m 7 gpppa  (New England Biolabs)


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    New England Biolabs m 7 gpppa
    The workflow and specificity of DO-seq (A) The workflow of DO-seq: First, yDcpS (in red) de-caps the <t>m</t> <t>7</t> G moiety from m 7 G-RNAs in total RNAs. Second, in the presence of ADPRC, NAD-RNAs can be biotinylated by HEEB, which subsequently are enriched by streptavidin beads. Enriched RNAs can be used for epitranscriptome-wide profiling as well as qRT-PCR analysis. (B) yDcpS was able to de-cap m 7 <t>GpppA-RNA</t> (38 nt) but not NAD-RNA (45 nt), as evidenced by a lower-sized band corresponding to the de-capped product in the TBE-Urea gel. (C) HEEB (200 mM) reacted with NAD-RNA (106 nt) and m 7 GpppA-RNA (106 nt), but not pppA-RNA (106 nt), resulting in a band retained by the streptavidin beads. (D) qRT-PCR analysis showed that NAD-RNA (106 nt), but not pppA-RNA (106 nt), could be enriched by DO-seq. qRT-PCR analysis showed that m 7 GpppA-RNA (106 nt) could be detected in the presence of ADPRC; pre-treatment of yDcpS eliminates potential enrichment of m 7 GpppA-RNA (106 nt). The difference between Ct of target RNA in enrichment and input was calculated, resulting in index conversion of ΔCt. Data were shown in mean ± s.e.m. (E) Epitranscriptome assessment of yDcpS to minimize the noise of m 7 G-RNAs. Two-fold enrichment of read counts was used as the cutoff. Standard DO-seq identified 2,460 NAD-RNAs, while 1,905 false-positive NAD-RNAs were found without the use of yDcpS, presumably derived from m 7 G-capped RNAs. Total RNAs were from liver tissues of 18-month mice.
    M 7 Gpppa, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Hidden features of NAD-RNA epitranscriptome in Drosophila life cycle"

    Article Title: Hidden features of NAD-RNA epitranscriptome in Drosophila life cycle

    Journal: iScience

    doi: 10.1016/j.isci.2023.108618

    The workflow and specificity of DO-seq (A) The workflow of DO-seq: First, yDcpS (in red) de-caps the m 7 G moiety from m 7 G-RNAs in total RNAs. Second, in the presence of ADPRC, NAD-RNAs can be biotinylated by HEEB, which subsequently are enriched by streptavidin beads. Enriched RNAs can be used for epitranscriptome-wide profiling as well as qRT-PCR analysis. (B) yDcpS was able to de-cap m 7 GpppA-RNA (38 nt) but not NAD-RNA (45 nt), as evidenced by a lower-sized band corresponding to the de-capped product in the TBE-Urea gel. (C) HEEB (200 mM) reacted with NAD-RNA (106 nt) and m 7 GpppA-RNA (106 nt), but not pppA-RNA (106 nt), resulting in a band retained by the streptavidin beads. (D) qRT-PCR analysis showed that NAD-RNA (106 nt), but not pppA-RNA (106 nt), could be enriched by DO-seq. qRT-PCR analysis showed that m 7 GpppA-RNA (106 nt) could be detected in the presence of ADPRC; pre-treatment of yDcpS eliminates potential enrichment of m 7 GpppA-RNA (106 nt). The difference between Ct of target RNA in enrichment and input was calculated, resulting in index conversion of ΔCt. Data were shown in mean ± s.e.m. (E) Epitranscriptome assessment of yDcpS to minimize the noise of m 7 G-RNAs. Two-fold enrichment of read counts was used as the cutoff. Standard DO-seq identified 2,460 NAD-RNAs, while 1,905 false-positive NAD-RNAs were found without the use of yDcpS, presumably derived from m 7 G-capped RNAs. Total RNAs were from liver tissues of 18-month mice.
    Figure Legend Snippet: The workflow and specificity of DO-seq (A) The workflow of DO-seq: First, yDcpS (in red) de-caps the m 7 G moiety from m 7 G-RNAs in total RNAs. Second, in the presence of ADPRC, NAD-RNAs can be biotinylated by HEEB, which subsequently are enriched by streptavidin beads. Enriched RNAs can be used for epitranscriptome-wide profiling as well as qRT-PCR analysis. (B) yDcpS was able to de-cap m 7 GpppA-RNA (38 nt) but not NAD-RNA (45 nt), as evidenced by a lower-sized band corresponding to the de-capped product in the TBE-Urea gel. (C) HEEB (200 mM) reacted with NAD-RNA (106 nt) and m 7 GpppA-RNA (106 nt), but not pppA-RNA (106 nt), resulting in a band retained by the streptavidin beads. (D) qRT-PCR analysis showed that NAD-RNA (106 nt), but not pppA-RNA (106 nt), could be enriched by DO-seq. qRT-PCR analysis showed that m 7 GpppA-RNA (106 nt) could be detected in the presence of ADPRC; pre-treatment of yDcpS eliminates potential enrichment of m 7 GpppA-RNA (106 nt). The difference between Ct of target RNA in enrichment and input was calculated, resulting in index conversion of ΔCt. Data were shown in mean ± s.e.m. (E) Epitranscriptome assessment of yDcpS to minimize the noise of m 7 G-RNAs. Two-fold enrichment of read counts was used as the cutoff. Standard DO-seq identified 2,460 NAD-RNAs, while 1,905 false-positive NAD-RNAs were found without the use of yDcpS, presumably derived from m 7 G-capped RNAs. Total RNAs were from liver tissues of 18-month mice.

    Techniques Used: Quantitative RT-PCR, Derivative Assay

    m 7 gpppa  (New England Biolabs)


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    New England Biolabs m 7 gpppa
    The workflow and specificity of DO-seq (A) The workflow of DO-seq: First, yDcpS (in red) de-caps the <t>m</t> <t>7</t> G moiety from m 7 G-RNAs in total RNAs. Second, in the presence of ADPRC, NAD-RNAs can be biotinylated by HEEB, which subsequently are enriched by streptavidin beads. Enriched RNAs can be used for epitranscriptome-wide profiling as well as qRT-PCR analysis. (B) yDcpS was able to de-cap m 7 <t>GpppA-RNA</t> (38 nt) but not NAD-RNA (45 nt), as evidenced by a lower-sized band corresponding to the de-capped product in the TBE-Urea gel. (C) HEEB (200 mM) reacted with NAD-RNA (106 nt) and m 7 GpppA-RNA (106 nt), but not pppA-RNA (106 nt), resulting in a band retained by the streptavidin beads. (D) qRT-PCR analysis showed that NAD-RNA (106 nt), but not pppA-RNA (106 nt), could be enriched by DO-seq. qRT-PCR analysis showed that m 7 GpppA-RNA (106 nt) could be detected in the presence of ADPRC; pre-treatment of yDcpS eliminates potential enrichment of m 7 GpppA-RNA (106 nt). The difference between Ct of target RNA in enrichment and input was calculated, resulting in index conversion of ΔCt. Data were shown in mean ± s.e.m. (E) Epitranscriptome assessment of yDcpS to minimize the noise of m 7 G-RNAs. Two-fold enrichment of read counts was used as the cutoff. Standard DO-seq identified 2,460 NAD-RNAs, while 1,905 false-positive NAD-RNAs were found without the use of yDcpS, presumably derived from m 7 G-capped RNAs. Total RNAs were from liver tissues of 18-month mice.
    M 7 Gpppa, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m 7 gpppa/product/New England Biolabs
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    1) Product Images from "Hidden features of NAD-RNA epitranscriptome in Drosophila life cycle"

    Article Title: Hidden features of NAD-RNA epitranscriptome in Drosophila life cycle

    Journal: iScience

    doi: 10.1016/j.isci.2023.108618

    The workflow and specificity of DO-seq (A) The workflow of DO-seq: First, yDcpS (in red) de-caps the m 7 G moiety from m 7 G-RNAs in total RNAs. Second, in the presence of ADPRC, NAD-RNAs can be biotinylated by HEEB, which subsequently are enriched by streptavidin beads. Enriched RNAs can be used for epitranscriptome-wide profiling as well as qRT-PCR analysis. (B) yDcpS was able to de-cap m 7 GpppA-RNA (38 nt) but not NAD-RNA (45 nt), as evidenced by a lower-sized band corresponding to the de-capped product in the TBE-Urea gel. (C) HEEB (200 mM) reacted with NAD-RNA (106 nt) and m 7 GpppA-RNA (106 nt), but not pppA-RNA (106 nt), resulting in a band retained by the streptavidin beads. (D) qRT-PCR analysis showed that NAD-RNA (106 nt), but not pppA-RNA (106 nt), could be enriched by DO-seq. qRT-PCR analysis showed that m 7 GpppA-RNA (106 nt) could be detected in the presence of ADPRC; pre-treatment of yDcpS eliminates potential enrichment of m 7 GpppA-RNA (106 nt). The difference between Ct of target RNA in enrichment and input was calculated, resulting in index conversion of ΔCt. Data were shown in mean ± s.e.m. (E) Epitranscriptome assessment of yDcpS to minimize the noise of m 7 G-RNAs. Two-fold enrichment of read counts was used as the cutoff. Standard DO-seq identified 2,460 NAD-RNAs, while 1,905 false-positive NAD-RNAs were found without the use of yDcpS, presumably derived from m 7 G-capped RNAs. Total RNAs were from liver tissues of 18-month mice.
    Figure Legend Snippet: The workflow and specificity of DO-seq (A) The workflow of DO-seq: First, yDcpS (in red) de-caps the m 7 G moiety from m 7 G-RNAs in total RNAs. Second, in the presence of ADPRC, NAD-RNAs can be biotinylated by HEEB, which subsequently are enriched by streptavidin beads. Enriched RNAs can be used for epitranscriptome-wide profiling as well as qRT-PCR analysis. (B) yDcpS was able to de-cap m 7 GpppA-RNA (38 nt) but not NAD-RNA (45 nt), as evidenced by a lower-sized band corresponding to the de-capped product in the TBE-Urea gel. (C) HEEB (200 mM) reacted with NAD-RNA (106 nt) and m 7 GpppA-RNA (106 nt), but not pppA-RNA (106 nt), resulting in a band retained by the streptavidin beads. (D) qRT-PCR analysis showed that NAD-RNA (106 nt), but not pppA-RNA (106 nt), could be enriched by DO-seq. qRT-PCR analysis showed that m 7 GpppA-RNA (106 nt) could be detected in the presence of ADPRC; pre-treatment of yDcpS eliminates potential enrichment of m 7 GpppA-RNA (106 nt). The difference between Ct of target RNA in enrichment and input was calculated, resulting in index conversion of ΔCt. Data were shown in mean ± s.e.m. (E) Epitranscriptome assessment of yDcpS to minimize the noise of m 7 G-RNAs. Two-fold enrichment of read counts was used as the cutoff. Standard DO-seq identified 2,460 NAD-RNAs, while 1,905 false-positive NAD-RNAs were found without the use of yDcpS, presumably derived from m 7 G-capped RNAs. Total RNAs were from liver tissues of 18-month mice.

    Techniques Used: Quantitative RT-PCR, Derivative Assay

    m 7 gpppa  (New England Biolabs)


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    New England Biolabs m 7 gpppa
    M 7 Gpppa, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    m 7 gpppa  (New England Biolabs)


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    New England Biolabs m 7 gpppa
    M 7 Gpppa, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    m 7 gpppa  (New England Biolabs)


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    New England Biolabs m 7 gpppa
    M 7 Gpppa, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    m 7 gpppa rna cap structure analog  (New England Biolabs)


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    New England Biolabs m 7 gpppa rna cap structure analog
    M 7 Gpppa Rna Cap Structure Analog, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    m 7 gpppa rna cap structure analog  (New England Biolabs)


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    New England Biolabs m 7 gpppa rna cap structure analog
    Identification of FAD cap binding proteins in budding yeast. ( A ) Schematic illustration of the FAD cap <t>-RNA</t> Affinity Purification (FcRAP). ( B ) Eluates from FcRAP were loaded on to a 4%-12% Bis-Tris gel and stained with SYPRO Ruby. The <t>m</t> <t>7</t> G cap affinity purification was used as a control. All protein bands labeled on the gel were excised from the gel and identified using mass spectrometry. ( C ) Electrophoretic mobility shift assay (EMSA) using increasing concentration of Xrn1 (2.5, 5 and 7.5 pmol) and a fixed concentration (5 pmol) of uniformly labeled in vitro transcribed FAD-capped or NAD-capped or triphosphate RNA (pppA–). ( D ) EMSA using equimolar concentration (5 pmol) of Xrn1 and uniformly labeled in vitro transcribed FAD-capped or NAD-capped or pppA–RNAs in the absence and presence of 7 pmol and 14 pmol total yeast RNA as a nonspecific competitor.
    M 7 Gpppa Rna Cap Structure Analog, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Identification of a novel deFADding activity in human, yeast and bacterial 5′ to 3′ exoribonucleases"

    Article Title: Identification of a novel deFADding activity in human, yeast and bacterial 5′ to 3′ exoribonucleases

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkac617

    Identification of FAD cap binding proteins in budding yeast. ( A ) Schematic illustration of the FAD cap -RNA Affinity Purification (FcRAP). ( B ) Eluates from FcRAP were loaded on to a 4%-12% Bis-Tris gel and stained with SYPRO Ruby. The m 7 G cap affinity purification was used as a control. All protein bands labeled on the gel were excised from the gel and identified using mass spectrometry. ( C ) Electrophoretic mobility shift assay (EMSA) using increasing concentration of Xrn1 (2.5, 5 and 7.5 pmol) and a fixed concentration (5 pmol) of uniformly labeled in vitro transcribed FAD-capped or NAD-capped or triphosphate RNA (pppA–). ( D ) EMSA using equimolar concentration (5 pmol) of Xrn1 and uniformly labeled in vitro transcribed FAD-capped or NAD-capped or pppA–RNAs in the absence and presence of 7 pmol and 14 pmol total yeast RNA as a nonspecific competitor.
    Figure Legend Snippet: Identification of FAD cap binding proteins in budding yeast. ( A ) Schematic illustration of the FAD cap -RNA Affinity Purification (FcRAP). ( B ) Eluates from FcRAP were loaded on to a 4%-12% Bis-Tris gel and stained with SYPRO Ruby. The m 7 G cap affinity purification was used as a control. All protein bands labeled on the gel were excised from the gel and identified using mass spectrometry. ( C ) Electrophoretic mobility shift assay (EMSA) using increasing concentration of Xrn1 (2.5, 5 and 7.5 pmol) and a fixed concentration (5 pmol) of uniformly labeled in vitro transcribed FAD-capped or NAD-capped or triphosphate RNA (pppA–). ( D ) EMSA using equimolar concentration (5 pmol) of Xrn1 and uniformly labeled in vitro transcribed FAD-capped or NAD-capped or pppA–RNAs in the absence and presence of 7 pmol and 14 pmol total yeast RNA as a nonspecific competitor.

    Techniques Used: Binding Assay, Affinity Purification, Staining, Labeling, Mass Spectrometry, Electrophoretic Mobility Shift Assay, Concentration Assay, In Vitro

    RNase AM is a novel deFADding enzyme in E. coli . ( A ) Reaction products of in vitro deFADding assays with 100 nM recombinant RNase AM. Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–), NAD-capped (NAD–) and m 7 G-capped (m 7 G–). Reaction products were resolved on 15% 7 M urea PAGE gels. ( B ) Time-course decay analysis of ∼300 nM uniformly 32 P-labeled monophosphate or FAD-capped RNA with the indicated amount of RNase AM protein are shown. Quantitation of RNA remaining is plotted from three independent experiments with ± SD denoted by error bars along with the one phase decay curve. ( C ) To demonstrate that RNase AM releases intact FAD upon deFADding like Xrn1 (in Figure ), FAD-capQ assay using different amount of in vitro transcribed FAD-capped RNA was used. Catalytic-dead mutant of RNase AM, D20A was used as control for the reaction. ( D ) Total RNA from WT and rnase AMΔ were subjected to the FAD-capQ assay to detect total levels of cellular FAD-capped RNA. Data represents an average from three independent biological experiments. Error bars represent ± SD with unpaired t -test; * P < 0.05, ** P < 0.001, *** P < 0.0001.
    Figure Legend Snippet: RNase AM is a novel deFADding enzyme in E. coli . ( A ) Reaction products of in vitro deFADding assays with 100 nM recombinant RNase AM. Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–), NAD-capped (NAD–) and m 7 G-capped (m 7 G–). Reaction products were resolved on 15% 7 M urea PAGE gels. ( B ) Time-course decay analysis of ∼300 nM uniformly 32 P-labeled monophosphate or FAD-capped RNA with the indicated amount of RNase AM protein are shown. Quantitation of RNA remaining is plotted from three independent experiments with ± SD denoted by error bars along with the one phase decay curve. ( C ) To demonstrate that RNase AM releases intact FAD upon deFADding like Xrn1 (in Figure ), FAD-capQ assay using different amount of in vitro transcribed FAD-capped RNA was used. Catalytic-dead mutant of RNase AM, D20A was used as control for the reaction. ( D ) Total RNA from WT and rnase AMΔ were subjected to the FAD-capQ assay to detect total levels of cellular FAD-capped RNA. Data represents an average from three independent biological experiments. Error bars represent ± SD with unpaired t -test; * P < 0.05, ** P < 0.001, *** P < 0.0001.

    Techniques Used: In Vitro, Recombinant, Labeling, Quantitation Assay, Mutagenesis

    m 7 gpppa rna cap structure analog  (New England Biolabs)


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    New England Biolabs m 7 gpppa rna cap structure analog
    (A) Schematic illustration of the FAD cap <t>-RNA</t> Affinity Purification (FcRAP). (B) Eluates from FcRAP were loaded on to a 4%-12% Bis-Tris gel and stained with SYPRO Ruby. The <t>m</t> <t>7</t> G cap affinity purification was used as a control. All protein bands labeled on the gel were excised from the gel and identified using mass spectrometry. (C) Electrophoretic mobility shift assay (EMSA) using increasing concentration of Xrn1 (2.5 pmol, 5 pmol and 7.5 pmol) and a fixed concentration (5 pmol) of uniformly labeled in vitro transcribed FADcapped or NAD-capped or triphosphate RNA (pppA–). (D) EMSA using equimolar concentration (5pmol) of Xrn1 and uniformly labeled in vitro transcribed FAD-capped or NAD-capped or pppA–RNAs in the presence of total yeast RNA as a nonspecific competitor.
    M 7 Gpppa Rna Cap Structure Analog, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m 7 gpppa rna cap structure analog/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    m 7 gpppa rna cap structure analog - by Bioz Stars, 2024-07
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    Images

    1) Product Images from "Identification of a novel deFADding activity in 5’ to 3’ exoribonucleases"

    Article Title: Identification of a novel deFADding activity in 5’ to 3’ exoribonucleases

    Journal: bioRxiv

    doi: 10.1101/2022.05.10.491372

    (A) Schematic illustration of the FAD cap -RNA Affinity Purification (FcRAP). (B) Eluates from FcRAP were loaded on to a 4%-12% Bis-Tris gel and stained with SYPRO Ruby. The m 7 G cap affinity purification was used as a control. All protein bands labeled on the gel were excised from the gel and identified using mass spectrometry. (C) Electrophoretic mobility shift assay (EMSA) using increasing concentration of Xrn1 (2.5 pmol, 5 pmol and 7.5 pmol) and a fixed concentration (5 pmol) of uniformly labeled in vitro transcribed FADcapped or NAD-capped or triphosphate RNA (pppA–). (D) EMSA using equimolar concentration (5pmol) of Xrn1 and uniformly labeled in vitro transcribed FAD-capped or NAD-capped or pppA–RNAs in the presence of total yeast RNA as a nonspecific competitor.
    Figure Legend Snippet: (A) Schematic illustration of the FAD cap -RNA Affinity Purification (FcRAP). (B) Eluates from FcRAP were loaded on to a 4%-12% Bis-Tris gel and stained with SYPRO Ruby. The m 7 G cap affinity purification was used as a control. All protein bands labeled on the gel were excised from the gel and identified using mass spectrometry. (C) Electrophoretic mobility shift assay (EMSA) using increasing concentration of Xrn1 (2.5 pmol, 5 pmol and 7.5 pmol) and a fixed concentration (5 pmol) of uniformly labeled in vitro transcribed FADcapped or NAD-capped or triphosphate RNA (pppA–). (D) EMSA using equimolar concentration (5pmol) of Xrn1 and uniformly labeled in vitro transcribed FAD-capped or NAD-capped or pppA–RNAs in the presence of total yeast RNA as a nonspecific competitor.

    Techniques Used: Affinity Purification, Staining, Labeling, Mass Spectrometry, Electrophoretic Mobility Shift Assay, Concentration Assay, In Vitro

    (A) Time-course decay analysis of uniformly 32 P-labeled m 7 G- or FAD-capped RNA in the presence of cell extract prepared from WT or xrn1Δ strains. The remaining RNA was quantified and plotted from three independent experiments with ± SD denoted by error bars. (B) FAD-capQ assay using different amounts of in vitro transcribed FAD-capped RNA to demonstrate the release of intact FAD upon Xrn1 mediated deFADding. Catalytic-dead mutant of Xrn1, E178Q was used as a control for the Xrn1 reaction and NAD-capped RNA was used as a control for specific detection of FAD in the assay (C) Total RNAs from WT and xrn1Δ were subjected to the FAD-capQ assay to detect total levels of cellular FAD-capped RNA. Data represents average from three independent experiments. Error bars represent ± SD with unpaired t -test; * p < 0.05, ** p < 0.001, *** p < 0.0001 (D) Reaction products of in vitro deFADding assays with 100nM recombinant human Xrn1(Hs_Xrn1) and catalytically inactive ( E178Q ). (E) Small RNAs (<200 nts) from HEK293T Ctrl cells and xrn1-KO were subjected to the FAD-capQ assay to detect total level of cellular FAD-capped RNAs. Data represents average from three independent experiments. Error bars represent ± SD with unpaired t -test; * p < 0.05, ** p < 0.001, *** p < 0.0001.
    Figure Legend Snippet: (A) Time-course decay analysis of uniformly 32 P-labeled m 7 G- or FAD-capped RNA in the presence of cell extract prepared from WT or xrn1Δ strains. The remaining RNA was quantified and plotted from three independent experiments with ± SD denoted by error bars. (B) FAD-capQ assay using different amounts of in vitro transcribed FAD-capped RNA to demonstrate the release of intact FAD upon Xrn1 mediated deFADding. Catalytic-dead mutant of Xrn1, E178Q was used as a control for the Xrn1 reaction and NAD-capped RNA was used as a control for specific detection of FAD in the assay (C) Total RNAs from WT and xrn1Δ were subjected to the FAD-capQ assay to detect total levels of cellular FAD-capped RNA. Data represents average from three independent experiments. Error bars represent ± SD with unpaired t -test; * p < 0.05, ** p < 0.001, *** p < 0.0001 (D) Reaction products of in vitro deFADding assays with 100nM recombinant human Xrn1(Hs_Xrn1) and catalytically inactive ( E178Q ). (E) Small RNAs (<200 nts) from HEK293T Ctrl cells and xrn1-KO were subjected to the FAD-capQ assay to detect total level of cellular FAD-capped RNAs. Data represents average from three independent experiments. Error bars represent ± SD with unpaired t -test; * p < 0.05, ** p < 0.001, *** p < 0.0001.

    Techniques Used: Labeling, In Vitro, Mutagenesis, Recombinant

    (A) Reaction products of in vitro deFADding assays with 100nM recombinant RNase AM. Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–), NAD-capped (NAD–), and m 7 G-capped (m 7 G–). The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels. (B) Time-course decay analysis of uniformly 32 P-labeled monophosphate or FAD-capped RNA with the indicated amount of RNase AM protein are shown. Quantitation of RNA remaining is plotted from three independent experiments with ± SD denoted by error bars. (C) To demonstrate that RNase AM releases intact FAD upon deFADding like Xrn1 (in ), FAD-capQ assay using different amount of in vitro transcribed FAD-capped RNA was used. Catalytic-dead mutant of RNase AM, D20A was used as control for the reaction. (D) Total RNA from WT and rnase AMΔ were subjected to the FAD-capQ assay to detect total levels of cellular FAD-capped RNA. Data represents an average from three independent experiments. Error bars represent ± SD with unpaired t -test; * p < 0.05, ** p < 0.001, *** p < 0.0001.
    Figure Legend Snippet: (A) Reaction products of in vitro deFADding assays with 100nM recombinant RNase AM. Uniformly 32 P-labeled monophosphate (pA–) or FAD-capped RNA (FAD–), NAD-capped (NAD–), and m 7 G-capped (m 7 G–). The asterisk represents the 32 P-labeling within the body of the RNA. Reaction products were resolved on 15% 7M urea PAGE gels. (B) Time-course decay analysis of uniformly 32 P-labeled monophosphate or FAD-capped RNA with the indicated amount of RNase AM protein are shown. Quantitation of RNA remaining is plotted from three independent experiments with ± SD denoted by error bars. (C) To demonstrate that RNase AM releases intact FAD upon deFADding like Xrn1 (in ), FAD-capQ assay using different amount of in vitro transcribed FAD-capped RNA was used. Catalytic-dead mutant of RNase AM, D20A was used as control for the reaction. (D) Total RNA from WT and rnase AMΔ were subjected to the FAD-capQ assay to detect total levels of cellular FAD-capped RNA. Data represents an average from three independent experiments. Error bars represent ± SD with unpaired t -test; * p < 0.05, ** p < 0.001, *** p < 0.0001.

    Techniques Used: In Vitro, Recombinant, Labeling, Quantitation Assay, Mutagenesis

    m 7 gpppa rna cap structure analog  (New England Biolabs)


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    Structured Review

    New England Biolabs m 7 gpppa rna cap structure analog
    a Schematic illustration of the NAD cap <t>-RNA</t> Affinity Purification (NcRAP). b Eluates from NcRAP were loaded on to a 4–12% Bis–Tris gel and stained with SYPRO Ruby. The <t>m</t> <t>7</t> G cap affinity purification was used as a control. All protein bands labeled on the gel were excised from the gel and identified using mass spectrometry.
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    Images

    1) Product Images from "Xrn1 is a deNADding enzyme modulating mitochondrial NAD-capped RNA"

    Article Title: Xrn1 is a deNADding enzyme modulating mitochondrial NAD-capped RNA

    Journal: Nature Communications

    doi: 10.1038/s41467-022-28555-7

    a Schematic illustration of the NAD cap -RNA Affinity Purification (NcRAP). b Eluates from NcRAP were loaded on to a 4–12% Bis–Tris gel and stained with SYPRO Ruby. The m 7 G cap affinity purification was used as a control. All protein bands labeled on the gel were excised from the gel and identified using mass spectrometry.
    Figure Legend Snippet: a Schematic illustration of the NAD cap -RNA Affinity Purification (NcRAP). b Eluates from NcRAP were loaded on to a 4–12% Bis–Tris gel and stained with SYPRO Ruby. The m 7 G cap affinity purification was used as a control. All protein bands labeled on the gel were excised from the gel and identified using mass spectrometry.

    Techniques Used: Affinity Purification, Staining, Labeling, Mass Spectrometry

    a Schematic illustration of in vitro 5′-end RNA decay. b Time-course decay analysis of uniformly 32 P-labeled m 7 G- or NAD-capped RNA in the presence of cell extract prepared from WT or xrn1Δ strains. The remaining RNA was quantified and plotted from n = 3 independent experiments (±SD denoted by error bars). Labeling as in the legend to Fig. . c Total NAD-capped RNA levels from WT and xrn1Δ were detected by NAD-capQ. Data represents average from n = 4 independent experiments. Error bars represent ±SEM; two sided unpaired t test; ** p = 0.0036). d qRT-PCR quantification of NAD-capped mRNA. NAD-capped RNA was enriched by NAD-capture, eluted from the beads, reverse transcribed and detected with gene-specific primers. Data are presented relative to the WT cells set to 1 and derived from n = 3 independent NAD-capture experiments; mean ± SEM; two-sided unpaired t test; ** p < 0.001, *** p < 0.0001. e Purified mitochondria derived from a strain harboring Xrn1 with a C-terminus Strep-tag II was analyzed by Western blot analysis with an anti-Strep-tag antibody. Western blotting analysis of whole cell extract (WCE) and extract prepared from purified mitochondria (mito) using Pgk1 (cytoplasmic protein) and Cox2 (mitochondrial protein) to determine the purity of mitochondria. Source data for panels b, c and d are provided in Source Data File . f Xrn1 knock-in strain containing the gene encoding green fluorescence protein (GFP) in frame with endogenous Xrn1 was generated into the strain carrying Edc3 fused to mCherry. Colocalization of Xrn1 with mitochondria (Mitotracker® Deep Red FM) and Edc3 (mCherry) by standard confocal (40X) microscopy is shown. g Reconstructed STORM images. Left panel shows images from a single optical section of RFP-labeled mitochondria and GFP-labeled Xrn1 in a single yeast cell obtained by spinning disk confocal (top) and STORM imaging (bottom). Merged image shows overlap of RFP and GFP signal. Right panel shows reconstructed STORM images obtained from sequentially acquired optical sections spaced 200 nm apart in the z axis. Merged images show colocalization of signals within the ~10 nm XY resolution. Scale bar is 100 nm.
    Figure Legend Snippet: a Schematic illustration of in vitro 5′-end RNA decay. b Time-course decay analysis of uniformly 32 P-labeled m 7 G- or NAD-capped RNA in the presence of cell extract prepared from WT or xrn1Δ strains. The remaining RNA was quantified and plotted from n = 3 independent experiments (±SD denoted by error bars). Labeling as in the legend to Fig. . c Total NAD-capped RNA levels from WT and xrn1Δ were detected by NAD-capQ. Data represents average from n = 4 independent experiments. Error bars represent ±SEM; two sided unpaired t test; ** p = 0.0036). d qRT-PCR quantification of NAD-capped mRNA. NAD-capped RNA was enriched by NAD-capture, eluted from the beads, reverse transcribed and detected with gene-specific primers. Data are presented relative to the WT cells set to 1 and derived from n = 3 independent NAD-capture experiments; mean ± SEM; two-sided unpaired t test; ** p < 0.001, *** p < 0.0001. e Purified mitochondria derived from a strain harboring Xrn1 with a C-terminus Strep-tag II was analyzed by Western blot analysis with an anti-Strep-tag antibody. Western blotting analysis of whole cell extract (WCE) and extract prepared from purified mitochondria (mito) using Pgk1 (cytoplasmic protein) and Cox2 (mitochondrial protein) to determine the purity of mitochondria. Source data for panels b, c and d are provided in Source Data File . f Xrn1 knock-in strain containing the gene encoding green fluorescence protein (GFP) in frame with endogenous Xrn1 was generated into the strain carrying Edc3 fused to mCherry. Colocalization of Xrn1 with mitochondria (Mitotracker® Deep Red FM) and Edc3 (mCherry) by standard confocal (40X) microscopy is shown. g Reconstructed STORM images. Left panel shows images from a single optical section of RFP-labeled mitochondria and GFP-labeled Xrn1 in a single yeast cell obtained by spinning disk confocal (top) and STORM imaging (bottom). Merged image shows overlap of RFP and GFP signal. Right panel shows reconstructed STORM images obtained from sequentially acquired optical sections spaced 200 nm apart in the z axis. Merged images show colocalization of signals within the ~10 nm XY resolution. Scale bar is 100 nm.

    Techniques Used: In Vitro, Labeling, Quantitative RT-PCR, Derivative Assay, Purification, Strep-tag, Western Blot, Knock-In, Fluorescence, Generated, Microscopy, Imaging

    m 7 gpppa cap analog  (New England Biolabs)


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    Structured Review

    New England Biolabs m 7 gpppa cap analog
    ( A ) Schematic representation of the 2′- O -methyl transfer reaction generating Cap-1-RNA from Cap-0-RNA. The methylated guanosine cap (m 7 G) moiety is colored in blue, and the methyl group added to the A 1 2′-OH position from the methyl donor SAM is in red. The remainder of the RNA structure with the first three labeled ribonucleotides is in black. ( B ) MTase-Glo luminescence assay results for Cap-0-RNA (m 7 GpppAUUAAA) and Cap-0 analog (m 7 <t>GpppA)</t> as substrates. n.d., no activity detected. The nsp16-nsp10 activity with Cap-0-RNA, SAM, and Mg 2+ was selected as a reference (100%), and all measured activities were normalized to this value. All values are means ± SD for biological triplicates conducted in two separate experiments using two independent preparations of nsp16-nsp10 ( n = 6). ( C ) The overall view of nsp16-nsp10 in complex with Mg 2+ , Cap-0-RNA, and SAM, (PDB 7JYY). The high-affinity binding site (HBS) and the low-affinity RNA binding (LBS) are labeled. ( D and E ) Close-up views of nsp16-nsp10 in complex with (D) Mg 2+ , Cap-0-RNA, and SAM (PDB 7JYY) or with (D) Mn 2+ , Cap-1-RNA, and SAH (PDB 7L6R). The nsp16 and nsp10 proteins are represented as solvent-exposed surfaces in tan and teal, respectively. Capped RNAs, SAM, and SAH are shown as sticks. Carbons are in gray for capped RNAs and in green for SAM and SAH; oxygens are in red; nitrogens are in blue; phosphates are in orange; sulfur is in yellow. Metal ions are shown as large spheres colored in purple for Mg 2+ and orange for Mn 2+ . Water molecules are small, cyan spheres. Hydrogen bonds between metal ions and waters from the first hydration sphere are shown as black dashed lines.
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    1) Product Images from "Mn 2+ coordinates Cap-0-RNA to align substrates for efficient 2′- O -methyl transfer by SARS-CoV-2 nsp16"

    Article Title: Mn 2+ coordinates Cap-0-RNA to align substrates for efficient 2′- O -methyl transfer by SARS-CoV-2 nsp16

    Journal: Science Signaling

    doi: 10.1126/scisignal.abh2071

    ( A ) Schematic representation of the 2′- O -methyl transfer reaction generating Cap-1-RNA from Cap-0-RNA. The methylated guanosine cap (m 7 G) moiety is colored in blue, and the methyl group added to the A 1 2′-OH position from the methyl donor SAM is in red. The remainder of the RNA structure with the first three labeled ribonucleotides is in black. ( B ) MTase-Glo luminescence assay results for Cap-0-RNA (m 7 GpppAUUAAA) and Cap-0 analog (m 7 GpppA) as substrates. n.d., no activity detected. The nsp16-nsp10 activity with Cap-0-RNA, SAM, and Mg 2+ was selected as a reference (100%), and all measured activities were normalized to this value. All values are means ± SD for biological triplicates conducted in two separate experiments using two independent preparations of nsp16-nsp10 ( n = 6). ( C ) The overall view of nsp16-nsp10 in complex with Mg 2+ , Cap-0-RNA, and SAM, (PDB 7JYY). The high-affinity binding site (HBS) and the low-affinity RNA binding (LBS) are labeled. ( D and E ) Close-up views of nsp16-nsp10 in complex with (D) Mg 2+ , Cap-0-RNA, and SAM (PDB 7JYY) or with (D) Mn 2+ , Cap-1-RNA, and SAH (PDB 7L6R). The nsp16 and nsp10 proteins are represented as solvent-exposed surfaces in tan and teal, respectively. Capped RNAs, SAM, and SAH are shown as sticks. Carbons are in gray for capped RNAs and in green for SAM and SAH; oxygens are in red; nitrogens are in blue; phosphates are in orange; sulfur is in yellow. Metal ions are shown as large spheres colored in purple for Mg 2+ and orange for Mn 2+ . Water molecules are small, cyan spheres. Hydrogen bonds between metal ions and waters from the first hydration sphere are shown as black dashed lines.
    Figure Legend Snippet: ( A ) Schematic representation of the 2′- O -methyl transfer reaction generating Cap-1-RNA from Cap-0-RNA. The methylated guanosine cap (m 7 G) moiety is colored in blue, and the methyl group added to the A 1 2′-OH position from the methyl donor SAM is in red. The remainder of the RNA structure with the first three labeled ribonucleotides is in black. ( B ) MTase-Glo luminescence assay results for Cap-0-RNA (m 7 GpppAUUAAA) and Cap-0 analog (m 7 GpppA) as substrates. n.d., no activity detected. The nsp16-nsp10 activity with Cap-0-RNA, SAM, and Mg 2+ was selected as a reference (100%), and all measured activities were normalized to this value. All values are means ± SD for biological triplicates conducted in two separate experiments using two independent preparations of nsp16-nsp10 ( n = 6). ( C ) The overall view of nsp16-nsp10 in complex with Mg 2+ , Cap-0-RNA, and SAM, (PDB 7JYY). The high-affinity binding site (HBS) and the low-affinity RNA binding (LBS) are labeled. ( D and E ) Close-up views of nsp16-nsp10 in complex with (D) Mg 2+ , Cap-0-RNA, and SAM (PDB 7JYY) or with (D) Mn 2+ , Cap-1-RNA, and SAH (PDB 7L6R). The nsp16 and nsp10 proteins are represented as solvent-exposed surfaces in tan and teal, respectively. Capped RNAs, SAM, and SAH are shown as sticks. Carbons are in gray for capped RNAs and in green for SAM and SAH; oxygens are in red; nitrogens are in blue; phosphates are in orange; sulfur is in yellow. Metal ions are shown as large spheres colored in purple for Mg 2+ and orange for Mn 2+ . Water molecules are small, cyan spheres. Hydrogen bonds between metal ions and waters from the first hydration sphere are shown as black dashed lines.

    Techniques Used: Methylation, Labeling, Luminescence Assay, Activity Assay, Binding Assay, RNA Binding Assay

    ( A ) A ball-and-stick representation of the hydrogen bonding network (dashed lines) between m 7 GpppA-RNA and nsp16 residues in crystal #2. Carbons are shown in black, nitrogens in blue, oxygens in red, phosphates in light green, sulfurs in yellow, and waters in cyan. ( B and C ) A detailed view of the hydration sphere of (B) Mn 2+ (orange sphere) in crystal #2 and (C) Mg 2+ (purple spheres) in crystal #3, mapped on the electrostatic surface of nsp16 (blue and red) and their interactions with water (cyan spheres) and nsp16 residues (beige sticks). The Cap-1-RNA is represented as sticks, with carbons in green, oxygens in red, nitrogens in blue, and phosphates in orange. Hydrogen bond interactions are shown as black dashed lines, and the omit electron density maps as blue mesh.
    Figure Legend Snippet: ( A ) A ball-and-stick representation of the hydrogen bonding network (dashed lines) between m 7 GpppA-RNA and nsp16 residues in crystal #2. Carbons are shown in black, nitrogens in blue, oxygens in red, phosphates in light green, sulfurs in yellow, and waters in cyan. ( B and C ) A detailed view of the hydration sphere of (B) Mn 2+ (orange sphere) in crystal #2 and (C) Mg 2+ (purple spheres) in crystal #3, mapped on the electrostatic surface of nsp16 (blue and red) and their interactions with water (cyan spheres) and nsp16 residues (beige sticks). The Cap-1-RNA is represented as sticks, with carbons in green, oxygens in red, nitrogens in blue, and phosphates in orange. Hydrogen bond interactions are shown as black dashed lines, and the omit electron density maps as blue mesh.

    Techniques Used:

    m 7 gpppa cap analog  (New England Biolabs)


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    New England Biolabs m 7 gpppa cap analog
    M 7 Gpppa Cap Analog, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs m 7 gpppa
    The workflow and specificity of DO-seq (A) The workflow of DO-seq: First, yDcpS (in red) de-caps the <t>m</t> <t>7</t> G moiety from m 7 G-RNAs in total RNAs. Second, in the presence of ADPRC, NAD-RNAs can be biotinylated by HEEB, which subsequently are enriched by streptavidin beads. Enriched RNAs can be used for epitranscriptome-wide profiling as well as qRT-PCR analysis. (B) yDcpS was able to de-cap m 7 <t>GpppA-RNA</t> (38 nt) but not NAD-RNA (45 nt), as evidenced by a lower-sized band corresponding to the de-capped product in the TBE-Urea gel. (C) HEEB (200 mM) reacted with NAD-RNA (106 nt) and m 7 GpppA-RNA (106 nt), but not pppA-RNA (106 nt), resulting in a band retained by the streptavidin beads. (D) qRT-PCR analysis showed that NAD-RNA (106 nt), but not pppA-RNA (106 nt), could be enriched by DO-seq. qRT-PCR analysis showed that m 7 GpppA-RNA (106 nt) could be detected in the presence of ADPRC; pre-treatment of yDcpS eliminates potential enrichment of m 7 GpppA-RNA (106 nt). The difference between Ct of target RNA in enrichment and input was calculated, resulting in index conversion of ΔCt. Data were shown in mean ± s.e.m. (E) Epitranscriptome assessment of yDcpS to minimize the noise of m 7 G-RNAs. Two-fold enrichment of read counts was used as the cutoff. Standard DO-seq identified 2,460 NAD-RNAs, while 1,905 false-positive NAD-RNAs were found without the use of yDcpS, presumably derived from m 7 G-capped RNAs. Total RNAs were from liver tissues of 18-month mice.
    M 7 Gpppa, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs m 7 gpppa rna cap structure analog
    The workflow and specificity of DO-seq (A) The workflow of DO-seq: First, yDcpS (in red) de-caps the <t>m</t> <t>7</t> G moiety from m 7 G-RNAs in total RNAs. Second, in the presence of ADPRC, NAD-RNAs can be biotinylated by HEEB, which subsequently are enriched by streptavidin beads. Enriched RNAs can be used for epitranscriptome-wide profiling as well as qRT-PCR analysis. (B) yDcpS was able to de-cap m 7 <t>GpppA-RNA</t> (38 nt) but not NAD-RNA (45 nt), as evidenced by a lower-sized band corresponding to the de-capped product in the TBE-Urea gel. (C) HEEB (200 mM) reacted with NAD-RNA (106 nt) and m 7 GpppA-RNA (106 nt), but not pppA-RNA (106 nt), resulting in a band retained by the streptavidin beads. (D) qRT-PCR analysis showed that NAD-RNA (106 nt), but not pppA-RNA (106 nt), could be enriched by DO-seq. qRT-PCR analysis showed that m 7 GpppA-RNA (106 nt) could be detected in the presence of ADPRC; pre-treatment of yDcpS eliminates potential enrichment of m 7 GpppA-RNA (106 nt). The difference between Ct of target RNA in enrichment and input was calculated, resulting in index conversion of ΔCt. Data were shown in mean ± s.e.m. (E) Epitranscriptome assessment of yDcpS to minimize the noise of m 7 G-RNAs. Two-fold enrichment of read counts was used as the cutoff. Standard DO-seq identified 2,460 NAD-RNAs, while 1,905 false-positive NAD-RNAs were found without the use of yDcpS, presumably derived from m 7 G-capped RNAs. Total RNAs were from liver tissues of 18-month mice.
    M 7 Gpppa Rna Cap Structure Analog, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m 7 gpppa rna cap structure analog/product/New England Biolabs
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    New England Biolabs m 7 gpppa cap analog
    ( A ) Schematic representation of the 2′- O -methyl transfer reaction generating Cap-1-RNA from Cap-0-RNA. The methylated guanosine cap (m 7 G) moiety is colored in blue, and the methyl group added to the A 1 2′-OH position from the methyl donor SAM is in red. The remainder of the RNA structure with the first three labeled ribonucleotides is in black. ( B ) MTase-Glo luminescence assay results for Cap-0-RNA (m 7 GpppAUUAAA) and Cap-0 analog (m 7 <t>GpppA)</t> as substrates. n.d., no activity detected. The nsp16-nsp10 activity with Cap-0-RNA, SAM, and Mg 2+ was selected as a reference (100%), and all measured activities were normalized to this value. All values are means ± SD for biological triplicates conducted in two separate experiments using two independent preparations of nsp16-nsp10 ( n = 6). ( C ) The overall view of nsp16-nsp10 in complex with Mg 2+ , Cap-0-RNA, and SAM, (PDB 7JYY). The high-affinity binding site (HBS) and the low-affinity RNA binding (LBS) are labeled. ( D and E ) Close-up views of nsp16-nsp10 in complex with (D) Mg 2+ , Cap-0-RNA, and SAM (PDB 7JYY) or with (D) Mn 2+ , Cap-1-RNA, and SAH (PDB 7L6R). The nsp16 and nsp10 proteins are represented as solvent-exposed surfaces in tan and teal, respectively. Capped RNAs, SAM, and SAH are shown as sticks. Carbons are in gray for capped RNAs and in green for SAM and SAH; oxygens are in red; nitrogens are in blue; phosphates are in orange; sulfur is in yellow. Metal ions are shown as large spheres colored in purple for Mg 2+ and orange for Mn 2+ . Water molecules are small, cyan spheres. Hydrogen bonds between metal ions and waters from the first hydration sphere are shown as black dashed lines.
    M 7 Gpppa Cap Analog, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The workflow and specificity of DO-seq (A) The workflow of DO-seq: First, yDcpS (in red) de-caps the m 7 G moiety from m 7 G-RNAs in total RNAs. Second, in the presence of ADPRC, NAD-RNAs can be biotinylated by HEEB, which subsequently are enriched by streptavidin beads. Enriched RNAs can be used for epitranscriptome-wide profiling as well as qRT-PCR analysis. (B) yDcpS was able to de-cap m 7 GpppA-RNA (38 nt) but not NAD-RNA (45 nt), as evidenced by a lower-sized band corresponding to the de-capped product in the TBE-Urea gel. (C) HEEB (200 mM) reacted with NAD-RNA (106 nt) and m 7 GpppA-RNA (106 nt), but not pppA-RNA (106 nt), resulting in a band retained by the streptavidin beads. (D) qRT-PCR analysis showed that NAD-RNA (106 nt), but not pppA-RNA (106 nt), could be enriched by DO-seq. qRT-PCR analysis showed that m 7 GpppA-RNA (106 nt) could be detected in the presence of ADPRC; pre-treatment of yDcpS eliminates potential enrichment of m 7 GpppA-RNA (106 nt). The difference between Ct of target RNA in enrichment and input was calculated, resulting in index conversion of ΔCt. Data were shown in mean ± s.e.m. (E) Epitranscriptome assessment of yDcpS to minimize the noise of m 7 G-RNAs. Two-fold enrichment of read counts was used as the cutoff. Standard DO-seq identified 2,460 NAD-RNAs, while 1,905 false-positive NAD-RNAs were found without the use of yDcpS, presumably derived from m 7 G-capped RNAs. Total RNAs were from liver tissues of 18-month mice.

    Journal: iScience

    Article Title: Hidden features of NAD-RNA epitranscriptome in Drosophila life cycle

    doi: 10.1016/j.isci.2023.108618

    Figure Lengend Snippet: The workflow and specificity of DO-seq (A) The workflow of DO-seq: First, yDcpS (in red) de-caps the m 7 G moiety from m 7 G-RNAs in total RNAs. Second, in the presence of ADPRC, NAD-RNAs can be biotinylated by HEEB, which subsequently are enriched by streptavidin beads. Enriched RNAs can be used for epitranscriptome-wide profiling as well as qRT-PCR analysis. (B) yDcpS was able to de-cap m 7 GpppA-RNA (38 nt) but not NAD-RNA (45 nt), as evidenced by a lower-sized band corresponding to the de-capped product in the TBE-Urea gel. (C) HEEB (200 mM) reacted with NAD-RNA (106 nt) and m 7 GpppA-RNA (106 nt), but not pppA-RNA (106 nt), resulting in a band retained by the streptavidin beads. (D) qRT-PCR analysis showed that NAD-RNA (106 nt), but not pppA-RNA (106 nt), could be enriched by DO-seq. qRT-PCR analysis showed that m 7 GpppA-RNA (106 nt) could be detected in the presence of ADPRC; pre-treatment of yDcpS eliminates potential enrichment of m 7 GpppA-RNA (106 nt). The difference between Ct of target RNA in enrichment and input was calculated, resulting in index conversion of ΔCt. Data were shown in mean ± s.e.m. (E) Epitranscriptome assessment of yDcpS to minimize the noise of m 7 G-RNAs. Two-fold enrichment of read counts was used as the cutoff. Standard DO-seq identified 2,460 NAD-RNAs, while 1,905 false-positive NAD-RNAs were found without the use of yDcpS, presumably derived from m 7 G-capped RNAs. Total RNAs were from liver tissues of 18-month mice.

    Article Snippet: For in vitro transcription,10 μM of double-stranded DNA (dsDNA) template in 100 μL 1×transcription buffer (Thermo Fisher Scientific, catalog: AM1334), along with 1 mM of each of GTP, CTP and UTP, with 1mM ATP (for pppA-RNA), 4 mM NAD (for NAD-RNA) or 4 mM m 7 GpppA (New England Biolabs, catalog: S1406S) (for m 7 G-RNA), 10 μL of T7 RNA polymerase (Thermo Fisher Scientific, catalog: AM1334), 5% DMSO, 5 mM DTT and 40 U RNase Inhibitor (Takara Bio, catalog: 2313B) were added and the transcription mixture was incubated at 37°C for 4 h. The reaction was incubated with 10 U DNase I (Thermo Fisher Scientific, catalog: AM1334) at 37°C for 30 min to remove the DNA template.

    Techniques: Quantitative RT-PCR, Derivative Assay

    ( A ) Schematic representation of the 2′- O -methyl transfer reaction generating Cap-1-RNA from Cap-0-RNA. The methylated guanosine cap (m 7 G) moiety is colored in blue, and the methyl group added to the A 1 2′-OH position from the methyl donor SAM is in red. The remainder of the RNA structure with the first three labeled ribonucleotides is in black. ( B ) MTase-Glo luminescence assay results for Cap-0-RNA (m 7 GpppAUUAAA) and Cap-0 analog (m 7 GpppA) as substrates. n.d., no activity detected. The nsp16-nsp10 activity with Cap-0-RNA, SAM, and Mg 2+ was selected as a reference (100%), and all measured activities were normalized to this value. All values are means ± SD for biological triplicates conducted in two separate experiments using two independent preparations of nsp16-nsp10 ( n = 6). ( C ) The overall view of nsp16-nsp10 in complex with Mg 2+ , Cap-0-RNA, and SAM, (PDB 7JYY). The high-affinity binding site (HBS) and the low-affinity RNA binding (LBS) are labeled. ( D and E ) Close-up views of nsp16-nsp10 in complex with (D) Mg 2+ , Cap-0-RNA, and SAM (PDB 7JYY) or with (D) Mn 2+ , Cap-1-RNA, and SAH (PDB 7L6R). The nsp16 and nsp10 proteins are represented as solvent-exposed surfaces in tan and teal, respectively. Capped RNAs, SAM, and SAH are shown as sticks. Carbons are in gray for capped RNAs and in green for SAM and SAH; oxygens are in red; nitrogens are in blue; phosphates are in orange; sulfur is in yellow. Metal ions are shown as large spheres colored in purple for Mg 2+ and orange for Mn 2+ . Water molecules are small, cyan spheres. Hydrogen bonds between metal ions and waters from the first hydration sphere are shown as black dashed lines.

    Journal: Science Signaling

    Article Title: Mn 2+ coordinates Cap-0-RNA to align substrates for efficient 2′- O -methyl transfer by SARS-CoV-2 nsp16

    doi: 10.1126/scisignal.abh2071

    Figure Lengend Snippet: ( A ) Schematic representation of the 2′- O -methyl transfer reaction generating Cap-1-RNA from Cap-0-RNA. The methylated guanosine cap (m 7 G) moiety is colored in blue, and the methyl group added to the A 1 2′-OH position from the methyl donor SAM is in red. The remainder of the RNA structure with the first three labeled ribonucleotides is in black. ( B ) MTase-Glo luminescence assay results for Cap-0-RNA (m 7 GpppAUUAAA) and Cap-0 analog (m 7 GpppA) as substrates. n.d., no activity detected. The nsp16-nsp10 activity with Cap-0-RNA, SAM, and Mg 2+ was selected as a reference (100%), and all measured activities were normalized to this value. All values are means ± SD for biological triplicates conducted in two separate experiments using two independent preparations of nsp16-nsp10 ( n = 6). ( C ) The overall view of nsp16-nsp10 in complex with Mg 2+ , Cap-0-RNA, and SAM, (PDB 7JYY). The high-affinity binding site (HBS) and the low-affinity RNA binding (LBS) are labeled. ( D and E ) Close-up views of nsp16-nsp10 in complex with (D) Mg 2+ , Cap-0-RNA, and SAM (PDB 7JYY) or with (D) Mn 2+ , Cap-1-RNA, and SAH (PDB 7L6R). The nsp16 and nsp10 proteins are represented as solvent-exposed surfaces in tan and teal, respectively. Capped RNAs, SAM, and SAH are shown as sticks. Carbons are in gray for capped RNAs and in green for SAM and SAH; oxygens are in red; nitrogens are in blue; phosphates are in orange; sulfur is in yellow. Metal ions are shown as large spheres colored in purple for Mg 2+ and orange for Mn 2+ . Water molecules are small, cyan spheres. Hydrogen bonds between metal ions and waters from the first hydration sphere are shown as black dashed lines.

    Article Snippet: SARS-CoV-2 samples of nsp10, nsp16, and nsp16-nsp10 were individually loaded into the sample cell and then titrated with either SAH, SAM, m 7 GpppA Cap analog or the m 7 GpppG Cap analog (New England Biolabs catalog #S1411L).

    Techniques: Methylation, Labeling, Luminescence Assay, Activity Assay, Binding Assay, RNA Binding Assay

    ( A ) A ball-and-stick representation of the hydrogen bonding network (dashed lines) between m 7 GpppA-RNA and nsp16 residues in crystal #2. Carbons are shown in black, nitrogens in blue, oxygens in red, phosphates in light green, sulfurs in yellow, and waters in cyan. ( B and C ) A detailed view of the hydration sphere of (B) Mn 2+ (orange sphere) in crystal #2 and (C) Mg 2+ (purple spheres) in crystal #3, mapped on the electrostatic surface of nsp16 (blue and red) and their interactions with water (cyan spheres) and nsp16 residues (beige sticks). The Cap-1-RNA is represented as sticks, with carbons in green, oxygens in red, nitrogens in blue, and phosphates in orange. Hydrogen bond interactions are shown as black dashed lines, and the omit electron density maps as blue mesh.

    Journal: Science Signaling

    Article Title: Mn 2+ coordinates Cap-0-RNA to align substrates for efficient 2′- O -methyl transfer by SARS-CoV-2 nsp16

    doi: 10.1126/scisignal.abh2071

    Figure Lengend Snippet: ( A ) A ball-and-stick representation of the hydrogen bonding network (dashed lines) between m 7 GpppA-RNA and nsp16 residues in crystal #2. Carbons are shown in black, nitrogens in blue, oxygens in red, phosphates in light green, sulfurs in yellow, and waters in cyan. ( B and C ) A detailed view of the hydration sphere of (B) Mn 2+ (orange sphere) in crystal #2 and (C) Mg 2+ (purple spheres) in crystal #3, mapped on the electrostatic surface of nsp16 (blue and red) and their interactions with water (cyan spheres) and nsp16 residues (beige sticks). The Cap-1-RNA is represented as sticks, with carbons in green, oxygens in red, nitrogens in blue, and phosphates in orange. Hydrogen bond interactions are shown as black dashed lines, and the omit electron density maps as blue mesh.

    Article Snippet: SARS-CoV-2 samples of nsp10, nsp16, and nsp16-nsp10 were individually loaded into the sample cell and then titrated with either SAH, SAM, m 7 GpppA Cap analog or the m 7 GpppG Cap analog (New England Biolabs catalog #S1411L).

    Techniques: