assay, Quantitative RT-PCR, Northern Blot

Journal: PLoS ONE

Article Title: Antisense Oligonucleotides Capable of Promoting Specific Target mRNA Reduction via Competing RNase H1-Dependent and Independent Mechanisms

doi: 10.1371/journal.pone.0108625

Figure Lengend Snippet: Activities of RNase H-independent 2′MOE ASOs. A) SOD/TO cells were transfected with a series of 38 2′MOE ASOs and gapmer ASOs of the same sequences (these gapmers were also tested in a separate experiment shown in Figure 2 ). Following ASO treatment, levels of SOD1 minigene mRNA were analyzed by qRT/PCR. Data are presented as percent spliced mRNA in treated vs. mock-treated cells. Δ = gapmer ASOs, ○ = 2′MOE ASOs. B) SOD/TO or SOD/TO-187 cells were transfected with 50 nM gapmer ASOs 440222 or 440282 and 2′MOE ASOs of the same sequence. Levels of spliced and pre-mRNA were assessed by qRT/PCR using the primer/probes depicted in Figure 1 . Activity is presented as percent expression relative to levels in mock-transfected cells. Solid bars, SOD/TO spliced mRNA; grey bars, SOD/TO pre-mRNA; striped bars, SOD/TO-187 spliced mRNA; hatched bars, SOD/TO-187 pre-mRNA. C) SOD/TO or SOD/TO-H1 cells were transfected with 30 nM gapmer ASOs 440282 or 440283 and 2′MOE ASOs of the same sequence. Levels of SOD1 minigene spliced and pre-mRNA were assessed by qRT/PCR as described above. Solid bars, SOD/TO spliced mRNA; grey bars, SOD/TO pre-mRNA; striped bars, SOD/TO-H1 spliced mRNA; hatched bars, SOD/TO-H1 pre-mRNA.

Article Snippet: For RNase H reduction experiments, 107 cells were seeded in 10-cm plates and treated for 4 h with 25 nM siRNA (Ambion Silencer siRNA, # s48358) in OptiMem media with 6 µg/ml Lipofectamine RNAiMax.

Techniques: Transfection, Allele-specific Oligonucleotide, Quantitative RT-PCR, Sequencing, Activity Assay, Expressing

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet: Gel analysis of RNase H/oligonucleotide accessibility assays. 30 fmol of internally labeled RNA were used. DNA oligonucleotides were used to a final concentration of 0 to 10 μM. Reactions were incubated for 30 min at 30°C in the presence or absence (C, control) of 0.25 U E. coli RNase H1. Reactions were stopped with 10 μl of 2X Loading Buffer and products were analyzed in an 8% denaturing PAA gel. See Figure 5C for relative band quantification. P1 and P2 indicate the two RNase H-oligonucleotide-mediated RNA cleavage products.

Article Snippet: Reactions were adjusted to a final volume of 10 μl with H2 O and incubated for 30 min at 30°C in the presence or absence (control) of 0.25 U E. coli RNase H1 (Ambion #AM2293).

Techniques: Labeling, Concentration Assay, Incubation