ribonuclease v1 Search Results


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  • 99
    Thermo Fisher rnase v1 s1 nuclease digested
    Schematic of RNA structure probing by PARS in zebrafish. Poly-A RNA from zebrafish is folded in-vitro. The folded RNA is cleaved by <t>RNase</t> V1 and <t>S1</t> nuclease separately. The enzyme cut sites generate 5’P ends and 3’ OH ends at the cleaved sites. Long fragments generated by single-hit kinetics are further fragmented by alkaline hydrolysis, which blocks the 3′ site of the enzyme-cut fragments. Sequencing adapters are ligated to the 5′ end followed by alkaline phosphatase treatment to 3’ P group. Adapters are ligated to 3’ends followed cDNA synthesis and PCR purification of the library. Appropriate size of the library is maintained by purification by nucleic acid beads. Sequenced reads are aligned back to the genome and only unique reads with the correct read start positions are considered for PARS score calculation
    Rnase V1 S1 Nuclease Digested, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher rnase v1
    RNA structure mapping results are shown with respect to the psbC 5′ UTR sequence. Asterisks indicate bases methylated by DMS in vitro and in vivo . Arrowheads indicate residues that were cleaved by <t>RNase</t> V1. Arrows indicate G residues that were cleaved by RNase T1. Most frequently methylated or cleaved positions are indicated by black asterisks, arrows and arrowheads. Less frequently methylated or cleaved sites are indicated in gray. Brackets indicate results with high accuracy and resolution. The 5′ terminal residue is designated +1, the translation initiation codon (GUG) is in bold, and the predicted Shine–Dalgarno sequence is underlined. Black and gray dots represent strong and weak RT pause sites, respectively.
    Rnase V1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 780 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rnase v1 - by Bioz Stars, 2021-01
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    99
    Thermo Fisher rnase v1 endonuclease protocol
    RNA structure mapping results are shown with respect to the psbC 5′ UTR sequence. Asterisks indicate bases methylated by DMS in vitro and in vivo . Arrowheads indicate residues that were cleaved by <t>RNase</t> V1. Arrows indicate G residues that were cleaved by RNase T1. Most frequently methylated or cleaved positions are indicated by black asterisks, arrows and arrowheads. Less frequently methylated or cleaved sites are indicated in gray. Brackets indicate results with high accuracy and resolution. The 5′ terminal residue is designated +1, the translation initiation codon (GUG) is in bold, and the predicted Shine–Dalgarno sequence is underlined. Black and gray dots represent strong and weak RT pause sites, respectively.
    Rnase V1 Endonuclease Protocol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rnase v1 structure mapping
    RNA structure mapping results are shown with respect to the psbC 5′ UTR sequence. Asterisks indicate bases methylated by DMS in vitro and in vivo . Arrowheads indicate residues that were cleaved by <t>RNase</t> V1. Arrows indicate G residues that were cleaved by RNase T1. Most frequently methylated or cleaved positions are indicated by black asterisks, arrows and arrowheads. Less frequently methylated or cleaved sites are indicated in gray. Brackets indicate results with high accuracy and resolution. The 5′ terminal residue is designated +1, the translation initiation codon (GUG) is in bold, and the predicted Shine–Dalgarno sequence is underlined. Black and gray dots represent strong and weak RT pause sites, respectively.
    Rnase V1 Structure Mapping, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Boehringer Mannheim rnase v1
    RNA structure mapping results are shown with respect to the psbC 5′ UTR sequence. Asterisks indicate bases methylated by DMS in vitro and in vivo . Arrowheads indicate residues that were cleaved by <t>RNase</t> V1. Arrows indicate G residues that were cleaved by RNase T1. Most frequently methylated or cleaved positions are indicated by black asterisks, arrows and arrowheads. Less frequently methylated or cleaved sites are indicated in gray. Brackets indicate results with high accuracy and resolution. The 5′ terminal residue is designated +1, the translation initiation codon (GUG) is in bold, and the predicted Shine–Dalgarno sequence is underlined. Black and gray dots represent strong and weak RT pause sites, respectively.
    Rnase V1, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase v1/product/Boehringer Mannheim
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rnase v1 - by Bioz Stars, 2021-01
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    Standard format Plasmid sent in bacteria as agar stab
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    Standard format Plasmid sent in bacteria as agar stab
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    Image Search Results


    Schematic of RNA structure probing by PARS in zebrafish. Poly-A RNA from zebrafish is folded in-vitro. The folded RNA is cleaved by RNase V1 and S1 nuclease separately. The enzyme cut sites generate 5’P ends and 3’ OH ends at the cleaved sites. Long fragments generated by single-hit kinetics are further fragmented by alkaline hydrolysis, which blocks the 3′ site of the enzyme-cut fragments. Sequencing adapters are ligated to the 5′ end followed by alkaline phosphatase treatment to 3’ P group. Adapters are ligated to 3’ends followed cDNA synthesis and PCR purification of the library. Appropriate size of the library is maintained by purification by nucleic acid beads. Sequenced reads are aligned back to the genome and only unique reads with the correct read start positions are considered for PARS score calculation

    Journal: BMC Genomics

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features

    doi: 10.1186/s12864-018-4497-0

    Figure Lengend Snippet: Schematic of RNA structure probing by PARS in zebrafish. Poly-A RNA from zebrafish is folded in-vitro. The folded RNA is cleaved by RNase V1 and S1 nuclease separately. The enzyme cut sites generate 5’P ends and 3’ OH ends at the cleaved sites. Long fragments generated by single-hit kinetics are further fragmented by alkaline hydrolysis, which blocks the 3′ site of the enzyme-cut fragments. Sequencing adapters are ligated to the 5′ end followed by alkaline phosphatase treatment to 3’ P group. Adapters are ligated to 3’ends followed cDNA synthesis and PCR purification of the library. Appropriate size of the library is maintained by purification by nucleic acid beads. Sequenced reads are aligned back to the genome and only unique reads with the correct read start positions are considered for PARS score calculation

    Article Snippet: Further, 20 μl of 3 M sodium acetate, 1 μl of glycogen and 300 μl of cold ethanol was added to precipitate RNA at -80 °C for 1 h. S1 Nuclease cleaved RNA pool in the top aqueous layer was extracted and 20 μl of 3 M sodium acetate, 1 μl of glycogen and 300 μl of cold ethanol was added to precipitate RNA at -80 °C for 1 h. Further, the purified RNase V1/S1 Nuclease digested samples were fragmented by alkaline hydrolysis buffer (Life Technologies, USA) containing 500 mM Sodium bicarbonate at 95 °C, for 1.5 min which generated 3’phosphate groups at the enzyme cleaved fragments.

    Techniques: In Vitro, Generated, Sequencing, Polymerase Chain Reaction, Purification

    Comparison of RNA structures of ubc 3’UTR as determined by PARS based pairing probability and enzymatic footprinting using RNase V1 and S1 Nuclease. a . Bar plot represents PARS scores of 3’UTR region of ubiquitin c (ubc) . Out of 105 positions, 87 positions are captured by PARS. b . Enzymatic footprinting of ubc 3’UTR probed by S1 Nuclease and RNase V1. Nucleotide positions are correlated with alkaline hydrolysis (AH) ladder and RNase T1 (G) ladder. Positions with similar structural pattern with PARS scores are highlighted. Red dots indicate unpaired positions; green indicates paired positions while yellow represents ambiguous regions. c . Heatmap representing secondary structure of 68 positions of ubc 3’UTR as determined by PARS and enzymatic footprinting (FP). Top panel represents PARS pairing probability; bottom panel indicates enzymatic footprinting pairing probability; middle panel represents the consensus between the two (PARS: FP). Red represents unpaired, green represents paired and yellow represents ambiguous regions

    Journal: BMC Genomics

    Article Title: RNA secondary structure profiling in zebrafish reveals unique regulatory features

    doi: 10.1186/s12864-018-4497-0

    Figure Lengend Snippet: Comparison of RNA structures of ubc 3’UTR as determined by PARS based pairing probability and enzymatic footprinting using RNase V1 and S1 Nuclease. a . Bar plot represents PARS scores of 3’UTR region of ubiquitin c (ubc) . Out of 105 positions, 87 positions are captured by PARS. b . Enzymatic footprinting of ubc 3’UTR probed by S1 Nuclease and RNase V1. Nucleotide positions are correlated with alkaline hydrolysis (AH) ladder and RNase T1 (G) ladder. Positions with similar structural pattern with PARS scores are highlighted. Red dots indicate unpaired positions; green indicates paired positions while yellow represents ambiguous regions. c . Heatmap representing secondary structure of 68 positions of ubc 3’UTR as determined by PARS and enzymatic footprinting (FP). Top panel represents PARS pairing probability; bottom panel indicates enzymatic footprinting pairing probability; middle panel represents the consensus between the two (PARS: FP). Red represents unpaired, green represents paired and yellow represents ambiguous regions

    Article Snippet: Further, 20 μl of 3 M sodium acetate, 1 μl of glycogen and 300 μl of cold ethanol was added to precipitate RNA at -80 °C for 1 h. S1 Nuclease cleaved RNA pool in the top aqueous layer was extracted and 20 μl of 3 M sodium acetate, 1 μl of glycogen and 300 μl of cold ethanol was added to precipitate RNA at -80 °C for 1 h. Further, the purified RNase V1/S1 Nuclease digested samples were fragmented by alkaline hydrolysis buffer (Life Technologies, USA) containing 500 mM Sodium bicarbonate at 95 °C, for 1.5 min which generated 3’phosphate groups at the enzyme cleaved fragments.

    Techniques: Footprinting

    RNA structure mapping results are shown with respect to the psbC 5′ UTR sequence. Asterisks indicate bases methylated by DMS in vitro and in vivo . Arrowheads indicate residues that were cleaved by RNase V1. Arrows indicate G residues that were cleaved by RNase T1. Most frequently methylated or cleaved positions are indicated by black asterisks, arrows and arrowheads. Less frequently methylated or cleaved sites are indicated in gray. Brackets indicate results with high accuracy and resolution. The 5′ terminal residue is designated +1, the translation initiation codon (GUG) is in bold, and the predicted Shine–Dalgarno sequence is underlined. Black and gray dots represent strong and weak RT pause sites, respectively.

    Journal: Frontiers in Plant Science

    Article Title: The RNA Structure of cis-acting Translational Elements of the Chloroplast psbC mRNA in Chlamydomonas reinhardtii

    doi: 10.3389/fpls.2016.00828

    Figure Lengend Snippet: RNA structure mapping results are shown with respect to the psbC 5′ UTR sequence. Asterisks indicate bases methylated by DMS in vitro and in vivo . Arrowheads indicate residues that were cleaved by RNase V1. Arrows indicate G residues that were cleaved by RNase T1. Most frequently methylated or cleaved positions are indicated by black asterisks, arrows and arrowheads. Less frequently methylated or cleaved sites are indicated in gray. Brackets indicate results with high accuracy and resolution. The 5′ terminal residue is designated +1, the translation initiation codon (GUG) is in bold, and the predicted Shine–Dalgarno sequence is underlined. Black and gray dots represent strong and weak RT pause sites, respectively.

    Article Snippet: Digestions with RNase T1 (Fermentas) and RNase V1 (Ambion) were performed with approximately 3 × 105 cpm RNA in 10 μl 10 mM Tris [pH 7], 100 mM KCl, 10 mM MgCl2 [Ambion] and either 1μg sheared yeast RNA (10 mg/ml, Ambion) and 1 mU of RNase V1 (1 U/μl, Ambion) for 5 min at 24°C, or 4 μg sheared yeast RNA (Sigma Chemicals) and 500 mU of RNase T1 (100 U/μl, Fermentas) for 2 min at 24°C.

    Techniques: Sequencing, Methylation, In Vitro, In Vivo

    In vitro analysis of stem-loop (SL) structures in the psbC 5′ UTR. Asterisks ( ∗ ) indicate A and C residues that are methylated by DMS in vitro . Arrows indicate G residues that are substrates for RNase T1 and arrowheads indicated cleavage sites for RNase V1. Most frequently methylated or cleaved positions are indicated by black asterisks, arrows and arrowheads. Less frequently methylated or cleaved sites are indicated in gray. Hexagons indicate locations of strong premature RT stops. Black dots indicate base-pairs that are strongly supported by the data, while gray dots are partially supported and could be present in a subpopulation of RNA substrate molecules. (A–C) DMS methylation and RNase cleavage sites on the three stem-loop structures (SL1, 2 and 3), which were seen in simulations using the mfold server ( Zuker, 2003 ). (D) Base-pairing of the nucleotides in the loop structure of SL2 to the sequences (367–383) 3′ to the SL2 structure giving rise to (E) pseudoknot tertiary structure. (F,G) DMS methylation, RNase cleavage and premature RT stop sites on the stem structure of SL2 from FuD34 and F34su1 mutant psbC 5′ UTR are shown. Note that DMS methylation could not be determined for the psbC-FuD34 mutant 5′ UTR and the absence of asterisks is due to this, not inaccessibility. (F,G) Mutant residues are shown in black boxes with white text.

    Journal: Frontiers in Plant Science

    Article Title: The RNA Structure of cis-acting Translational Elements of the Chloroplast psbC mRNA in Chlamydomonas reinhardtii

    doi: 10.3389/fpls.2016.00828

    Figure Lengend Snippet: In vitro analysis of stem-loop (SL) structures in the psbC 5′ UTR. Asterisks ( ∗ ) indicate A and C residues that are methylated by DMS in vitro . Arrows indicate G residues that are substrates for RNase T1 and arrowheads indicated cleavage sites for RNase V1. Most frequently methylated or cleaved positions are indicated by black asterisks, arrows and arrowheads. Less frequently methylated or cleaved sites are indicated in gray. Hexagons indicate locations of strong premature RT stops. Black dots indicate base-pairs that are strongly supported by the data, while gray dots are partially supported and could be present in a subpopulation of RNA substrate molecules. (A–C) DMS methylation and RNase cleavage sites on the three stem-loop structures (SL1, 2 and 3), which were seen in simulations using the mfold server ( Zuker, 2003 ). (D) Base-pairing of the nucleotides in the loop structure of SL2 to the sequences (367–383) 3′ to the SL2 structure giving rise to (E) pseudoknot tertiary structure. (F,G) DMS methylation, RNase cleavage and premature RT stop sites on the stem structure of SL2 from FuD34 and F34su1 mutant psbC 5′ UTR are shown. Note that DMS methylation could not be determined for the psbC-FuD34 mutant 5′ UTR and the absence of asterisks is due to this, not inaccessibility. (F,G) Mutant residues are shown in black boxes with white text.

    Article Snippet: Digestions with RNase T1 (Fermentas) and RNase V1 (Ambion) were performed with approximately 3 × 105 cpm RNA in 10 μl 10 mM Tris [pH 7], 100 mM KCl, 10 mM MgCl2 [Ambion] and either 1μg sheared yeast RNA (10 mg/ml, Ambion) and 1 mU of RNase V1 (1 U/μl, Ambion) for 5 min at 24°C, or 4 μg sheared yeast RNA (Sigma Chemicals) and 500 mU of RNase T1 (100 U/μl, Fermentas) for 2 min at 24°C.

    Techniques: In Vitro, Methylation, Mutagenesis

    RNA structure analyses of the psbC 5′ UTR by enzymatic probing. In vitro transcribed RNA corresponding to the wild-type and mutant psbC 5′ UTR were untreated (0) or digested with RNase T1 (T1) or RNase V1 (V1), which cleave unpaired G residues or RNA helices, respectively. Digestion products were resolved by denaturing PAGE and revealed by autoradiography. The positions of cleavage sites are indicated with respect to their positions on the 5′ UTR, based on the motilities of molecular size markers (not shown). (A) The substrate RNA was 3′- 32 P-labeled wild-type psbC 5′ UTR. (B,C) The substrates were 5′- 32 P-end-labeled RNAs corresponding to the psb C 5′ UTR with wild-type sequence (wt), or carrying one of the mutations; psbC-FuD34 (m), or psbC-F34suI (s). The exact position of certain digestion products could not be determined with certainty ( ∗ ).

    Journal: Frontiers in Plant Science

    Article Title: The RNA Structure of cis-acting Translational Elements of the Chloroplast psbC mRNA in Chlamydomonas reinhardtii

    doi: 10.3389/fpls.2016.00828

    Figure Lengend Snippet: RNA structure analyses of the psbC 5′ UTR by enzymatic probing. In vitro transcribed RNA corresponding to the wild-type and mutant psbC 5′ UTR were untreated (0) or digested with RNase T1 (T1) or RNase V1 (V1), which cleave unpaired G residues or RNA helices, respectively. Digestion products were resolved by denaturing PAGE and revealed by autoradiography. The positions of cleavage sites are indicated with respect to their positions on the 5′ UTR, based on the motilities of molecular size markers (not shown). (A) The substrate RNA was 3′- 32 P-labeled wild-type psbC 5′ UTR. (B,C) The substrates were 5′- 32 P-end-labeled RNAs corresponding to the psb C 5′ UTR with wild-type sequence (wt), or carrying one of the mutations; psbC-FuD34 (m), or psbC-F34suI (s). The exact position of certain digestion products could not be determined with certainty ( ∗ ).

    Article Snippet: Digestions with RNase T1 (Fermentas) and RNase V1 (Ambion) were performed with approximately 3 × 105 cpm RNA in 10 μl 10 mM Tris [pH 7], 100 mM KCl, 10 mM MgCl2 [Ambion] and either 1μg sheared yeast RNA (10 mg/ml, Ambion) and 1 mU of RNase V1 (1 U/μl, Ambion) for 5 min at 24°C, or 4 μg sheared yeast RNA (Sigma Chemicals) and 500 mU of RNase T1 (100 U/μl, Fermentas) for 2 min at 24°C.

    Techniques: In Vitro, Mutagenesis, Polyacrylamide Gel Electrophoresis, Autoradiography, Labeling, Sequencing

    ( A ) Sequence and secondary structure for the 5′ and the 3′ ends of the HCV genome. The 5′ UTR plus domains V and VI located at the core coding sequence are included. The minimum region for IRES activity is shown. The 3′ end of the viral genomic RNA is organized into two structural elements: the CRE region and the 3′X-tail, separated by a hypervariable sequence and the polyU/UC stretch. Numbers refer to the nucleotide positions of the HCV Con1 isolate. Residues accessible to RNase T1, RNase V1, or lead processing under nondenaturing conditions are indicated by an asterisk, an arrow, or underlined, respectively. Start and stop translation codons placed at positions 342 and 9371, respectively, are shown in bold. ( B ) Diagram of the transcripts encompassing different functional domains of both the 5′ and the 3′ ends of the HCV genome used in this study.

    Journal: RNA

    Article Title: A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

    doi: 10.1261/rna.1680809

    Figure Lengend Snippet: ( A ) Sequence and secondary structure for the 5′ and the 3′ ends of the HCV genome. The 5′ UTR plus domains V and VI located at the core coding sequence are included. The minimum region for IRES activity is shown. The 3′ end of the viral genomic RNA is organized into two structural elements: the CRE region and the 3′X-tail, separated by a hypervariable sequence and the polyU/UC stretch. Numbers refer to the nucleotide positions of the HCV Con1 isolate. Residues accessible to RNase T1, RNase V1, or lead processing under nondenaturing conditions are indicated by an asterisk, an arrow, or underlined, respectively. Start and stop translation codons placed at positions 342 and 9371, respectively, are shown in bold. ( B ) Diagram of the transcripts encompassing different functional domains of both the 5′ and the 3′ ends of the HCV genome used in this study.

    Article Snippet: Briefly, complex formation was accomplished as noted above and subjected to partial digestion with 0.1 units of cobra venom RNase V1 (Pierce Biotechnology) or 0.1 units of RNase T1 (Industrial Research) at 4°C for 10 and 5 min, respectively.

    Techniques: Sequencing, Activity Assay, Functional Assay

    Secondary structure analysis of the 5′ and the 3′ ends of the HCV genome and identification of the interacting residues. ( A ) 32 P-5′-end-labeled 5′HCV-691 was partially digested with RNase T1 or Pb 2+ , either in the absence (−) or presence (+) of the 3′HCV-9181. The right panel shows a different run length aimed to resolve the higher molecular weight cleavage products. The functional subdomains of the IRES region are indicated. C, 5′HCV-691 incubated in binding buffer. T1L, T1 cleavage ladder. ( B ) Primer extension analysis of the 3′HCV-9181 transcript treated with RNase T1 or RNase V1 in the absence (−) or presence (+) of the 5′-end RNA. cDNA products were analyzed in 6% denaturing polyacrylamide gels in parallel with a sequence ladder obtained with the same labeled primer. The autoradiograph shows the results obtained for the CRE region.

    Journal: RNA

    Article Title: A long-range RNA–RNA interaction between the 5′ and 3′ ends of the HCV genome

    doi: 10.1261/rna.1680809

    Figure Lengend Snippet: Secondary structure analysis of the 5′ and the 3′ ends of the HCV genome and identification of the interacting residues. ( A ) 32 P-5′-end-labeled 5′HCV-691 was partially digested with RNase T1 or Pb 2+ , either in the absence (−) or presence (+) of the 3′HCV-9181. The right panel shows a different run length aimed to resolve the higher molecular weight cleavage products. The functional subdomains of the IRES region are indicated. C, 5′HCV-691 incubated in binding buffer. T1L, T1 cleavage ladder. ( B ) Primer extension analysis of the 3′HCV-9181 transcript treated with RNase T1 or RNase V1 in the absence (−) or presence (+) of the 5′-end RNA. cDNA products were analyzed in 6% denaturing polyacrylamide gels in parallel with a sequence ladder obtained with the same labeled primer. The autoradiograph shows the results obtained for the CRE region.

    Article Snippet: Briefly, complex formation was accomplished as noted above and subjected to partial digestion with 0.1 units of cobra venom RNase V1 (Pierce Biotechnology) or 0.1 units of RNase T1 (Industrial Research) at 4°C for 10 and 5 min, respectively.

    Techniques: Labeling, Molecular Weight, Functional Assay, Incubation, Binding Assay, Sequencing, Autoradiography

    Chemical and enzymatic probing of the 3′UTR of hA3G mRNA and RNase footprinting of Vif. ( A–C ) Representative gels of structure probing with CMCT (A) and footprinting using RNase A (B) or RNAse V1 (C). Nucleotides modified by CMCT (A) and RNase cleavages protected by Vif (B and C) are indicated by red bars or dots. ( D ) Secondary structure model of the 3′UTR of hA3G mRNA summarizing the experimental data.

    Journal: Nucleic Acids Research

    Article Title: HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation

    doi: 10.1093/nar/gkp1009

    Figure Lengend Snippet: Chemical and enzymatic probing of the 3′UTR of hA3G mRNA and RNase footprinting of Vif. ( A–C ) Representative gels of structure probing with CMCT (A) and footprinting using RNase A (B) or RNAse V1 (C). Nucleotides modified by CMCT (A) and RNase cleavages protected by Vif (B and C) are indicated by red bars or dots. ( D ) Secondary structure model of the 3′UTR of hA3G mRNA summarizing the experimental data.

    Article Snippet: Enzymatic footprinting experiments were performed on hA3G-5′UTR and hA3G-3′UTR RNAs in the presence of increasing concentrations of Vif using ribonuclease (RNase) V1, T1 and A (Ambion).

    Techniques: Footprinting, Modification

    Chemical and enzymatic probing of the 5′UTR of hA3G mRNA and RNase footprinting of Vif. ( A–C ) Representative gels of structure probing with DMS (A) and RNAse V1 footprinting (B and C). Nucleotides modified by DMS (A) and RNase V1 cleavages protected by Vif (B and C) are indicated by red bars or dots. ( D ) Secondary structure model of the 5′-UTR of hA3G mRNA summarizing the experimental data.

    Journal: Nucleic Acids Research

    Article Title: HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation

    doi: 10.1093/nar/gkp1009

    Figure Lengend Snippet: Chemical and enzymatic probing of the 5′UTR of hA3G mRNA and RNase footprinting of Vif. ( A–C ) Representative gels of structure probing with DMS (A) and RNAse V1 footprinting (B and C). Nucleotides modified by DMS (A) and RNase V1 cleavages protected by Vif (B and C) are indicated by red bars or dots. ( D ) Secondary structure model of the 5′-UTR of hA3G mRNA summarizing the experimental data.

    Article Snippet: Enzymatic footprinting experiments were performed on hA3G-5′UTR and hA3G-3′UTR RNAs in the presence of increasing concentrations of Vif using ribonuclease (RNase) V1, T1 and A (Ambion).

    Techniques: Footprinting, Modification

    AFM image of G4 dendriplexes prepared for 20 minutes attacked by RNase enzyme. (A) AFM image of hexagonal G4 dendriplexes prepared by mixing of G4 dendrimers with 0.7 µg of anti-GAPDH siRNA at N/P ratio of 2/1 for 20 minutes at room temperature before loading onto the surface of freshly cleaved mica. AFM images of G4 dendriplexes after incubation with RNase V1 enzyme for 1–28 minutes (B–F) shows separation of the adsorbed dendriplexes and degradation of the complexed siRNA molecules (dark spots) that increased with the increase in incubation time. Two dendriplexes (defined wit dotted circles) remained intact throughout the incubation time with RNase V1 enzyme suggesting the formation of individual compact particles. Scale bar in these images is 200 nm and the Z scale is 15 nm.

    Journal: PLoS ONE

    Article Title: Visualizing the Attack of RNase Enzymes on Dendriplexes and Naked RNA Using Atomic Force Microscopy

    doi: 10.1371/journal.pone.0061710

    Figure Lengend Snippet: AFM image of G4 dendriplexes prepared for 20 minutes attacked by RNase enzyme. (A) AFM image of hexagonal G4 dendriplexes prepared by mixing of G4 dendrimers with 0.7 µg of anti-GAPDH siRNA at N/P ratio of 2/1 for 20 minutes at room temperature before loading onto the surface of freshly cleaved mica. AFM images of G4 dendriplexes after incubation with RNase V1 enzyme for 1–28 minutes (B–F) shows separation of the adsorbed dendriplexes and degradation of the complexed siRNA molecules (dark spots) that increased with the increase in incubation time. Two dendriplexes (defined wit dotted circles) remained intact throughout the incubation time with RNase V1 enzyme suggesting the formation of individual compact particles. Scale bar in these images is 200 nm and the Z scale is 15 nm.

    Article Snippet: Anti-GAPDH siRNA and RNase V1 enzyme were purchased from Ambion Inc. (Austin, TX).

    Techniques: Incubation

    AFM image of G5 dendriplexes prepared for 20 minutes attacked by RNase enzyme. (A) AFM image of hexagonal G5 dendriplexes prepared by mixing of G5 dendrimers with 0.7 µg of anti-GAPDH siRNA at N/P ratio of 2/1 for 20 minutes at room temperature before loading onto the surface of freshly cleaved mica. AFM images of G5 dendriplexes after incubation with RNase V1 enzyme for 1–60 minutes (B–F) shows separation of the adsorbed dendriplexes and degradation of the complexed siRNA molecules (dark spots) that increased with the increase in incubation time. Scale bar in these images is 200 nm and the Z scale is 17 nm.

    Journal: PLoS ONE

    Article Title: Visualizing the Attack of RNase Enzymes on Dendriplexes and Naked RNA Using Atomic Force Microscopy

    doi: 10.1371/journal.pone.0061710

    Figure Lengend Snippet: AFM image of G5 dendriplexes prepared for 20 minutes attacked by RNase enzyme. (A) AFM image of hexagonal G5 dendriplexes prepared by mixing of G5 dendrimers with 0.7 µg of anti-GAPDH siRNA at N/P ratio of 2/1 for 20 minutes at room temperature before loading onto the surface of freshly cleaved mica. AFM images of G5 dendriplexes after incubation with RNase V1 enzyme for 1–60 minutes (B–F) shows separation of the adsorbed dendriplexes and degradation of the complexed siRNA molecules (dark spots) that increased with the increase in incubation time. Scale bar in these images is 200 nm and the Z scale is 17 nm.

    Article Snippet: Anti-GAPDH siRNA and RNase V1 enzyme were purchased from Ambion Inc. (Austin, TX).

    Techniques: Incubation

    AFM image of free siRNAs before and after attack by RNase enzyme. (A) AFM image of free anti-GAPDH siRNA dissolved in 1 mM PBS containing 2 mM MgCl 2 after adding to the surface of freshly cleaved mica, which shows rod-, sphere-, and bead-like arrangements. (B) AFM image taken 1.5 minutes after adding RNase V1 enzyme, which shows rapid fragmentation of adsorbed siRNA molecules. (C) Time-lapse images showing a single siRNA molecule denoted by the white arrow ( t = 0 min), the attack of RNase V1 enzyme on free siRNA molecule ( t = 1.5 min), and complete siRNA degradation ( t = 3 min). The scale bar in images A and B is 100 nm and the Z scale is 9 nm. The scale bar in image C is 35 nm and Z scale is 7 nm.

    Journal: PLoS ONE

    Article Title: Visualizing the Attack of RNase Enzymes on Dendriplexes and Naked RNA Using Atomic Force Microscopy

    doi: 10.1371/journal.pone.0061710

    Figure Lengend Snippet: AFM image of free siRNAs before and after attack by RNase enzyme. (A) AFM image of free anti-GAPDH siRNA dissolved in 1 mM PBS containing 2 mM MgCl 2 after adding to the surface of freshly cleaved mica, which shows rod-, sphere-, and bead-like arrangements. (B) AFM image taken 1.5 minutes after adding RNase V1 enzyme, which shows rapid fragmentation of adsorbed siRNA molecules. (C) Time-lapse images showing a single siRNA molecule denoted by the white arrow ( t = 0 min), the attack of RNase V1 enzyme on free siRNA molecule ( t = 1.5 min), and complete siRNA degradation ( t = 3 min). The scale bar in images A and B is 100 nm and the Z scale is 9 nm. The scale bar in image C is 35 nm and Z scale is 7 nm.

    Article Snippet: Anti-GAPDH siRNA and RNase V1 enzyme were purchased from Ambion Inc. (Austin, TX).

    Techniques:

    AFM image of G5 dendriplexes prepared for 24 hours attacked by RNase enzyme. (A) AFM image of G5 dendriplexes prepared by mixing of G5 dendrimers with 0.7 µg of anti-GAPDH siRNA at N/P ratio of 2/1 for 24 hours at room temperature before loading onto the surface of freshly cleaved mica. G5 dendriplexes remain intact upon incubating with RNase V1 enzyme for 30 (B) and 60 minutes (C). Scale bar in these AFM images is 140 nm and the Z scale is 5 nm.

    Journal: PLoS ONE

    Article Title: Visualizing the Attack of RNase Enzymes on Dendriplexes and Naked RNA Using Atomic Force Microscopy

    doi: 10.1371/journal.pone.0061710

    Figure Lengend Snippet: AFM image of G5 dendriplexes prepared for 24 hours attacked by RNase enzyme. (A) AFM image of G5 dendriplexes prepared by mixing of G5 dendrimers with 0.7 µg of anti-GAPDH siRNA at N/P ratio of 2/1 for 24 hours at room temperature before loading onto the surface of freshly cleaved mica. G5 dendriplexes remain intact upon incubating with RNase V1 enzyme for 30 (B) and 60 minutes (C). Scale bar in these AFM images is 140 nm and the Z scale is 5 nm.

    Article Snippet: Anti-GAPDH siRNA and RNase V1 enzyme were purchased from Ambion Inc. (Austin, TX).

    Techniques:

    MVA recombinants expressing excess early dsRNA from neo or EGFP transgenes induce increased IFN-β expression. (A) Schematic representation of the two types of MVA recombinants generating excess early dsRNA either from two neo inserts (top) or from two EGFP inserts (bottom), each with the corresponding control and reference constructs. IGR, intergenic region. (B) Total RNA from murine BALB/3T3-A31 cells infected with the indicated viruses (MOI 10) or mock infected for 6 h was digested with RNase A/T1 (ssRNase digest) or RNase A/T1/V1 (ss+dsRNase digest) or not digested, and duplicate RT-qPCR quantification of total EGFP transcript (both sense and antisense) was performed as described in Materials and Methods. The mean of the fold induction values of EGFP or C7L transcripts over mock in undigested samples was set to 100%, and the mean percentage of the remaining qPCR signals after the indicated RNase digests was calculated for EGFP and C7L transcripts. Shown is one out two independent experiments. Where error bars are not visible, the standard error was negligible. (C) MEFs in 6-well plates were mock infected or infected with crude stocks of the indicated MVA recombinants at an MOI of 10 in duplicate. Fold induction of IFN-β mRNA over mock was determined by duplicate RT-qPCR per sample using total RNA isolated from cells at 6 h p.i. using a commercially available TaqMan assay (Life Technologies) for the murine IFN-β gene. Poly(I·C) was transfected using Fugene HD at 2 μg/well. 18S rRNA served as the endogenous control in all RT-qPCR analyses. Where error bars are not visible, the standard error was negligible. (D) IFN-β amounts in supernatants of MEF cultures infected in parallel to those shown in panel C were determined at 14 h p.i. by ELISA. (E) Murine A31 cells were either preincubated with 40 μg/ml of AraC for 1 h or left untreated and infected in duplicate at an MOI of 10 with the indicated MVAs with either 40 μg/ml AraC throughout infection or without AraC. Cells were harvested at 6 h p.i. for isolation of total RNA. Messenger RNAs for murine IFN-β and the late F17R VACV gene were quantified by qRT-PCR analysis as described above.

    Journal: Journal of Virology

    Article Title: Recombinant Modified Vaccinia Virus Ankara Generating Excess Early Double-Stranded RNA Transiently Activates Protein Kinase R and Triggers Enhanced Innate Immune Responses

    doi: 10.1128/JVI.02082-14

    Figure Lengend Snippet: MVA recombinants expressing excess early dsRNA from neo or EGFP transgenes induce increased IFN-β expression. (A) Schematic representation of the two types of MVA recombinants generating excess early dsRNA either from two neo inserts (top) or from two EGFP inserts (bottom), each with the corresponding control and reference constructs. IGR, intergenic region. (B) Total RNA from murine BALB/3T3-A31 cells infected with the indicated viruses (MOI 10) or mock infected for 6 h was digested with RNase A/T1 (ssRNase digest) or RNase A/T1/V1 (ss+dsRNase digest) or not digested, and duplicate RT-qPCR quantification of total EGFP transcript (both sense and antisense) was performed as described in Materials and Methods. The mean of the fold induction values of EGFP or C7L transcripts over mock in undigested samples was set to 100%, and the mean percentage of the remaining qPCR signals after the indicated RNase digests was calculated for EGFP and C7L transcripts. Shown is one out two independent experiments. Where error bars are not visible, the standard error was negligible. (C) MEFs in 6-well plates were mock infected or infected with crude stocks of the indicated MVA recombinants at an MOI of 10 in duplicate. Fold induction of IFN-β mRNA over mock was determined by duplicate RT-qPCR per sample using total RNA isolated from cells at 6 h p.i. using a commercially available TaqMan assay (Life Technologies) for the murine IFN-β gene. Poly(I·C) was transfected using Fugene HD at 2 μg/well. 18S rRNA served as the endogenous control in all RT-qPCR analyses. Where error bars are not visible, the standard error was negligible. (D) IFN-β amounts in supernatants of MEF cultures infected in parallel to those shown in panel C were determined at 14 h p.i. by ELISA. (E) Murine A31 cells were either preincubated with 40 μg/ml of AraC for 1 h or left untreated and infected in duplicate at an MOI of 10 with the indicated MVAs with either 40 μg/ml AraC throughout infection or without AraC. Cells were harvested at 6 h p.i. for isolation of total RNA. Messenger RNAs for murine IFN-β and the late F17R VACV gene were quantified by qRT-PCR analysis as described above.

    Article Snippet: DNase-treated total RNA samples were digested with single-strand-specific RNases A and T1 (Ambion) or with RNase A/T1 plus dsRNA-specific RNase V1 (Ambion) in a total volume of 20 μl for 1 h at 37°C.

    Techniques: Expressing, Construct, Infection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Isolation, TaqMan Assay, Transfection, Enzyme-linked Immunosorbent Assay