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  • 99
    Thermo Fisher rnase v1 enzyme
    AFM image of G4 dendriplexes prepared for 20 minutes attacked by <t>RNase</t> 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 <t>V1</t> 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.
    Rnase V1 Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GE Healthcare rnase v1
    Analysis of the complex formed between HTNV N protein and deletion RNAs by UV cross-linking analysis. The concentration of N protein used in each binding reaction was 3.5 × 10 −6 M. Reaction mixtures were assembled in 100 mM NaCl with 5 mM MgCl 2 in addition to standard reaction components as described in Material and Methods. Binding reactions were separated by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis, and unbound RNA was digested by adding 1 U of <t>RNase</t> V1. Signals were imaged with the Molecular Dynamics Storm PhosphorImager and quantified using ImageQuaNT version 4.2 software (Molecular Dynamics). The RNAs used to form the complexes are HTNV S-segment vRNA (lane 1), ORF RNA (lane 2), minipan RNA (lane 3), and Δ12 RNA (lane 4).
    Rnase V1, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher rnase v1
    ( A ) ‘G’ sequence determination of 3′ end-labeled 1–570 RNA in our standard conditions of cleavage by parallel running of RNA degradation with <t>RNase</t> T1 0.0005 µg/µl in denaturing conditions (lane 3), in standard buffer (lane 4) and in the presence of miR-122 at 15 nM (lane 5). Lanes 1 is an alkali ladder degradation and lane 2, the RNA incubated in standard buffer alone. Gs are identified at the right of the gel. ( B ) Secondary structure of stem loop VI and its boundary regions summarizing the position with differential reactivity for RNase T1. Increased resistance is indicated by solid triangles and increased sensitivity by blank triangles. Nucleotide numbering is used, as in Figure 1 . ( C ) Evaluation of the effect of increasing concentrations of probes on the T1 nuclease pattern of cleavage. Lane 1 is a MW marker. Control incubation of 1–570 RNA in the buffer (lane 2), or after addition 0.0005 µg/µl of RNase T1 (lane 3). In subsequent lanes, before addition of the T1 RNase, RNAs were pre-incubated for 1 h with increasing concentrations of ODN 22(−) of 15 nM, 150 nM and 1500 nM (lanes 4–6), or miR-122 at final concentrations of 1.5 nM, 15 nM and 150 nM (lanes 7–9) and a 10-mer oligoribonucleotide carrying the 7 nucleotides of the miR-122 seed sequence to a final concentration of 1.5 nM, 15 nM and 150 nM (lanes 10–12). ( D ) Same as panel B, but the RNase used was double-stranded RNase V1. Arrows indicate the changes in sensitivity to the nuclease.
    Rnase V1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 779 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche ribonuclease rnase v1
    Secondary structure analysis of the HIV-1/BRU RNA region from positions 5303 to 5559. ( A ) Schematic representation of the HIV-1 genome and of the RNAs used for secondary structure analysis. In the HIV-1 genome, the 5′ss (D) and 3′ss (A) are shown; boxes, open reading frames. In the four RNA transcripts (L3 to A3C), numbering of HIV-1/BRU RNA sequences is according to Ratner et al. (25), exon sequences are represented by rectangles, introns by thin lines, the thick horizontal lines at the beginning of the transcripts correspond to sequences generated by plasmid pBluescriptKSII + . The 5′ss, 3′ss and ESS2 are indicated. In transcripts C3 and L3, the junction between the two HIV-1 RNA regions within the intron is indicated by a vertical broken line. ( B ) Examples of primer extension analyses of enzymatically digested and chemically modified L3 transcript. Lanes marked by V, S and CMCT correspond to <t>V1</t> RNase digestion, S1 nuclease digestion and CMCT modification, respectively. Conditions for digestion and modification are given in Materials and Methods. Lanes marked by c correspond to control experiments with the untreated RNA transcript, lanes UGCA to the sequencing ladders. Numbering of nucleotides in the HIV-1/BRU RNA is on the right of the autoradiogram, as well as the positions of the 3′ss (A3, A4c, A4a, A4b and A5). Positions of the various stem–loop structures identified on the basis of this analysis (Fig. 1C) and of ESS2 are shown on the left. The primers used for extension with reverse transcriptase are indicated below each autoradiogram. ( C ) The results of enzymatic probing are schematically represented on the proposed secondary structure model. Cleavages by enzymes are shown by arrows surmounted with circles for S1 nuclease and squares for V1 RNase. Three red circles or squares indicate a strong cleavage, two orange circles or squares a medium cleavage, one green circle or square a low cleavage. The free energy of the proposed stem–loop structures at 37°C, in 1 M NaCl were calculated with the MFold software. Positions of nucleotides in the HIV-1/BRU RNA are given. Stem–loop structures are designated as SLS1–5. The various 3′ss are indicated by blue arrows. The ESS2 inhibitory element is squared. Results of CMCT modification of SLS1 and SLS2 are shown in the inset. Red circles indicate a strong, orange a medium and green a low level of modification. Squared nucleotides were not modified.
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    85
    Thermo Fisher rnase v1 digestion buffer
    Secondary structure analysis of the HIV-1/BRU RNA region from positions 5303 to 5559. ( A ) Schematic representation of the HIV-1 genome and of the RNAs used for secondary structure analysis. In the HIV-1 genome, the 5′ss (D) and 3′ss (A) are shown; boxes, open reading frames. In the four RNA transcripts (L3 to A3C), numbering of HIV-1/BRU RNA sequences is according to Ratner et al. (25), exon sequences are represented by rectangles, introns by thin lines, the thick horizontal lines at the beginning of the transcripts correspond to sequences generated by plasmid pBluescriptKSII + . The 5′ss, 3′ss and ESS2 are indicated. In transcripts C3 and L3, the junction between the two HIV-1 RNA regions within the intron is indicated by a vertical broken line. ( B ) Examples of primer extension analyses of enzymatically digested and chemically modified L3 transcript. Lanes marked by V, S and CMCT correspond to <t>V1</t> RNase digestion, S1 nuclease digestion and CMCT modification, respectively. Conditions for digestion and modification are given in Materials and Methods. Lanes marked by c correspond to control experiments with the untreated RNA transcript, lanes UGCA to the sequencing ladders. Numbering of nucleotides in the HIV-1/BRU RNA is on the right of the autoradiogram, as well as the positions of the 3′ss (A3, A4c, A4a, A4b and A5). Positions of the various stem–loop structures identified on the basis of this analysis (Fig. 1C) and of ESS2 are shown on the left. The primers used for extension with reverse transcriptase are indicated below each autoradiogram. ( C ) The results of enzymatic probing are schematically represented on the proposed secondary structure model. Cleavages by enzymes are shown by arrows surmounted with circles for S1 nuclease and squares for V1 RNase. Three red circles or squares indicate a strong cleavage, two orange circles or squares a medium cleavage, one green circle or square a low cleavage. The free energy of the proposed stem–loop structures at 37°C, in 1 M NaCl were calculated with the MFold software. Positions of nucleotides in the HIV-1/BRU RNA are given. Stem–loop structures are designated as SLS1–5. The various 3′ss are indicated by blue arrows. The ESS2 inhibitory element is squared. Results of CMCT modification of SLS1 and SLS2 are shown in the inset. Red circles indicate a strong, orange a medium and green a low level of modification. Squared nucleotides were not modified.
    Rnase V1 Digestion Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher dsrna specific rnase v1
    MVA recombinants expressing excess early <t>dsRNA</t> 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 <t>RNase</t> 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.
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    91
    Boehringer Mannheim rnase v1
    MVA recombinants expressing excess early <t>dsRNA</t> 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 <t>RNase</t> 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.
    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
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    85
    Thermo Fisher cobra venom rnase v1
    ( 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 <t>RNase</t> 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.
    Cobra Venom Rnase V1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche mu rnase v1
    ( 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 <t>RNase</t> 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.
    Mu Rnase V1, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher rnase v1 1u
    ( 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 <t>RNase</t> 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.
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    99
    Thermo Fisher rnase v1 structure mapping
    ( 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 <t>RNase</t> 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.
    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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    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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    99
    Thermo Fisher rnase v1 endonuclease protocol
    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 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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    90
    Thermo Fisher te rnase
    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
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    95
    Thermo Fisher rnase mix
    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
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    Image Search Results


    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:

    Analysis of the complex formed between HTNV N protein and deletion RNAs by UV cross-linking analysis. The concentration of N protein used in each binding reaction was 3.5 × 10 −6 M. Reaction mixtures were assembled in 100 mM NaCl with 5 mM MgCl 2 in addition to standard reaction components as described in Material and Methods. Binding reactions were separated by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis, and unbound RNA was digested by adding 1 U of RNase V1. Signals were imaged with the Molecular Dynamics Storm PhosphorImager and quantified using ImageQuaNT version 4.2 software (Molecular Dynamics). The RNAs used to form the complexes are HTNV S-segment vRNA (lane 1), ORF RNA (lane 2), minipan RNA (lane 3), and Δ12 RNA (lane 4).

    Journal: Journal of Virology

    Article Title: cis-Acting Signals in Encapsidation of Hantaan Virus S-Segment Viral Genomic RNA by Its N Protein

    doi: 10.1128/JVI.75.6.2646-2652.2001

    Figure Lengend Snippet: Analysis of the complex formed between HTNV N protein and deletion RNAs by UV cross-linking analysis. The concentration of N protein used in each binding reaction was 3.5 × 10 −6 M. Reaction mixtures were assembled in 100 mM NaCl with 5 mM MgCl 2 in addition to standard reaction components as described in Material and Methods. Binding reactions were separated by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis, and unbound RNA was digested by adding 1 U of RNase V1. Signals were imaged with the Molecular Dynamics Storm PhosphorImager and quantified using ImageQuaNT version 4.2 software (Molecular Dynamics). The RNAs used to form the complexes are HTNV S-segment vRNA (lane 1), ORF RNA (lane 2), minipan RNA (lane 3), and Δ12 RNA (lane 4).

    Article Snippet: Unbound RNA was digested by adding 1 U of RNase V1 (Amersham Pharmacia Biotech) or 50 U of RNase T1 (Ambion) and incubating for 30 min at 37°C.

    Techniques: Concentration Assay, Binding Assay, Polyacrylamide Gel Electrophoresis, Software

    Secondary structure analysis of the HIV-1/BRU RNA region from positions 5303 to 5559. ( A ) Schematic representation of the HIV-1 genome and of the RNAs used for secondary structure analysis. In the HIV-1 genome, the 5′ss (D) and 3′ss (A) are shown; boxes, open reading frames. In the four RNA transcripts (L3 to A3C), numbering of HIV-1/BRU RNA sequences is according to Ratner et al. (25), exon sequences are represented by rectangles, introns by thin lines, the thick horizontal lines at the beginning of the transcripts correspond to sequences generated by plasmid pBluescriptKSII + . The 5′ss, 3′ss and ESS2 are indicated. In transcripts C3 and L3, the junction between the two HIV-1 RNA regions within the intron is indicated by a vertical broken line. ( B ) Examples of primer extension analyses of enzymatically digested and chemically modified L3 transcript. Lanes marked by V, S and CMCT correspond to V1 RNase digestion, S1 nuclease digestion and CMCT modification, respectively. Conditions for digestion and modification are given in Materials and Methods. Lanes marked by c correspond to control experiments with the untreated RNA transcript, lanes UGCA to the sequencing ladders. Numbering of nucleotides in the HIV-1/BRU RNA is on the right of the autoradiogram, as well as the positions of the 3′ss (A3, A4c, A4a, A4b and A5). Positions of the various stem–loop structures identified on the basis of this analysis (Fig. 1C) and of ESS2 are shown on the left. The primers used for extension with reverse transcriptase are indicated below each autoradiogram. ( C ) The results of enzymatic probing are schematically represented on the proposed secondary structure model. Cleavages by enzymes are shown by arrows surmounted with circles for S1 nuclease and squares for V1 RNase. Three red circles or squares indicate a strong cleavage, two orange circles or squares a medium cleavage, one green circle or square a low cleavage. The free energy of the proposed stem–loop structures at 37°C, in 1 M NaCl were calculated with the MFold software. Positions of nucleotides in the HIV-1/BRU RNA are given. Stem–loop structures are designated as SLS1–5. The various 3′ss are indicated by blue arrows. The ESS2 inhibitory element is squared. Results of CMCT modification of SLS1 and SLS2 are shown in the inset. Red circles indicate a strong, orange a medium and green a low level of modification. Squared nucleotides were not modified.

    Journal: Nucleic Acids Research

    Article Title: Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3? splice site and its cis-regulatory element: possible involvement in RNA splicing

    doi:

    Figure Lengend Snippet: Secondary structure analysis of the HIV-1/BRU RNA region from positions 5303 to 5559. ( A ) Schematic representation of the HIV-1 genome and of the RNAs used for secondary structure analysis. In the HIV-1 genome, the 5′ss (D) and 3′ss (A) are shown; boxes, open reading frames. In the four RNA transcripts (L3 to A3C), numbering of HIV-1/BRU RNA sequences is according to Ratner et al. (25), exon sequences are represented by rectangles, introns by thin lines, the thick horizontal lines at the beginning of the transcripts correspond to sequences generated by plasmid pBluescriptKSII + . The 5′ss, 3′ss and ESS2 are indicated. In transcripts C3 and L3, the junction between the two HIV-1 RNA regions within the intron is indicated by a vertical broken line. ( B ) Examples of primer extension analyses of enzymatically digested and chemically modified L3 transcript. Lanes marked by V, S and CMCT correspond to V1 RNase digestion, S1 nuclease digestion and CMCT modification, respectively. Conditions for digestion and modification are given in Materials and Methods. Lanes marked by c correspond to control experiments with the untreated RNA transcript, lanes UGCA to the sequencing ladders. Numbering of nucleotides in the HIV-1/BRU RNA is on the right of the autoradiogram, as well as the positions of the 3′ss (A3, A4c, A4a, A4b and A5). Positions of the various stem–loop structures identified on the basis of this analysis (Fig. 1C) and of ESS2 are shown on the left. The primers used for extension with reverse transcriptase are indicated below each autoradiogram. ( C ) The results of enzymatic probing are schematically represented on the proposed secondary structure model. Cleavages by enzymes are shown by arrows surmounted with circles for S1 nuclease and squares for V1 RNase. Three red circles or squares indicate a strong cleavage, two orange circles or squares a medium cleavage, one green circle or square a low cleavage. The free energy of the proposed stem–loop structures at 37°C, in 1 M NaCl were calculated with the MFold software. Positions of nucleotides in the HIV-1/BRU RNA are given. Stem–loop structures are designated as SLS1–5. The various 3′ss are indicated by blue arrows. The ESS2 inhibitory element is squared. Results of CMCT modification of SLS1 and SLS2 are shown in the inset. Red circles indicate a strong, orange a medium and green a low level of modification. Squared nucleotides were not modified.

    Article Snippet: V1 RNase was prepared from Naja oxiana venom ( ) and S1 nuclease was purchased from Amersham Pharmacia Biotech.

    Techniques: Generated, Plasmid Preparation, Modification, Sequencing, Software

    Conservation of SLS2 and SLS1 in the groups M and O of HIV-1 strains and in the SIVcpz strains. Nucleotide sequences in this figure are from the HIV sequence database (http://hiv-web.lanl.gov/, Los Alamos National Laboratory, Los Alamos, NM). They were identified by alignment of the HIV-1/BRU RNA sequence with sequences from HIV-1 RNAs of other strains and the SIVcpz strains using the ClustalW program (43). ( A ) SLS2s are drawn for various strains of the group M of HIV-1 strains, for the ANT70C strain of group O and for the SIVcpzGAB strain. The name of the strains, the clade and the group are indicated below each structure. The free energy of each SLS2, at 37°C, in 1 M NaCl, as calculated by the MFold program is indicated. Sequence variations as compared to BRU are indicated by red nucleotides. Base pairs within green rectangles are conserved by semi-compensatory mutations. ( B ) Schematic representation of the transcript used for the experimental study of the HIV-1/MAL RNA region containing site A3. The same symbols were used as in Figure 1A. ( C ) Probing of the secondary structure of the L3-MAL RNA with V1 RNase (lane V) and S1 nuclease (lane S). Primer 1458 was used for reverse transcriptase analysis of cleaved and intact RNA (lane c) and for generating the sequencing ladders (lanes UGCA). Numbering of the HIV-1/MAL RNA sequence on the right of the autoradiogram is according to the Los Alamos HIV sequence database. Beside the autoradiogram, a schematic representation of the cleavages observed is given on the MAL SLS2 model (squares for V1 cleavages; dots for S1 cleavages; red, orange and green for strong, medium and low cleavages, respectively). Helix and loop positions are indicated on the left of the autoradiogram. ( D ) Based on the experimentally identified SLS1 of the BRU RNA, ‘SLS1-like’ structures were drawn for the HIV-1/ANT70C, SIVcpzGAB and SIVcpzANT strains. Base pairs in red and green rectangles are conserved by compensatory and semi-compensatory mutations, respectively. The upper part of SLS1, that is not conserved in the SIVcpzANT RNA, is represented by a discontinuous line.

    Journal: Nucleic Acids Research

    Article Title: Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3? splice site and its cis-regulatory element: possible involvement in RNA splicing

    doi:

    Figure Lengend Snippet: Conservation of SLS2 and SLS1 in the groups M and O of HIV-1 strains and in the SIVcpz strains. Nucleotide sequences in this figure are from the HIV sequence database (http://hiv-web.lanl.gov/, Los Alamos National Laboratory, Los Alamos, NM). They were identified by alignment of the HIV-1/BRU RNA sequence with sequences from HIV-1 RNAs of other strains and the SIVcpz strains using the ClustalW program (43). ( A ) SLS2s are drawn for various strains of the group M of HIV-1 strains, for the ANT70C strain of group O and for the SIVcpzGAB strain. The name of the strains, the clade and the group are indicated below each structure. The free energy of each SLS2, at 37°C, in 1 M NaCl, as calculated by the MFold program is indicated. Sequence variations as compared to BRU are indicated by red nucleotides. Base pairs within green rectangles are conserved by semi-compensatory mutations. ( B ) Schematic representation of the transcript used for the experimental study of the HIV-1/MAL RNA region containing site A3. The same symbols were used as in Figure 1A. ( C ) Probing of the secondary structure of the L3-MAL RNA with V1 RNase (lane V) and S1 nuclease (lane S). Primer 1458 was used for reverse transcriptase analysis of cleaved and intact RNA (lane c) and for generating the sequencing ladders (lanes UGCA). Numbering of the HIV-1/MAL RNA sequence on the right of the autoradiogram is according to the Los Alamos HIV sequence database. Beside the autoradiogram, a schematic representation of the cleavages observed is given on the MAL SLS2 model (squares for V1 cleavages; dots for S1 cleavages; red, orange and green for strong, medium and low cleavages, respectively). Helix and loop positions are indicated on the left of the autoradiogram. ( D ) Based on the experimentally identified SLS1 of the BRU RNA, ‘SLS1-like’ structures were drawn for the HIV-1/ANT70C, SIVcpzGAB and SIVcpzANT strains. Base pairs in red and green rectangles are conserved by compensatory and semi-compensatory mutations, respectively. The upper part of SLS1, that is not conserved in the SIVcpzANT RNA, is represented by a discontinuous line.

    Article Snippet: V1 RNase was prepared from Naja oxiana venom ( ) and S1 nuclease was purchased from Amersham Pharmacia Biotech.

    Techniques: Sequencing

     The HIV-1/BRU SLS3  is formed in a nuclear extract and two structural motifs A and B  are protected by association with nuclear components. ( A )  The S3 transcript used for the experimental analysis is shown. ( B ) Examples of primer extension analyses of the  S3 transcript cleaved by V1, T1 or T2 nucleases (lanes marked by  V, T1, T2, respectively) or modified by kethoxal or DMS (lanes marked  by K or D, respectively) are presented. Enzymatic and chemical reactions  were either performed in the splicing buffer D (B) or in a nuclear  extract (NE). As a control, a primer extension was made with the  intact RNA transcript incubated in the absence of reagent, either  in buffer D or in the nuclear extract (lanes marked by c). Lanes  UGCA correspond to the sequencing ladders. Numbering of the nucleotides  in the HIV-1/BRU RNA is indicated on the right. Positions  of the loops and helices 3 and of ESS2 are shown on the left. ( C ) Schematic representation of results of chemical  and enzymatic probing of S3, in the splicing buffer D (C1) or in  the nuclear extract (C2). In Panel C1, the circled nucleotides were  modified by DMS or kethoxal. Cleavages by RNases are shown by arrows,  surmounted with a circle for T2 RNase, a star for T1 RNase and a  square for V1 RNase. Green, orange and red symbols indicate low,  medium and strong modifications or cleavages, respectively. In C2,  protection against the action of chemical reagents and nucleases  are shown in blue, the intensity of the blue color reflects the  level of protection. Increased sensitivity to chemical reagents  and nucleases is indicated in yellow (low increase) or orange (medium  increase). In both C1 and C2, the oligonucleotide primer 954 used  for the reverse transcriptase analysis is indicated. The 3G residues  at the 5′-end of the S3 transcript were  generated by the T7 RNA polymerase promoter. The portion of the  S3 RNA that was analyzed is delimited by the two broken arrows in  C1. Due to pause of reverse transcriptase at some of the V1 cleavage  sites in the region from positions 5397 to 5438 in the nuclear extract, no  estimation of their variation of intensity as compared to naked  RNA is given for this part of SLS3. In C3, the HIV-1/BRU  functional sequences contained in SLS3 are indicated, namely: the  tat  start codon and  vpr  stop codon  (squared in green), the putative SC35 binding site (red letters),  the branched sites (circled in blue) and PPT (in blue rectangles)  of the A4c 3′ss and the branched sites  for A4a and b 3′ss (circled in orange).  The limits of the RNA fragments used by Caputi  et al.  (11)  and Del Gatto-Konczak  et al . (32) for hnRNP A/B  crosslinking experiments are indicated by black and red arrows,  respectively.

    Journal: Nucleic Acids Research

    Article Title: Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3? splice site and its cis-regulatory element: possible involvement in RNA splicing

    doi:

    Figure Lengend Snippet: The HIV-1/BRU SLS3 is formed in a nuclear extract and two structural motifs A and B are protected by association with nuclear components. ( A ) The S3 transcript used for the experimental analysis is shown. ( B ) Examples of primer extension analyses of the S3 transcript cleaved by V1, T1 or T2 nucleases (lanes marked by V, T1, T2, respectively) or modified by kethoxal or DMS (lanes marked by K or D, respectively) are presented. Enzymatic and chemical reactions were either performed in the splicing buffer D (B) or in a nuclear extract (NE). As a control, a primer extension was made with the intact RNA transcript incubated in the absence of reagent, either in buffer D or in the nuclear extract (lanes marked by c). Lanes UGCA correspond to the sequencing ladders. Numbering of the nucleotides in the HIV-1/BRU RNA is indicated on the right. Positions of the loops and helices 3 and of ESS2 are shown on the left. ( C ) Schematic representation of results of chemical and enzymatic probing of S3, in the splicing buffer D (C1) or in the nuclear extract (C2). In Panel C1, the circled nucleotides were modified by DMS or kethoxal. Cleavages by RNases are shown by arrows, surmounted with a circle for T2 RNase, a star for T1 RNase and a square for V1 RNase. Green, orange and red symbols indicate low, medium and strong modifications or cleavages, respectively. In C2, protection against the action of chemical reagents and nucleases are shown in blue, the intensity of the blue color reflects the level of protection. Increased sensitivity to chemical reagents and nucleases is indicated in yellow (low increase) or orange (medium increase). In both C1 and C2, the oligonucleotide primer 954 used for the reverse transcriptase analysis is indicated. The 3G residues at the 5′-end of the S3 transcript were generated by the T7 RNA polymerase promoter. The portion of the S3 RNA that was analyzed is delimited by the two broken arrows in C1. Due to pause of reverse transcriptase at some of the V1 cleavage sites in the region from positions 5397 to 5438 in the nuclear extract, no estimation of their variation of intensity as compared to naked RNA is given for this part of SLS3. In C3, the HIV-1/BRU functional sequences contained in SLS3 are indicated, namely: the tat start codon and vpr stop codon (squared in green), the putative SC35 binding site (red letters), the branched sites (circled in blue) and PPT (in blue rectangles) of the A4c 3′ss and the branched sites for A4a and b 3′ss (circled in orange). The limits of the RNA fragments used by Caputi et al. (11) and Del Gatto-Konczak et al . (32) for hnRNP A/B crosslinking experiments are indicated by black and red arrows, respectively.

    Article Snippet: V1 RNase was prepared from Naja oxiana venom ( ) and S1 nuclease was purchased from Amersham Pharmacia Biotech.

    Techniques: Modification, Incubation, Sequencing, Generated, Functional Assay, Binding Assay

    ( A ) ‘G’ sequence determination of 3′ end-labeled 1–570 RNA in our standard conditions of cleavage by parallel running of RNA degradation with RNase T1 0.0005 µg/µl in denaturing conditions (lane 3), in standard buffer (lane 4) and in the presence of miR-122 at 15 nM (lane 5). Lanes 1 is an alkali ladder degradation and lane 2, the RNA incubated in standard buffer alone. Gs are identified at the right of the gel. ( B ) Secondary structure of stem loop VI and its boundary regions summarizing the position with differential reactivity for RNase T1. Increased resistance is indicated by solid triangles and increased sensitivity by blank triangles. Nucleotide numbering is used, as in Figure 1 . ( C ) Evaluation of the effect of increasing concentrations of probes on the T1 nuclease pattern of cleavage. Lane 1 is a MW marker. Control incubation of 1–570 RNA in the buffer (lane 2), or after addition 0.0005 µg/µl of RNase T1 (lane 3). In subsequent lanes, before addition of the T1 RNase, RNAs were pre-incubated for 1 h with increasing concentrations of ODN 22(−) of 15 nM, 150 nM and 1500 nM (lanes 4–6), or miR-122 at final concentrations of 1.5 nM, 15 nM and 150 nM (lanes 7–9) and a 10-mer oligoribonucleotide carrying the 7 nucleotides of the miR-122 seed sequence to a final concentration of 1.5 nM, 15 nM and 150 nM (lanes 10–12). ( D ) Same as panel B, but the RNase used was double-stranded RNase V1. Arrows indicate the changes in sensitivity to the nuclease.

    Journal: Nucleic Acids Research

    Article Title: In vitro characterization of a miR-122-sensitive double-helical switch element in the 5? region of hepatitis C virus RNA

    doi: 10.1093/nar/gkp553

    Figure Lengend Snippet: ( A ) ‘G’ sequence determination of 3′ end-labeled 1–570 RNA in our standard conditions of cleavage by parallel running of RNA degradation with RNase T1 0.0005 µg/µl in denaturing conditions (lane 3), in standard buffer (lane 4) and in the presence of miR-122 at 15 nM (lane 5). Lanes 1 is an alkali ladder degradation and lane 2, the RNA incubated in standard buffer alone. Gs are identified at the right of the gel. ( B ) Secondary structure of stem loop VI and its boundary regions summarizing the position with differential reactivity for RNase T1. Increased resistance is indicated by solid triangles and increased sensitivity by blank triangles. Nucleotide numbering is used, as in Figure 1 . ( C ) Evaluation of the effect of increasing concentrations of probes on the T1 nuclease pattern of cleavage. Lane 1 is a MW marker. Control incubation of 1–570 RNA in the buffer (lane 2), or after addition 0.0005 µg/µl of RNase T1 (lane 3). In subsequent lanes, before addition of the T1 RNase, RNAs were pre-incubated for 1 h with increasing concentrations of ODN 22(−) of 15 nM, 150 nM and 1500 nM (lanes 4–6), or miR-122 at final concentrations of 1.5 nM, 15 nM and 150 nM (lanes 7–9) and a 10-mer oligoribonucleotide carrying the 7 nucleotides of the miR-122 seed sequence to a final concentration of 1.5 nM, 15 nM and 150 nM (lanes 10–12). ( D ) Same as panel B, but the RNase used was double-stranded RNase V1. Arrows indicate the changes in sensitivity to the nuclease.

    Article Snippet: Structural mapping with the single- or double-stranded RNases, T1 and V1 Various concentrations were prepared of RNase T1 (Calbiochem) (0.01, 0.001, 0.0001, 0.00001 μg/μl) and RNase V1 (Ambion) (0.0005 and 0.001 U/μl).

    Techniques: Sequencing, Labeling, Incubation, Marker, Concentration Assay

    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

    M. tuberculosis H37Rv RNA altered human monocyte's ability to control M. tuberculosis infection. CFUs were determined for human monocytes infected with M. tuberculosis H37Rv and incubated for four days in the presence of 1 µg/ml of M. tuberculosis H37Rv CF, purified RNA (gpRNA), or purified RNA digested with RNaseV1 (gpRNA+RNaseV1). The presence of CF or gpRNA resulted in a significant increase in CFUs as compared to the untreated infected monocytes (Control). Data represent the mean ± SEM of 3 replicates of the same experiment (*p

    Journal: PLoS ONE

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism

    doi: 10.1371/journal.pone.0029970

    Figure Lengend Snippet: M. tuberculosis H37Rv RNA altered human monocyte's ability to control M. tuberculosis infection. CFUs were determined for human monocytes infected with M. tuberculosis H37Rv and incubated for four days in the presence of 1 µg/ml of M. tuberculosis H37Rv CF, purified RNA (gpRNA), or purified RNA digested with RNaseV1 (gpRNA+RNaseV1). The presence of CF or gpRNA resulted in a significant increase in CFUs as compared to the untreated infected monocytes (Control). Data represent the mean ± SEM of 3 replicates of the same experiment (*p

    Article Snippet: The gel purified RNA was incubated overnight at 37° C with or without 0.1 U/µl RNaseV1 (Ambion, Austin, TX) and lyophilized.

    Techniques: Infection, Incubation, Purification

    RNA in DEAE-Sepharose Fraction 7 induces apoptosis in human monocytes. A. Apoptosis induced by DEAE-Sepharose Fraction 7 (F7), F7 treated with proteinase K (F7+ProtK), F7 treated with DNase1 (F7+DNase1), F7 treated with RNaseV1 (F7+RNaseV1), CF, CF treated with RNaseV1 (CF+RNaseV1), CF-Man, CF-Man treated with RNaseV1 (CF-Man+RNaseV1). Apoptosis was measured by flow cytometry and presented as percentage of human monocytes that stained annexinV positive. *** significance p

    Journal: PLoS ONE

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism

    doi: 10.1371/journal.pone.0029970

    Figure Lengend Snippet: RNA in DEAE-Sepharose Fraction 7 induces apoptosis in human monocytes. A. Apoptosis induced by DEAE-Sepharose Fraction 7 (F7), F7 treated with proteinase K (F7+ProtK), F7 treated with DNase1 (F7+DNase1), F7 treated with RNaseV1 (F7+RNaseV1), CF, CF treated with RNaseV1 (CF+RNaseV1), CF-Man, CF-Man treated with RNaseV1 (CF-Man+RNaseV1). Apoptosis was measured by flow cytometry and presented as percentage of human monocytes that stained annexinV positive. *** significance p

    Article Snippet: The gel purified RNA was incubated overnight at 37° C with or without 0.1 U/µl RNaseV1 (Ambion, Austin, TX) and lyophilized.

    Techniques: Flow Cytometry, Cytometry, Staining

    Human monocyte apoptosis is specifically induced by gel-purified mycobacterial RNA. A. SDS-PAGE with ethidium bromide (left) and silver stained (right) gel-purified RNA (gpRNA) from F7 and treated with RNaseV1 (gpRNA+RNaseV1). B. Monocyte apoptosis induced by gpRNA untreated or treated with RNaseV1, anti-CD95 untreated or treated with RNaseV1 (anti-CD95+RNaseV1) and rabbit mRNA. Significance as described above.

    Journal: PLoS ONE

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism

    doi: 10.1371/journal.pone.0029970

    Figure Lengend Snippet: Human monocyte apoptosis is specifically induced by gel-purified mycobacterial RNA. A. SDS-PAGE with ethidium bromide (left) and silver stained (right) gel-purified RNA (gpRNA) from F7 and treated with RNaseV1 (gpRNA+RNaseV1). B. Monocyte apoptosis induced by gpRNA untreated or treated with RNaseV1, anti-CD95 untreated or treated with RNaseV1 (anti-CD95+RNaseV1) and rabbit mRNA. Significance as described above.

    Article Snippet: The gel purified RNA was incubated overnight at 37° C with or without 0.1 U/µl RNaseV1 (Ambion, Austin, TX) and lyophilized.

    Techniques: Purification, SDS Page, Staining

    PIP-seq is reproducible and captures known RBP–RNA interactions. (A) Correlation in read counts between two formaldehyde-cross-linked ssRNase-treated PIP-seq replicates (footprint sample on left, RNase digestion control on right). (B) As (A) , but for formaldehyde-cross-linked dsRNase-treated replicates. (C) Overlap in PPS calls between formaldehyde-cross-linked ssRNase-treated (top, blue), and formaldehyde-cross-linked dsRNase-treated (bottom, green) PIP-seq replicates. (D) Overlap between PPSs identified from three formaldehyde-treated PIP-seq samples and various CLIP datasets. Values are shown as log 2 enrichment over shuffled background distributions. *** denotes P

    Journal: Genome Biology

    Article Title: RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome

    doi: 10.1186/gb-2014-15-1-r3

    Figure Lengend Snippet: PIP-seq is reproducible and captures known RBP–RNA interactions. (A) Correlation in read counts between two formaldehyde-cross-linked ssRNase-treated PIP-seq replicates (footprint sample on left, RNase digestion control on right). (B) As (A) , but for formaldehyde-cross-linked dsRNase-treated replicates. (C) Overlap in PPS calls between formaldehyde-cross-linked ssRNase-treated (top, blue), and formaldehyde-cross-linked dsRNase-treated (bottom, green) PIP-seq replicates. (D) Overlap between PPSs identified from three formaldehyde-treated PIP-seq samples and various CLIP datasets. Values are shown as log 2 enrichment over shuffled background distributions. *** denotes P

    Article Snippet: Subsequently, these DNA-depleted lysates were split and treated with either 100 U/mL of a single-stranded RNase (ssRNase) (RNaseONE (Promega, Madison, WI)) with 200 μg/mL BSA in 1× RNaseONE buffer for 1 hour at room temperature, or 2.5 U/mL of a double-stranded RNase (dsRNase) (RNaseV1 (Ambion, Austin, TX)) in 1× RNA structure buffer for 1 hour at 37°C as previously described [ , ] (see Figure A for a schematic description).

    Techniques: Cross-linking Immunoprecipitation

    Functional analysis and characterization of protein-binding sites. (A) Distribution of ssRNase-treated (light blue bars) and dsRNase-treated (green bars) PPS sizes from formaldehyde-cross-linked samples. Dashed lines represent median PPS sizes (ssRNase, blue line and dsRNase, green line). (B) Genomic distribution of PPS density, measured as PPS base coverage normalized to RNase digestion control read counts per genomic region. Proximal intron refers to 500 nucleotides at the 5′ and 3′ ends of introns. (C) Cumulative distribution of average SiPhy-π scores in PPSs (red line) versus similarly sized flanking sequences (gray line). (D) Comparison of average SiPhy-π scores between PPSs (red bars) and flanking sequences (gray bars) for various genomic regions. (E) Average SiPhy-π score profiles across the first and last 25 nucleotides of PPSs as well as 50 nucleotides upstream and downstream of exonic (green line), intronic (blue line) and lncRNA (orange line) PPSs. *** denotes P

    Journal: Genome Biology

    Article Title: RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome

    doi: 10.1186/gb-2014-15-1-r3

    Figure Lengend Snippet: Functional analysis and characterization of protein-binding sites. (A) Distribution of ssRNase-treated (light blue bars) and dsRNase-treated (green bars) PPS sizes from formaldehyde-cross-linked samples. Dashed lines represent median PPS sizes (ssRNase, blue line and dsRNase, green line). (B) Genomic distribution of PPS density, measured as PPS base coverage normalized to RNase digestion control read counts per genomic region. Proximal intron refers to 500 nucleotides at the 5′ and 3′ ends of introns. (C) Cumulative distribution of average SiPhy-π scores in PPSs (red line) versus similarly sized flanking sequences (gray line). (D) Comparison of average SiPhy-π scores between PPSs (red bars) and flanking sequences (gray bars) for various genomic regions. (E) Average SiPhy-π score profiles across the first and last 25 nucleotides of PPSs as well as 50 nucleotides upstream and downstream of exonic (green line), intronic (blue line) and lncRNA (orange line) PPSs. *** denotes P

    Article Snippet: Subsequently, these DNA-depleted lysates were split and treated with either 100 U/mL of a single-stranded RNase (ssRNase) (RNaseONE (Promega, Madison, WI)) with 200 μg/mL BSA in 1× RNaseONE buffer for 1 hour at room temperature, or 2.5 U/mL of a double-stranded RNase (dsRNase) (RNaseV1 (Ambion, Austin, TX)) in 1× RNA structure buffer for 1 hour at 37°C as previously described [ , ] (see Figure A for a schematic description).

    Techniques: Functional Assay, Protein Binding

    Overview of the PIP-seq method. (A) In the PIP-seq method, cells are cross-linked with formaldehyde or 254-nm UV light, or not cross-linked. They are lysed and divided into footprint and RNase digestion control samples. The footprint sample is treated with an RNase (ss- or dsRNase), which results in a population of RNase-protected RNA–RBP complexes. The protein cross-links are then reversed (by heating for formaldehyde cross-links or by proteinase K treatment for UV cross-links), leaving only the footprints where the RNA was protein-bound. For the RNase digestion control sample, which is designed to control for RNase insensitive regions, the order of operations is reversed; bound proteins are first removed by treatment with SDS and proteinase K, and then the unprotected RNA sample is subjected to RNase treatment. Strand-specific high-throughput sequencing libraries are prepared from both footprint and RNase digestion control samples and normalized using rehybridization and duplex-specific nuclease (DSN) treatment. PPSs are identified from the sequencing data using a Poisson model. Screenshots show UCSC browser views of sequencing reads from the footprint and RNase digestion control sample (same scale) and PPSs identified from the regions of the genes listed. (B,C) Absolute distribution of PPSs throughout RNA species for formaldehyde (B) and UV (C) cross-linked PIP-seq experiments. (D,E) Average PPS count per RNA molecule (classified by RNA type (mRNA and lncRNA) and transcript region (for example, 5′ UTR)) for formaldehyde (D) and UV (E) cross-linked PIP-seq experiments. Percentages indicate the fraction of each RNA type or region that contains PPS information. (F) Average expression ( y -axis) of human mRNAs separated by total number of PPSs identified in their sequence ( x -axis) for PPSs identified using formaldehyde cross-linking. CDS, coding sequence; DSN, duplex-specific nuclease; dsRNase, double-stranded RNase; lncRNA, long non-coding RNA; PIP-seq, protein interaction profile sequencing; PPS, protein-protected site; ssRNase, single-stranded RNase; UTR, untranslated region.

    Journal: Genome Biology

    Article Title: RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome

    doi: 10.1186/gb-2014-15-1-r3

    Figure Lengend Snippet: Overview of the PIP-seq method. (A) In the PIP-seq method, cells are cross-linked with formaldehyde or 254-nm UV light, or not cross-linked. They are lysed and divided into footprint and RNase digestion control samples. The footprint sample is treated with an RNase (ss- or dsRNase), which results in a population of RNase-protected RNA–RBP complexes. The protein cross-links are then reversed (by heating for formaldehyde cross-links or by proteinase K treatment for UV cross-links), leaving only the footprints where the RNA was protein-bound. For the RNase digestion control sample, which is designed to control for RNase insensitive regions, the order of operations is reversed; bound proteins are first removed by treatment with SDS and proteinase K, and then the unprotected RNA sample is subjected to RNase treatment. Strand-specific high-throughput sequencing libraries are prepared from both footprint and RNase digestion control samples and normalized using rehybridization and duplex-specific nuclease (DSN) treatment. PPSs are identified from the sequencing data using a Poisson model. Screenshots show UCSC browser views of sequencing reads from the footprint and RNase digestion control sample (same scale) and PPSs identified from the regions of the genes listed. (B,C) Absolute distribution of PPSs throughout RNA species for formaldehyde (B) and UV (C) cross-linked PIP-seq experiments. (D,E) Average PPS count per RNA molecule (classified by RNA type (mRNA and lncRNA) and transcript region (for example, 5′ UTR)) for formaldehyde (D) and UV (E) cross-linked PIP-seq experiments. Percentages indicate the fraction of each RNA type or region that contains PPS information. (F) Average expression ( y -axis) of human mRNAs separated by total number of PPSs identified in their sequence ( x -axis) for PPSs identified using formaldehyde cross-linking. CDS, coding sequence; DSN, duplex-specific nuclease; dsRNase, double-stranded RNase; lncRNA, long non-coding RNA; PIP-seq, protein interaction profile sequencing; PPS, protein-protected site; ssRNase, single-stranded RNase; UTR, untranslated region.

    Article Snippet: Subsequently, these DNA-depleted lysates were split and treated with either 100 U/mL of a single-stranded RNase (ssRNase) (RNaseONE (Promega, Madison, WI)) with 200 μg/mL BSA in 1× RNaseONE buffer for 1 hour at room temperature, or 2.5 U/mL of a double-stranded RNase (dsRNase) (RNaseV1 (Ambion, Austin, TX)) in 1× RNA structure buffer for 1 hour at 37°C as previously described [ , ] (see Figure A for a schematic description).

    Techniques: Next-Generation Sequencing, Sequencing, Expressing

    Secondary structure analysis of the HIV-1/BRU RNA region from positions 5303 to 5559. ( A ) Schematic representation of the HIV-1 genome and of the RNAs used for secondary structure analysis. In the HIV-1 genome, the 5′ss (D) and 3′ss (A) are shown; boxes, open reading frames. In the four RNA transcripts (L3 to A3C), numbering of HIV-1/BRU RNA sequences is according to Ratner et al. (25), exon sequences are represented by rectangles, introns by thin lines, the thick horizontal lines at the beginning of the transcripts correspond to sequences generated by plasmid pBluescriptKSII + . The 5′ss, 3′ss and ESS2 are indicated. In transcripts C3 and L3, the junction between the two HIV-1 RNA regions within the intron is indicated by a vertical broken line. ( B ) Examples of primer extension analyses of enzymatically digested and chemically modified L3 transcript. Lanes marked by V, S and CMCT correspond to V1 RNase digestion, S1 nuclease digestion and CMCT modification, respectively. Conditions for digestion and modification are given in Materials and Methods. Lanes marked by c correspond to control experiments with the untreated RNA transcript, lanes UGCA to the sequencing ladders. Numbering of nucleotides in the HIV-1/BRU RNA is on the right of the autoradiogram, as well as the positions of the 3′ss (A3, A4c, A4a, A4b and A5). Positions of the various stem–loop structures identified on the basis of this analysis (Fig. 1C) and of ESS2 are shown on the left. The primers used for extension with reverse transcriptase are indicated below each autoradiogram. ( C ) The results of enzymatic probing are schematically represented on the proposed secondary structure model. Cleavages by enzymes are shown by arrows surmounted with circles for S1 nuclease and squares for V1 RNase. Three red circles or squares indicate a strong cleavage, two orange circles or squares a medium cleavage, one green circle or square a low cleavage. The free energy of the proposed stem–loop structures at 37°C, in 1 M NaCl were calculated with the MFold software. Positions of nucleotides in the HIV-1/BRU RNA are given. Stem–loop structures are designated as SLS1–5. The various 3′ss are indicated by blue arrows. The ESS2 inhibitory element is squared. Results of CMCT modification of SLS1 and SLS2 are shown in the inset. Red circles indicate a strong, orange a medium and green a low level of modification. Squared nucleotides were not modified.

    Journal: Nucleic Acids Research

    Article Title: Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3? splice site and its cis-regulatory element: possible involvement in RNA splicing

    doi:

    Figure Lengend Snippet: Secondary structure analysis of the HIV-1/BRU RNA region from positions 5303 to 5559. ( A ) Schematic representation of the HIV-1 genome and of the RNAs used for secondary structure analysis. In the HIV-1 genome, the 5′ss (D) and 3′ss (A) are shown; boxes, open reading frames. In the four RNA transcripts (L3 to A3C), numbering of HIV-1/BRU RNA sequences is according to Ratner et al. (25), exon sequences are represented by rectangles, introns by thin lines, the thick horizontal lines at the beginning of the transcripts correspond to sequences generated by plasmid pBluescriptKSII + . The 5′ss, 3′ss and ESS2 are indicated. In transcripts C3 and L3, the junction between the two HIV-1 RNA regions within the intron is indicated by a vertical broken line. ( B ) Examples of primer extension analyses of enzymatically digested and chemically modified L3 transcript. Lanes marked by V, S and CMCT correspond to V1 RNase digestion, S1 nuclease digestion and CMCT modification, respectively. Conditions for digestion and modification are given in Materials and Methods. Lanes marked by c correspond to control experiments with the untreated RNA transcript, lanes UGCA to the sequencing ladders. Numbering of nucleotides in the HIV-1/BRU RNA is on the right of the autoradiogram, as well as the positions of the 3′ss (A3, A4c, A4a, A4b and A5). Positions of the various stem–loop structures identified on the basis of this analysis (Fig. 1C) and of ESS2 are shown on the left. The primers used for extension with reverse transcriptase are indicated below each autoradiogram. ( C ) The results of enzymatic probing are schematically represented on the proposed secondary structure model. Cleavages by enzymes are shown by arrows surmounted with circles for S1 nuclease and squares for V1 RNase. Three red circles or squares indicate a strong cleavage, two orange circles or squares a medium cleavage, one green circle or square a low cleavage. The free energy of the proposed stem–loop structures at 37°C, in 1 M NaCl were calculated with the MFold software. Positions of nucleotides in the HIV-1/BRU RNA are given. Stem–loop structures are designated as SLS1–5. The various 3′ss are indicated by blue arrows. The ESS2 inhibitory element is squared. Results of CMCT modification of SLS1 and SLS2 are shown in the inset. Red circles indicate a strong, orange a medium and green a low level of modification. Squared nucleotides were not modified.

    Article Snippet: Enzymatic digestions with V1 RNase and S1 nuclease were carried out, as previously described , using 1 µl of in vitro synthesized RNA in the presence of 1.25 µg of a commercial yeast tRNA mixture (Roche Diagnostics) for V1 RNase digestion and 2.5 µg of yeast tRNA for S1 nuclease digestion.

    Techniques: Generated, Plasmid Preparation, Modification, Sequencing, Software

    Conservation of SLS2 and SLS1 in the groups M and O of HIV-1 strains and in the SIVcpz strains. Nucleotide sequences in this figure are from the HIV sequence database (http://hiv-web.lanl.gov/, Los Alamos National Laboratory, Los Alamos, NM). They were identified by alignment of the HIV-1/BRU RNA sequence with sequences from HIV-1 RNAs of other strains and the SIVcpz strains using the ClustalW program (43). ( A ) SLS2s are drawn for various strains of the group M of HIV-1 strains, for the ANT70C strain of group O and for the SIVcpzGAB strain. The name of the strains, the clade and the group are indicated below each structure. The free energy of each SLS2, at 37°C, in 1 M NaCl, as calculated by the MFold program is indicated. Sequence variations as compared to BRU are indicated by red nucleotides. Base pairs within green rectangles are conserved by semi-compensatory mutations. ( B ) Schematic representation of the transcript used for the experimental study of the HIV-1/MAL RNA region containing site A3. The same symbols were used as in Figure 1A. ( C ) Probing of the secondary structure of the L3-MAL RNA with V1 RNase (lane V) and S1 nuclease (lane S). Primer 1458 was used for reverse transcriptase analysis of cleaved and intact RNA (lane c) and for generating the sequencing ladders (lanes UGCA). Numbering of the HIV-1/MAL RNA sequence on the right of the autoradiogram is according to the Los Alamos HIV sequence database. Beside the autoradiogram, a schematic representation of the cleavages observed is given on the MAL SLS2 model (squares for V1 cleavages; dots for S1 cleavages; red, orange and green for strong, medium and low cleavages, respectively). Helix and loop positions are indicated on the left of the autoradiogram. ( D ) Based on the experimentally identified SLS1 of the BRU RNA, ‘SLS1-like’ structures were drawn for the HIV-1/ANT70C, SIVcpzGAB and SIVcpzANT strains. Base pairs in red and green rectangles are conserved by compensatory and semi-compensatory mutations, respectively. The upper part of SLS1, that is not conserved in the SIVcpzANT RNA, is represented by a discontinuous line.

    Journal: Nucleic Acids Research

    Article Title: Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3? splice site and its cis-regulatory element: possible involvement in RNA splicing

    doi:

    Figure Lengend Snippet: Conservation of SLS2 and SLS1 in the groups M and O of HIV-1 strains and in the SIVcpz strains. Nucleotide sequences in this figure are from the HIV sequence database (http://hiv-web.lanl.gov/, Los Alamos National Laboratory, Los Alamos, NM). They were identified by alignment of the HIV-1/BRU RNA sequence with sequences from HIV-1 RNAs of other strains and the SIVcpz strains using the ClustalW program (43). ( A ) SLS2s are drawn for various strains of the group M of HIV-1 strains, for the ANT70C strain of group O and for the SIVcpzGAB strain. The name of the strains, the clade and the group are indicated below each structure. The free energy of each SLS2, at 37°C, in 1 M NaCl, as calculated by the MFold program is indicated. Sequence variations as compared to BRU are indicated by red nucleotides. Base pairs within green rectangles are conserved by semi-compensatory mutations. ( B ) Schematic representation of the transcript used for the experimental study of the HIV-1/MAL RNA region containing site A3. The same symbols were used as in Figure 1A. ( C ) Probing of the secondary structure of the L3-MAL RNA with V1 RNase (lane V) and S1 nuclease (lane S). Primer 1458 was used for reverse transcriptase analysis of cleaved and intact RNA (lane c) and for generating the sequencing ladders (lanes UGCA). Numbering of the HIV-1/MAL RNA sequence on the right of the autoradiogram is according to the Los Alamos HIV sequence database. Beside the autoradiogram, a schematic representation of the cleavages observed is given on the MAL SLS2 model (squares for V1 cleavages; dots for S1 cleavages; red, orange and green for strong, medium and low cleavages, respectively). Helix and loop positions are indicated on the left of the autoradiogram. ( D ) Based on the experimentally identified SLS1 of the BRU RNA, ‘SLS1-like’ structures were drawn for the HIV-1/ANT70C, SIVcpzGAB and SIVcpzANT strains. Base pairs in red and green rectangles are conserved by compensatory and semi-compensatory mutations, respectively. The upper part of SLS1, that is not conserved in the SIVcpzANT RNA, is represented by a discontinuous line.

    Article Snippet: Enzymatic digestions with V1 RNase and S1 nuclease were carried out, as previously described , using 1 µl of in vitro synthesized RNA in the presence of 1.25 µg of a commercial yeast tRNA mixture (Roche Diagnostics) for V1 RNase digestion and 2.5 µg of yeast tRNA for S1 nuclease digestion.

    Techniques: Sequencing

     The HIV-1/BRU SLS3  is formed in a nuclear extract and two structural motifs A and B  are protected by association with nuclear components. ( A )  The S3 transcript used for the experimental analysis is shown. ( B ) Examples of primer extension analyses of the  S3 transcript cleaved by V1, T1 or T2 nucleases (lanes marked by  V, T1, T2, respectively) or modified by kethoxal or DMS (lanes marked  by K or D, respectively) are presented. Enzymatic and chemical reactions  were either performed in the splicing buffer D (B) or in a nuclear  extract (NE). As a control, a primer extension was made with the  intact RNA transcript incubated in the absence of reagent, either  in buffer D or in the nuclear extract (lanes marked by c). Lanes  UGCA correspond to the sequencing ladders. Numbering of the nucleotides  in the HIV-1/BRU RNA is indicated on the right. Positions  of the loops and helices 3 and of ESS2 are shown on the left. ( C ) Schematic representation of results of chemical  and enzymatic probing of S3, in the splicing buffer D (C1) or in  the nuclear extract (C2). In Panel C1, the circled nucleotides were  modified by DMS or kethoxal. Cleavages by RNases are shown by arrows,  surmounted with a circle for T2 RNase, a star for T1 RNase and a  square for V1 RNase. Green, orange and red symbols indicate low,  medium and strong modifications or cleavages, respectively. In C2,  protection against the action of chemical reagents and nucleases  are shown in blue, the intensity of the blue color reflects the  level of protection. Increased sensitivity to chemical reagents  and nucleases is indicated in yellow (low increase) or orange (medium  increase). In both C1 and C2, the oligonucleotide primer 954 used  for the reverse transcriptase analysis is indicated. The 3G residues  at the 5′-end of the S3 transcript were  generated by the T7 RNA polymerase promoter. The portion of the  S3 RNA that was analyzed is delimited by the two broken arrows in  C1. Due to pause of reverse transcriptase at some of the V1 cleavage  sites in the region from positions 5397 to 5438 in the nuclear extract, no  estimation of their variation of intensity as compared to naked  RNA is given for this part of SLS3. In C3, the HIV-1/BRU  functional sequences contained in SLS3 are indicated, namely: the  tat  start codon and  vpr  stop codon  (squared in green), the putative SC35 binding site (red letters),  the branched sites (circled in blue) and PPT (in blue rectangles)  of the A4c 3′ss and the branched sites  for A4a and b 3′ss (circled in orange).  The limits of the RNA fragments used by Caputi  et al.  (11)  and Del Gatto-Konczak  et al . (32) for hnRNP A/B  crosslinking experiments are indicated by black and red arrows,  respectively.

    Journal: Nucleic Acids Research

    Article Title: Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3? splice site and its cis-regulatory element: possible involvement in RNA splicing

    doi:

    Figure Lengend Snippet: The HIV-1/BRU SLS3 is formed in a nuclear extract and two structural motifs A and B are protected by association with nuclear components. ( A ) The S3 transcript used for the experimental analysis is shown. ( B ) Examples of primer extension analyses of the S3 transcript cleaved by V1, T1 or T2 nucleases (lanes marked by V, T1, T2, respectively) or modified by kethoxal or DMS (lanes marked by K or D, respectively) are presented. Enzymatic and chemical reactions were either performed in the splicing buffer D (B) or in a nuclear extract (NE). As a control, a primer extension was made with the intact RNA transcript incubated in the absence of reagent, either in buffer D or in the nuclear extract (lanes marked by c). Lanes UGCA correspond to the sequencing ladders. Numbering of the nucleotides in the HIV-1/BRU RNA is indicated on the right. Positions of the loops and helices 3 and of ESS2 are shown on the left. ( C ) Schematic representation of results of chemical and enzymatic probing of S3, in the splicing buffer D (C1) or in the nuclear extract (C2). In Panel C1, the circled nucleotides were modified by DMS or kethoxal. Cleavages by RNases are shown by arrows, surmounted with a circle for T2 RNase, a star for T1 RNase and a square for V1 RNase. Green, orange and red symbols indicate low, medium and strong modifications or cleavages, respectively. In C2, protection against the action of chemical reagents and nucleases are shown in blue, the intensity of the blue color reflects the level of protection. Increased sensitivity to chemical reagents and nucleases is indicated in yellow (low increase) or orange (medium increase). In both C1 and C2, the oligonucleotide primer 954 used for the reverse transcriptase analysis is indicated. The 3G residues at the 5′-end of the S3 transcript were generated by the T7 RNA polymerase promoter. The portion of the S3 RNA that was analyzed is delimited by the two broken arrows in C1. Due to pause of reverse transcriptase at some of the V1 cleavage sites in the region from positions 5397 to 5438 in the nuclear extract, no estimation of their variation of intensity as compared to naked RNA is given for this part of SLS3. In C3, the HIV-1/BRU functional sequences contained in SLS3 are indicated, namely: the tat start codon and vpr stop codon (squared in green), the putative SC35 binding site (red letters), the branched sites (circled in blue) and PPT (in blue rectangles) of the A4c 3′ss and the branched sites for A4a and b 3′ss (circled in orange). The limits of the RNA fragments used by Caputi et al. (11) and Del Gatto-Konczak et al . (32) for hnRNP A/B crosslinking experiments are indicated by black and red arrows, respectively.

    Article Snippet: Enzymatic digestions with V1 RNase and S1 nuclease were carried out, as previously described , using 1 µl of in vitro synthesized RNA in the presence of 1.25 µg of a commercial yeast tRNA mixture (Roche Diagnostics) for V1 RNase digestion and 2.5 µg of yeast tRNA for S1 nuclease digestion.

    Techniques: Modification, Incubation, Sequencing, Generated, Functional Assay, Binding Assay

    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

    ( 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

    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