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    RNA Loading Dye 2x
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    New England Biolabs rna
    RNA Loading Dye 2x
    RNA Loading Dye 2x 4 0 ml
    https://www.bioz.com/result/rna/product/New England Biolabs
    Average 99 stars, based on 81 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Allosteric regulation of Csx1, a type IIIB-associated CARF domain ribonuclease by RNAs carrying a tetraadenylate tail"

    Article Title: Allosteric regulation of Csx1, a type IIIB-associated CARF domain ribonuclease by RNAs carrying a tetraadenylate tail

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx726

    UV cross-link analysis of the interaction between SisCsx1 and different RNA oligos. ( A ) SisCsx1 (3 μM) was incubated with increasing concentrations of S 3 A 7 (0, 0.1, 1, 10, 50 μM) at 70°C for 10 min and exposed to UV irradiation for 30 min. Then, the samples were resolved by SDS-loading buffer, analyzed by SDS-PAGE and visualized by Coomassie staining. ( B and C ) Labeled indicated RNA oligos ( Supplementary Table S2 ) were incubated with 3 μM SisCsx1 at 70°C for 10 min and exposed to UV irradiation for 30 min. Then the samples were loaded onto a SDS-polyacrylamide gel and analyzed by autoradiography (upper panel) and Coomassie staining (lower panel). The right panels show the relative amount of the indicated RNA oligos bound by SisCsx1. Only the signal from the main band was calculated and the amount of Csx1-associated S 3 A 7 and CA 4 was set as ‘1’ in (B) and (C), respectively.
    Figure Legend Snippet: UV cross-link analysis of the interaction between SisCsx1 and different RNA oligos. ( A ) SisCsx1 (3 μM) was incubated with increasing concentrations of S 3 A 7 (0, 0.1, 1, 10, 50 μM) at 70°C for 10 min and exposed to UV irradiation for 30 min. Then, the samples were resolved by SDS-loading buffer, analyzed by SDS-PAGE and visualized by Coomassie staining. ( B and C ) Labeled indicated RNA oligos ( Supplementary Table S2 ) were incubated with 3 μM SisCsx1 at 70°C for 10 min and exposed to UV irradiation for 30 min. Then the samples were loaded onto a SDS-polyacrylamide gel and analyzed by autoradiography (upper panel) and Coomassie staining (lower panel). The right panels show the relative amount of the indicated RNA oligos bound by SisCsx1. Only the signal from the main band was calculated and the amount of Csx1-associated S 3 A 7 and CA 4 was set as ‘1’ in (B) and (C), respectively.

    Techniques Used: Incubation, Irradiation, SDS Page, Staining, Labeling, Autoradiography

    2) Product Images from "Homodimerisation-independent cleavage of dsRNA by a pestiviral nicking endoribonuclease"

    Article Title: Homodimerisation-independent cleavage of dsRNA by a pestiviral nicking endoribonuclease

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26557-4

    E rns is a nicking endoribonuclease. ( a ) Schematic representation of the RNase activity assay with the modified hybrids. A single-stranded RNA of positive polarity (ssRNA+) labeled in red together with either a single-stranded methylated RNA (metRNA) or a ssDNA of negative polarity, both labeled in green, were boiled and cooled down at room temperature for hybridization (RNA/metRNA or RNA/DNA). Strep-tag purified wild-type (C171), monomeric (R171) and RNase-inactive mutant (H30F) of E rns were incubated at the concentrations indicated with 625 nM single-stranded metRNA or DNA ( b ), double-stranded RNA/DNA− ( c ) or double-stranded RNA/metRNA-hybrids ( d ). Samples were separated by 14% SDS-PAGE and fluorescence was analysed with a Li-Cor Odyssey system. Due to the known, defined length of the directly labeled fragments (30 b for ssRNA, 30 bp for dsRNA), no size ladder was applied. Non-cropped gels as representative experiment out of three ( b ) or four ( c , d ) are shown.
    Figure Legend Snippet: E rns is a nicking endoribonuclease. ( a ) Schematic representation of the RNase activity assay with the modified hybrids. A single-stranded RNA of positive polarity (ssRNA+) labeled in red together with either a single-stranded methylated RNA (metRNA) or a ssDNA of negative polarity, both labeled in green, were boiled and cooled down at room temperature for hybridization (RNA/metRNA or RNA/DNA). Strep-tag purified wild-type (C171), monomeric (R171) and RNase-inactive mutant (H30F) of E rns were incubated at the concentrations indicated with 625 nM single-stranded metRNA or DNA ( b ), double-stranded RNA/DNA− ( c ) or double-stranded RNA/metRNA-hybrids ( d ). Samples were separated by 14% SDS-PAGE and fluorescence was analysed with a Li-Cor Odyssey system. Due to the known, defined length of the directly labeled fragments (30 b for ssRNA, 30 bp for dsRNA), no size ladder was applied. Non-cropped gels as representative experiment out of three ( b ) or four ( c , d ) are shown.

    Techniques Used: Activity Assay, Modification, Labeling, Methylation, Hybridization, Strep-tag, Purification, Mutagenesis, Incubation, SDS Page, Fluorescence

    3) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: RNA

    doi: 10.1261/rna.073858.119

    3′-extended RNA by-products are formed during IVT reactions. ( A ) dsRNA immunoblot assay using a dsRNA-specific antibody (J2) on three different mRNA sequences (CLuc, RFP, and GFP). Poly(I:C) and RNase III treatment is used as a control to detect and validate dsRNA, respectively. For the immunoblots, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses, 0.05 µg of RNA was loaded in each well. ( B , C ) IVT reactions using short templates (30 bp) run on denaturing gels under standard conditions (four rNTPs), and under conditions where one rNTP was eliminated to prevent formation of the dsRNA products (−rUTP for B and −rCTP for C ). RNase III digestion control to confirm the dsRNA nature of the extended by-product. Chemically synthesized RNA was used as a control. ( D , E ) Intact mass spectrometry of IVT products when all four rNTPs are present (+rUTP for D and +rCTP for E ), and when one rNTP is eliminated to prevent formation of the dsRNA products (−rUTP for D and −rCTP for E ). Expected mass of extended products is calculated based on average rNTP values.
    Figure Legend Snippet: 3′-extended RNA by-products are formed during IVT reactions. ( A ) dsRNA immunoblot assay using a dsRNA-specific antibody (J2) on three different mRNA sequences (CLuc, RFP, and GFP). Poly(I:C) and RNase III treatment is used as a control to detect and validate dsRNA, respectively. For the immunoblots, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses, 0.05 µg of RNA was loaded in each well. ( B , C ) IVT reactions using short templates (30 bp) run on denaturing gels under standard conditions (four rNTPs), and under conditions where one rNTP was eliminated to prevent formation of the dsRNA products (−rUTP for B and −rCTP for C ). RNase III digestion control to confirm the dsRNA nature of the extended by-product. Chemically synthesized RNA was used as a control. ( D , E ) Intact mass spectrometry of IVT products when all four rNTPs are present (+rUTP for D and +rCTP for E ), and when one rNTP is eliminated to prevent formation of the dsRNA products (−rUTP for D and −rCTP for E ). Expected mass of extended products is calculated based on average rNTP values.

    Techniques Used: Western Blot, Nucleic Acid Electrophoresis, Synthesized, Mass Spectrometry

    High-temperature IVT with TsT7-1 leads to a reduction in 3′-extended by-products. ( A ) Denaturing gel analysis of IVT products synthesized from short IVT template-1 using TsT7-1 performed at a temperature range of 37°C to 55°C. Chemically synthesized short IVT template 1 RNA was used as a control. Equal amounts were loaded in each lane. ( B ) Intact mass spectrometry analysis of Template IVT short-1 and Template IVT short-2 using TsT7-1 under standard conditions (all four rNTPs; +rUTP or rCTP) or lacking one rNTP (−rUTP or −rCTP) performed at either 37°C or 50°C. Expected run-off transcript (including n + 1 and n + 2 products) is highlighted in orange, and products longer than the run-off transcript are highlighted in gray. ( C ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized with TsT7-1 or wild-type RNAP at a temperature range from 37°C to 55°C. For the immunoblots with TsT7-1, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses with TsT7-1, 0.05 µg of RNA was loaded in each well. ( D ) Intact mass spectrometry analysis of IVT reactions in the presence or absence of TsT7-1 on chemically synthesized RNA ( n , n + 1, n + 2) performed at either 37°C or 50°C. Products longer than the run-off transcript are highlighted in gray.
    Figure Legend Snippet: High-temperature IVT with TsT7-1 leads to a reduction in 3′-extended by-products. ( A ) Denaturing gel analysis of IVT products synthesized from short IVT template-1 using TsT7-1 performed at a temperature range of 37°C to 55°C. Chemically synthesized short IVT template 1 RNA was used as a control. Equal amounts were loaded in each lane. ( B ) Intact mass spectrometry analysis of Template IVT short-1 and Template IVT short-2 using TsT7-1 under standard conditions (all four rNTPs; +rUTP or rCTP) or lacking one rNTP (−rUTP or −rCTP) performed at either 37°C or 50°C. Expected run-off transcript (including n + 1 and n + 2 products) is highlighted in orange, and products longer than the run-off transcript are highlighted in gray. ( C ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized with TsT7-1 or wild-type RNAP at a temperature range from 37°C to 55°C. For the immunoblots with TsT7-1, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses with TsT7-1, 0.05 µg of RNA was loaded in each well. ( D ) Intact mass spectrometry analysis of IVT reactions in the presence or absence of TsT7-1 on chemically synthesized RNA ( n , n + 1, n + 2) performed at either 37°C or 50°C. Products longer than the run-off transcript are highlighted in gray.

    Techniques Used: Synthesized, Mass Spectrometry, Nucleic Acid Electrophoresis, Western Blot

    Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.
    Figure Legend Snippet: Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    Thermostable (Ts) RNAPs are active at high temperatures and do not affect 3′-extended RNA by-product formation. ( A ) Melting temperatures of wild-type T7 RNAP (orange), TsT7-1 (blue), and TsT7-2 (red) determined by nano-differential scanning fluorimetry. ( B ) Molecular beacon assay for the efficiency of IVT with TsT7-1 or TsT7-2 compared to wild-type RNAP at temperatures ranging from 37°C to 62°C. ( C ) Gel electrophoreses analyses of CLuc RNA synthesized with either wild-type RNAP, TsT7-1, or TsT7-2 RNAPs at temperatures ranging from 37°C to 62°C. Equal volume of IVT reactions were loaded in each well to reflect the differences in RNA yield at each temperature. ( D ) dsRNA immunoblot using J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from IVT reactions performed at 37°C using wild-type T7, TsT7-1, and TsT7-2 RNAPs. For the immunoblots, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses, 0.05 µg of RNA was loaded in each well. ( E ) Denaturing gel analysis of IVT reactions using Template IVT short-1 with Ts RNAPs compared to wild-type RNAP. Reactions were performed with either four NTPs (+rUTP) or with three NTPs (−rUTP). ( F ) Intact mass spectrometry of short IVT products from TsT7-1 under standard conditions (37°C) with products longer than the run-off transcript highlighted in gray and the expected run-off transcripts in orange (including n + 1, n + 2 products from nontemplated additions).
    Figure Legend Snippet: Thermostable (Ts) RNAPs are active at high temperatures and do not affect 3′-extended RNA by-product formation. ( A ) Melting temperatures of wild-type T7 RNAP (orange), TsT7-1 (blue), and TsT7-2 (red) determined by nano-differential scanning fluorimetry. ( B ) Molecular beacon assay for the efficiency of IVT with TsT7-1 or TsT7-2 compared to wild-type RNAP at temperatures ranging from 37°C to 62°C. ( C ) Gel electrophoreses analyses of CLuc RNA synthesized with either wild-type RNAP, TsT7-1, or TsT7-2 RNAPs at temperatures ranging from 37°C to 62°C. Equal volume of IVT reactions were loaded in each well to reflect the differences in RNA yield at each temperature. ( D ) dsRNA immunoblot using J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from IVT reactions performed at 37°C using wild-type T7, TsT7-1, and TsT7-2 RNAPs. For the immunoblots, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses, 0.05 µg of RNA was loaded in each well. ( E ) Denaturing gel analysis of IVT reactions using Template IVT short-1 with Ts RNAPs compared to wild-type RNAP. Reactions were performed with either four NTPs (+rUTP) or with three NTPs (−rUTP). ( F ) Intact mass spectrometry of short IVT products from TsT7-1 under standard conditions (37°C) with products longer than the run-off transcript highlighted in gray and the expected run-off transcripts in orange (including n + 1, n + 2 products from nontemplated additions).

    Techniques Used: Nano Differential Scanning Fluorimetry, Synthesized, Nucleic Acid Electrophoresis, Western Blot, Mass Spectrometry

    mRNAs generated by high-temperature IVT with TsT7-1 are functional and have reduced immunogenicity in vivo. ( A ) Luciferase activity from HEK293 cells transfected with CLuc mRNA (unmodified or modified with pseudouridine) synthesized with wild-type T7 or TsT7-1 RNAP at 37°C or 50°C. ( B ) Representative chromatogram for CLuc mRNA separated on an HPLC column. Absorbance at 260 nm representing the RNA products in the IVT reaction. dsRNA immunoblot with J2 antibody on fractions collected from the HPLC purification. ( C ) IFN-α levels in supernatant collected from DCs transfected with either crude IVT CLuc mRNA or CLuc mRNA purified with HPLC. IFN-α levels in the supernatant were measured 24 h post-transfection. Error bars are standard error of the mean. Poly(I:C), a synthetic analog of dsRNA and Resiquimod (R848), an activator of Toll-like receptors were used as controls for interferon activation.
    Figure Legend Snippet: mRNAs generated by high-temperature IVT with TsT7-1 are functional and have reduced immunogenicity in vivo. ( A ) Luciferase activity from HEK293 cells transfected with CLuc mRNA (unmodified or modified with pseudouridine) synthesized with wild-type T7 or TsT7-1 RNAP at 37°C or 50°C. ( B ) Representative chromatogram for CLuc mRNA separated on an HPLC column. Absorbance at 260 nm representing the RNA products in the IVT reaction. dsRNA immunoblot with J2 antibody on fractions collected from the HPLC purification. ( C ) IFN-α levels in supernatant collected from DCs transfected with either crude IVT CLuc mRNA or CLuc mRNA purified with HPLC. IFN-α levels in the supernatant were measured 24 h post-transfection. Error bars are standard error of the mean. Poly(I:C), a synthetic analog of dsRNA and Resiquimod (R848), an activator of Toll-like receptors were used as controls for interferon activation.

    Techniques Used: Generated, Functional Assay, In Vivo, Luciferase, Activity Assay, Transfection, Modification, Synthesized, High Performance Liquid Chromatography, Purification, Activation Assay

    4) Product Images from "Synthesis of low immunogenicity RNA with high-temperature in vitro transcription"

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    Journal: RNA

    doi: 10.1261/rna.073858.119

    3′-extended RNA by-products are formed during IVT reactions. ( A ) dsRNA immunoblot assay using a dsRNA-specific antibody (J2) on three different mRNA sequences (CLuc, RFP, and GFP). Poly(I:C) and RNase III treatment is used as a control to detect and validate dsRNA, respectively. For the immunoblots, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses, 0.05 µg of RNA was loaded in each well. ( B , C ) IVT reactions using short templates (30 bp) run on denaturing gels under standard conditions (four rNTPs), and under conditions where one rNTP was eliminated to prevent formation of the dsRNA products (−rUTP for B and −rCTP for C ). RNase III digestion control to confirm the dsRNA nature of the extended by-product. Chemically synthesized RNA was used as a control. ( D , E ) Intact mass spectrometry of IVT products when all four rNTPs are present (+rUTP for D and +rCTP for E ), and when one rNTP is eliminated to prevent formation of the dsRNA products (−rUTP for D and −rCTP for E ). Expected mass of extended products is calculated based on average rNTP values.
    Figure Legend Snippet: 3′-extended RNA by-products are formed during IVT reactions. ( A ) dsRNA immunoblot assay using a dsRNA-specific antibody (J2) on three different mRNA sequences (CLuc, RFP, and GFP). Poly(I:C) and RNase III treatment is used as a control to detect and validate dsRNA, respectively. For the immunoblots, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses, 0.05 µg of RNA was loaded in each well. ( B , C ) IVT reactions using short templates (30 bp) run on denaturing gels under standard conditions (four rNTPs), and under conditions where one rNTP was eliminated to prevent formation of the dsRNA products (−rUTP for B and −rCTP for C ). RNase III digestion control to confirm the dsRNA nature of the extended by-product. Chemically synthesized RNA was used as a control. ( D , E ) Intact mass spectrometry of IVT products when all four rNTPs are present (+rUTP for D and +rCTP for E ), and when one rNTP is eliminated to prevent formation of the dsRNA products (−rUTP for D and −rCTP for E ). Expected mass of extended products is calculated based on average rNTP values.

    Techniques Used: Western Blot, Nucleic Acid Electrophoresis, Synthesized, Mass Spectrometry

    High-temperature IVT with TsT7-1 leads to a reduction in 3′-extended by-products. ( A ) Denaturing gel analysis of IVT products synthesized from short IVT template-1 using TsT7-1 performed at a temperature range of 37°C to 55°C. Chemically synthesized short IVT template 1 RNA was used as a control. Equal amounts were loaded in each lane. ( B ) Intact mass spectrometry analysis of Template IVT short-1 and Template IVT short-2 using TsT7-1 under standard conditions (all four rNTPs; +rUTP or rCTP) or lacking one rNTP (−rUTP or −rCTP) performed at either 37°C or 50°C. Expected run-off transcript (including n + 1 and n + 2 products) is highlighted in orange, and products longer than the run-off transcript are highlighted in gray. ( C ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized with TsT7-1 or wild-type RNAP at a temperature range from 37°C to 55°C. For the immunoblots with TsT7-1, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses with TsT7-1, 0.05 µg of RNA was loaded in each well. ( D ) Intact mass spectrometry analysis of IVT reactions in the presence or absence of TsT7-1 on chemically synthesized RNA ( n , n + 1, n + 2) performed at either 37°C or 50°C. Products longer than the run-off transcript are highlighted in gray.
    Figure Legend Snippet: High-temperature IVT with TsT7-1 leads to a reduction in 3′-extended by-products. ( A ) Denaturing gel analysis of IVT products synthesized from short IVT template-1 using TsT7-1 performed at a temperature range of 37°C to 55°C. Chemically synthesized short IVT template 1 RNA was used as a control. Equal amounts were loaded in each lane. ( B ) Intact mass spectrometry analysis of Template IVT short-1 and Template IVT short-2 using TsT7-1 under standard conditions (all four rNTPs; +rUTP or rCTP) or lacking one rNTP (−rUTP or −rCTP) performed at either 37°C or 50°C. Expected run-off transcript (including n + 1 and n + 2 products) is highlighted in orange, and products longer than the run-off transcript are highlighted in gray. ( C ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized with TsT7-1 or wild-type RNAP at a temperature range from 37°C to 55°C. For the immunoblots with TsT7-1, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses with TsT7-1, 0.05 µg of RNA was loaded in each well. ( D ) Intact mass spectrometry analysis of IVT reactions in the presence or absence of TsT7-1 on chemically synthesized RNA ( n , n + 1, n + 2) performed at either 37°C or 50°C. Products longer than the run-off transcript are highlighted in gray.

    Techniques Used: Synthesized, Mass Spectrometry, Nucleic Acid Electrophoresis, Western Blot

    Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.
    Figure Legend Snippet: Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.

    Techniques Used: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    Thermostable (Ts) RNAPs are active at high temperatures and do not affect 3′-extended RNA by-product formation. ( A ) Melting temperatures of wild-type T7 RNAP (orange), TsT7-1 (blue), and TsT7-2 (red) determined by nano-differential scanning fluorimetry. ( B ) Molecular beacon assay for the efficiency of IVT with TsT7-1 or TsT7-2 compared to wild-type RNAP at temperatures ranging from 37°C to 62°C. ( C ) Gel electrophoreses analyses of CLuc RNA synthesized with either wild-type RNAP, TsT7-1, or TsT7-2 RNAPs at temperatures ranging from 37°C to 62°C. Equal volume of IVT reactions were loaded in each well to reflect the differences in RNA yield at each temperature. ( D ) dsRNA immunoblot using J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from IVT reactions performed at 37°C using wild-type T7, TsT7-1, and TsT7-2 RNAPs. For the immunoblots, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses, 0.05 µg of RNA was loaded in each well. ( E ) Denaturing gel analysis of IVT reactions using Template IVT short-1 with Ts RNAPs compared to wild-type RNAP. Reactions were performed with either four NTPs (+rUTP) or with three NTPs (−rUTP). ( F ) Intact mass spectrometry of short IVT products from TsT7-1 under standard conditions (37°C) with products longer than the run-off transcript highlighted in gray and the expected run-off transcripts in orange (including n + 1, n + 2 products from nontemplated additions).
    Figure Legend Snippet: Thermostable (Ts) RNAPs are active at high temperatures and do not affect 3′-extended RNA by-product formation. ( A ) Melting temperatures of wild-type T7 RNAP (orange), TsT7-1 (blue), and TsT7-2 (red) determined by nano-differential scanning fluorimetry. ( B ) Molecular beacon assay for the efficiency of IVT with TsT7-1 or TsT7-2 compared to wild-type RNAP at temperatures ranging from 37°C to 62°C. ( C ) Gel electrophoreses analyses of CLuc RNA synthesized with either wild-type RNAP, TsT7-1, or TsT7-2 RNAPs at temperatures ranging from 37°C to 62°C. Equal volume of IVT reactions were loaded in each well to reflect the differences in RNA yield at each temperature. ( D ) dsRNA immunoblot using J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from IVT reactions performed at 37°C using wild-type T7, TsT7-1, and TsT7-2 RNAPs. For the immunoblots, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses, 0.05 µg of RNA was loaded in each well. ( E ) Denaturing gel analysis of IVT reactions using Template IVT short-1 with Ts RNAPs compared to wild-type RNAP. Reactions were performed with either four NTPs (+rUTP) or with three NTPs (−rUTP). ( F ) Intact mass spectrometry of short IVT products from TsT7-1 under standard conditions (37°C) with products longer than the run-off transcript highlighted in gray and the expected run-off transcripts in orange (including n + 1, n + 2 products from nontemplated additions).

    Techniques Used: Nano Differential Scanning Fluorimetry, Synthesized, Nucleic Acid Electrophoresis, Western Blot, Mass Spectrometry

    mRNAs generated by high-temperature IVT with TsT7-1 are functional and have reduced immunogenicity in vivo. ( A ) Luciferase activity from HEK293 cells transfected with CLuc mRNA (unmodified or modified with pseudouridine) synthesized with wild-type T7 or TsT7-1 RNAP at 37°C or 50°C. ( B ) Representative chromatogram for CLuc mRNA separated on an HPLC column. Absorbance at 260 nm representing the RNA products in the IVT reaction. dsRNA immunoblot with J2 antibody on fractions collected from the HPLC purification. ( C ) IFN-α levels in supernatant collected from DCs transfected with either crude IVT CLuc mRNA or CLuc mRNA purified with HPLC. IFN-α levels in the supernatant were measured 24 h post-transfection. Error bars are standard error of the mean. Poly(I:C), a synthetic analog of dsRNA and Resiquimod (R848), an activator of Toll-like receptors were used as controls for interferon activation.
    Figure Legend Snippet: mRNAs generated by high-temperature IVT with TsT7-1 are functional and have reduced immunogenicity in vivo. ( A ) Luciferase activity from HEK293 cells transfected with CLuc mRNA (unmodified or modified with pseudouridine) synthesized with wild-type T7 or TsT7-1 RNAP at 37°C or 50°C. ( B ) Representative chromatogram for CLuc mRNA separated on an HPLC column. Absorbance at 260 nm representing the RNA products in the IVT reaction. dsRNA immunoblot with J2 antibody on fractions collected from the HPLC purification. ( C ) IFN-α levels in supernatant collected from DCs transfected with either crude IVT CLuc mRNA or CLuc mRNA purified with HPLC. IFN-α levels in the supernatant were measured 24 h post-transfection. Error bars are standard error of the mean. Poly(I:C), a synthetic analog of dsRNA and Resiquimod (R848), an activator of Toll-like receptors were used as controls for interferon activation.

    Techniques Used: Generated, Functional Assay, In Vivo, Luciferase, Activity Assay, Transfection, Modification, Synthesized, High Performance Liquid Chromatography, Purification, Activation Assay

    5) Product Images from "Effect of UV Radiation on Fluorescent RNA Aptamers’ Functional and Templating Ability"

    Article Title: Effect of UV Radiation on Fluorescent RNA Aptamers’ Functional and Templating Ability

    Journal: Chembiochem

    doi: 10.1002/cbic.201900261

    Partial unfolding of a UV‐damaged MG aptamer. A) Denaturing PAGE of the MG aptamer after exposure to UV for 6 h (+UV) or incubated in the dark for 6 h (−UV). B) Native PAGE of the MG aptamer, without exposure (incubated in the dark for 6 h, left) or with exposure (right 3 lanes, with increasing amount of loaded RNA) to UV for 6 h, illustrating the presence of a more slowly migrating band after irradiation. Irradiated samples were ethanol‐precipitated and annealed before native PAGE.
    Figure Legend Snippet: Partial unfolding of a UV‐damaged MG aptamer. A) Denaturing PAGE of the MG aptamer after exposure to UV for 6 h (+UV) or incubated in the dark for 6 h (−UV). B) Native PAGE of the MG aptamer, without exposure (incubated in the dark for 6 h, left) or with exposure (right 3 lanes, with increasing amount of loaded RNA) to UV for 6 h, illustrating the presence of a more slowly migrating band after irradiation. Irradiated samples were ethanol‐precipitated and annealed before native PAGE.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Incubation, Clear Native PAGE, Irradiation

    Characterization of damaged sites on the MG aptamer. A) Reverse transcription with a fluorescently tagged primer of RNA exposed to UV for various times (from right to left: 0, 0.5, 1, 2, 3, 4, 5 h) at room temperature was analyzed by PAGE. The lanes labeled A, U, and C correspond to sequencing reactions performed by using ddTTP, ddATP, and ddGTP as chain terminators, respectively. UV exposure was also carried out at 80 °C (left two lanes) for the times indicated. The site of damage is identified along the right side of the gel; the site listed is immediately downstream of the last nucleotide of the stalled DNA copy. B) Disappearance of full‐length cDNA during UV exposure. The single‐exponential decay has a rate constant of ≈0.8 h −1 (1.0 and 0.61 h −1 in two replicates). C) UV reactivity measured as the ratio of band intensity at a particular site divided by the band intensity of the full‐length cDNA ( R I ) after 4 h of exposure to UV. D) UV reactivity shown on the secondary structure of the MG aptamer in the context of the reverse transcription construct; the areas of the blue circles are proportional to the reactivity (see part (C)). The position of MG is shown in red.
    Figure Legend Snippet: Characterization of damaged sites on the MG aptamer. A) Reverse transcription with a fluorescently tagged primer of RNA exposed to UV for various times (from right to left: 0, 0.5, 1, 2, 3, 4, 5 h) at room temperature was analyzed by PAGE. The lanes labeled A, U, and C correspond to sequencing reactions performed by using ddTTP, ddATP, and ddGTP as chain terminators, respectively. UV exposure was also carried out at 80 °C (left two lanes) for the times indicated. The site of damage is identified along the right side of the gel; the site listed is immediately downstream of the last nucleotide of the stalled DNA copy. B) Disappearance of full‐length cDNA during UV exposure. The single‐exponential decay has a rate constant of ≈0.8 h −1 (1.0 and 0.61 h −1 in two replicates). C) UV reactivity measured as the ratio of band intensity at a particular site divided by the band intensity of the full‐length cDNA ( R I ) after 4 h of exposure to UV. D) UV reactivity shown on the secondary structure of the MG aptamer in the context of the reverse transcription construct; the areas of the blue circles are proportional to the reactivity (see part (C)). The position of MG is shown in red.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Labeling, Sequencing, Construct

    6) Product Images from "Homodimerisation-independent cleavage of dsRNA by a pestiviral nicking endoribonuclease"

    Article Title: Homodimerisation-independent cleavage of dsRNA by a pestiviral nicking endoribonuclease

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-26557-4

    E rns is a nicking endoribonuclease. ( a ) Schematic representation of the RNase activity assay with the modified hybrids. A single-stranded RNA of positive polarity (ssRNA+) labeled in red together with either a single-stranded methylated RNA (metRNA) or a ssDNA of negative polarity, both labeled in green, were boiled and cooled down at room temperature for hybridization (RNA/metRNA or RNA/DNA). Strep-tag purified wild-type (C171), monomeric (R171) and RNase-inactive mutant (H30F) of E rns were incubated at the concentrations indicated with 625 nM single-stranded metRNA or DNA ( b ), double-stranded RNA/DNA− ( c ) or double-stranded RNA/metRNA-hybrids ( d ). Samples were separated by 14% SDS-PAGE and fluorescence was analysed with a Li-Cor Odyssey system. Due to the known, defined length of the directly labeled fragments (30 b for ssRNA, 30 bp for dsRNA), no size ladder was applied. Non-cropped gels as representative experiment out of three ( b ) or four ( c , d ) are shown.
    Figure Legend Snippet: E rns is a nicking endoribonuclease. ( a ) Schematic representation of the RNase activity assay with the modified hybrids. A single-stranded RNA of positive polarity (ssRNA+) labeled in red together with either a single-stranded methylated RNA (metRNA) or a ssDNA of negative polarity, both labeled in green, were boiled and cooled down at room temperature for hybridization (RNA/metRNA or RNA/DNA). Strep-tag purified wild-type (C171), monomeric (R171) and RNase-inactive mutant (H30F) of E rns were incubated at the concentrations indicated with 625 nM single-stranded metRNA or DNA ( b ), double-stranded RNA/DNA− ( c ) or double-stranded RNA/metRNA-hybrids ( d ). Samples were separated by 14% SDS-PAGE and fluorescence was analysed with a Li-Cor Odyssey system. Due to the known, defined length of the directly labeled fragments (30 b for ssRNA, 30 bp for dsRNA), no size ladder was applied. Non-cropped gels as representative experiment out of three ( b ) or four ( c , d ) are shown.

    Techniques Used: Activity Assay, Modification, Labeling, Methylation, Hybridization, Strep-tag, Purification, Mutagenesis, Incubation, SDS Page, Fluorescence

    7) Product Images from "Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase"

    Article Title: Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase

    Journal: bioRxiv

    doi: 10.1101/2019.12.17.879585

    Effect of LdCsm3 mutations on the ssDNA cleavage and binding of LdCsm. ( a ) Target RNA cleavage of LdCsm3 mutated derivatives. 50 nM of S1-46 RNA were incubated with 50 nM of LdCsm or the indicated mutant derivatives for 10 min and the samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. ( b ) RNA-activated ssDNA cleavage by effectors carrying one of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4b . ( c ) ssDNA binding by effectors carrying each of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4c . ( d ) Relative ssDNA binding between the wild-type LdCsm effector and its LdCsm3 mutated derivatives. The ssDNA activity of LdCsm in non-homologous RNA was used as the standard and set up as 1. Results shown are average of three independent assays, bars represent the mean standard deviation (± SD).
    Figure Legend Snippet: Effect of LdCsm3 mutations on the ssDNA cleavage and binding of LdCsm. ( a ) Target RNA cleavage of LdCsm3 mutated derivatives. 50 nM of S1-46 RNA were incubated with 50 nM of LdCsm or the indicated mutant derivatives for 10 min and the samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. ( b ) RNA-activated ssDNA cleavage by effectors carrying one of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4b . ( c ) ssDNA binding by effectors carrying each of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4c . ( d ) Relative ssDNA binding between the wild-type LdCsm effector and its LdCsm3 mutated derivatives. The ssDNA activity of LdCsm in non-homologous RNA was used as the standard and set up as 1. Results shown are average of three independent assays, bars represent the mean standard deviation (± SD).

    Techniques Used: Binding Assay, Incubation, Mutagenesis, Polyacrylamide Gel Electrophoresis, Construct, Activity Assay, Standard Deviation

    Model of allosteric activation and repression of the LdCsm DNase. The previous works have proposed that the initial recognition of nascent transcript at the 5′ end of target RNA for type III complex, since both of Csm5 subunit in Csm complex and Cmr1 subunit in Cmr complex are crucial for target RNA binding 36 , 37 , 68 . These suggested that the binary LdCsm effector complex interacts with target transcript initially at the 5′ end of target RNA and further via sequence complementarity between the protospacer and the corresponding crRNA, leading to the formation of a ternary effector complex with a major conformational change. Addition of a single nucleotide at the 3′-end of protospacer RNA results in an important allosteric change in the LdCsm DNase, giving an active enzyme. CTR-bound LdCsm exhibits the full level of substrate binding and DNA cleavage whereas NTR-bound LdCsm closes the substrate-binding pocket, which deactivates the DNase. Finally, multiple Csm3 subunits cleave the target transcripts, and release of target RNA cleavage products restores the binary conformation, completing the spatiotemporal regulation of LdCsm systems.
    Figure Legend Snippet: Model of allosteric activation and repression of the LdCsm DNase. The previous works have proposed that the initial recognition of nascent transcript at the 5′ end of target RNA for type III complex, since both of Csm5 subunit in Csm complex and Cmr1 subunit in Cmr complex are crucial for target RNA binding 36 , 37 , 68 . These suggested that the binary LdCsm effector complex interacts with target transcript initially at the 5′ end of target RNA and further via sequence complementarity between the protospacer and the corresponding crRNA, leading to the formation of a ternary effector complex with a major conformational change. Addition of a single nucleotide at the 3′-end of protospacer RNA results in an important allosteric change in the LdCsm DNase, giving an active enzyme. CTR-bound LdCsm exhibits the full level of substrate binding and DNA cleavage whereas NTR-bound LdCsm closes the substrate-binding pocket, which deactivates the DNase. Finally, multiple Csm3 subunits cleave the target transcripts, and release of target RNA cleavage products restores the binary conformation, completing the spatiotemporal regulation of LdCsm systems.

    Techniques Used: Activation Assay, RNA Binding Assay, Sequencing, Binding Assay

    Biochemical characterization of the LdCsm effector complex. ( a ) Schematic of three different homologous target RNAs: CTR, cognate target RNA carrying 6-nt 3′ anti-tag with mismatch to the 5′ tag of the corresponding crRNA; NTR, noncognate target RNA containing 8-nt 3′ anti-tag that is complementary to the 5′ tag of the corresponding crRNA, and PTR, 40 nt protospacer target RNA completely lacking 3′ anti-tag. ( b ) Analysis of target RNA cleavage by LdCsm. Different target RNAs (50 nM) were individually mixed with 50 nM LdCsm and incubated for 10 min. The resulting samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. ( c ) Analysis of RNA-activated ssDNA cleavage by LdCsm. 50 nM S10-60 ssDNA substrate was mixed with 50 nM LdCsm and 500 nM of each of the target RNA and incubated for 10 min. Samples were analyzed by denaturing PAGE. ( d ) Analysis of cOA synthesis by LdCsm. ~2 nM [α- 32 P]-ATP was mixed with a range of cold ATP (48 nM – 1 mM) and incubated with 50 nM LdCsm in the presence of 500 nM CTR for 120 min, the S. islandicus Cmr-α complex was used as the positive reference.
    Figure Legend Snippet: Biochemical characterization of the LdCsm effector complex. ( a ) Schematic of three different homologous target RNAs: CTR, cognate target RNA carrying 6-nt 3′ anti-tag with mismatch to the 5′ tag of the corresponding crRNA; NTR, noncognate target RNA containing 8-nt 3′ anti-tag that is complementary to the 5′ tag of the corresponding crRNA, and PTR, 40 nt protospacer target RNA completely lacking 3′ anti-tag. ( b ) Analysis of target RNA cleavage by LdCsm. Different target RNAs (50 nM) were individually mixed with 50 nM LdCsm and incubated for 10 min. The resulting samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. ( c ) Analysis of RNA-activated ssDNA cleavage by LdCsm. 50 nM S10-60 ssDNA substrate was mixed with 50 nM LdCsm and 500 nM of each of the target RNA and incubated for 10 min. Samples were analyzed by denaturing PAGE. ( d ) Analysis of cOA synthesis by LdCsm. ~2 nM [α- 32 P]-ATP was mixed with a range of cold ATP (48 nM – 1 mM) and incubated with 50 nM LdCsm in the presence of 500 nM CTR for 120 min, the S. islandicus Cmr-α complex was used as the positive reference.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    8) Product Images from "Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase"

    Article Title: Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase

    Journal: Cell Discovery

    doi: 10.1038/s41421-020-0160-4

    Effect of LdCsm3 mutations on the ssDNA cleavage and binding of LdCsm. a Target RNA cleavage of LdCsm3 mutated derivatives. Fifty nM of S1–46 RNA were incubated with 50 nM of LdCsm or the indicated mutant derivatives for 10 min, and the samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. b RNA-activated ssDNA cleavage by effectors carrying one of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4b . c ssDNA binding by effectors carrying each of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4c . d Relative ssDNA binding between the wild-type LdCsm effector and its LdCsm3 mutated derivatives. The ssDNA activity of LdCsm in non-homologous RNA was used as the standard and set up as 1. Results shown are average of three independent assays; bars represent the mean standard deviation (±SD). The red arrow indicates the ssDNA-LdCsm complex.
    Figure Legend Snippet: Effect of LdCsm3 mutations on the ssDNA cleavage and binding of LdCsm. a Target RNA cleavage of LdCsm3 mutated derivatives. Fifty nM of S1–46 RNA were incubated with 50 nM of LdCsm or the indicated mutant derivatives for 10 min, and the samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. b RNA-activated ssDNA cleavage by effectors carrying one of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4b . c ssDNA binding by effectors carrying each of the constructed LdCsm3 mutants. Reaction conditions were the same as in Fig. 4c . d Relative ssDNA binding between the wild-type LdCsm effector and its LdCsm3 mutated derivatives. The ssDNA activity of LdCsm in non-homologous RNA was used as the standard and set up as 1. Results shown are average of three independent assays; bars represent the mean standard deviation (±SD). The red arrow indicates the ssDNA-LdCsm complex.

    Techniques Used: Binding Assay, Incubation, Mutagenesis, Polyacrylamide Gel Electrophoresis, Construct, Activity Assay, Standard Deviation

    Model of allosteric activation and repression of the LdCsm DNase. The previous works have proposed the initial recognition of nascent transcript at the 5ʹ end of target RNA for type III complex, since both of Csm5 subunit in Csm complex and Cmr1 subunit in Cmr complex are crucial for target RNA binding 36 , 37 , 68 . These suggested that the binary LdCsm effector complex interacts with target transcript initially at the 5ʹ end of target RNA and further via sequence complementarity between the protospacer and the corresponding crRNA, leading to the formation of a ternary effector complex with a major conformational change. Addition of a single nucleotide at the 3ʹ-end of protospacer RNA results in an important allosteric change in the LdCsm DNase, giving an active enzyme. CTR-bound LdCsm exhibits the full level of substrate binding and DNA cleavage, whereas NTR-bound LdCsm closes the substrate-binding pocket, which deactivates the DNase. Finally, multiple Csm3 subunits cleave the target transcripts, and release of target RNA cleavage products restores the binary conformation, completing the spatiotemporal regulation of LdCsm systems.
    Figure Legend Snippet: Model of allosteric activation and repression of the LdCsm DNase. The previous works have proposed the initial recognition of nascent transcript at the 5ʹ end of target RNA for type III complex, since both of Csm5 subunit in Csm complex and Cmr1 subunit in Cmr complex are crucial for target RNA binding 36 , 37 , 68 . These suggested that the binary LdCsm effector complex interacts with target transcript initially at the 5ʹ end of target RNA and further via sequence complementarity between the protospacer and the corresponding crRNA, leading to the formation of a ternary effector complex with a major conformational change. Addition of a single nucleotide at the 3ʹ-end of protospacer RNA results in an important allosteric change in the LdCsm DNase, giving an active enzyme. CTR-bound LdCsm exhibits the full level of substrate binding and DNA cleavage, whereas NTR-bound LdCsm closes the substrate-binding pocket, which deactivates the DNase. Finally, multiple Csm3 subunits cleave the target transcripts, and release of target RNA cleavage products restores the binary conformation, completing the spatiotemporal regulation of LdCsm systems.

    Techniques Used: Activation Assay, RNA Binding Assay, Sequencing, Binding Assay

    Biochemical characterization of the LdCsm effector complex. a Schematic of three different homologous target RNAs: CTR cognate target RNA carrying 6-nt 3ʹ anti-tag with mismatch to the 5ʹ tag of the corresponding crRNA, NTR noncognate target RNA containing 8-nt 3ʹ anti-tag that is complementary to the 5ʹ tag of the corresponding crRNA, PTR 40 nt protospacer target RNA completely lacking 3ʹ anti-tag. b Analysis of target RNA cleavage by LdCsm. Different target RNAs (50 nM) were individually mixed with 50 nM LdCsm and incubated for 10 min. The resulting samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. c Analysis of RNA-activated ssDNA cleavage by LdCsm. 50 nM S10–60 ssDNA substrate was mixed with 50 nM LdCsm and 500 nM of each of the target RNA and incubated for 10 min. Samples were analyzed by denaturing PAGE. d Analysis of cOA synthesis by LdCsm. Approximately 2 nM [α- 32 P]-ATP was mixed with a range of cold ATP (48 nM–1 mM) and incubated with 50 nM LdCsm in the presence of 500 nM CTR for 120 min; the S. islandicus Cmr-α complex was used as the positive reference.
    Figure Legend Snippet: Biochemical characterization of the LdCsm effector complex. a Schematic of three different homologous target RNAs: CTR cognate target RNA carrying 6-nt 3ʹ anti-tag with mismatch to the 5ʹ tag of the corresponding crRNA, NTR noncognate target RNA containing 8-nt 3ʹ anti-tag that is complementary to the 5ʹ tag of the corresponding crRNA, PTR 40 nt protospacer target RNA completely lacking 3ʹ anti-tag. b Analysis of target RNA cleavage by LdCsm. Different target RNAs (50 nM) were individually mixed with 50 nM LdCsm and incubated for 10 min. The resulting samples were analyzed by denaturing PAGE. Duplex: Duplex of crRNA and substrate. c Analysis of RNA-activated ssDNA cleavage by LdCsm. 50 nM S10–60 ssDNA substrate was mixed with 50 nM LdCsm and 500 nM of each of the target RNA and incubated for 10 min. Samples were analyzed by denaturing PAGE. d Analysis of cOA synthesis by LdCsm. Approximately 2 nM [α- 32 P]-ATP was mixed with a range of cold ATP (48 nM–1 mM) and incubated with 50 nM LdCsm in the presence of 500 nM CTR for 120 min; the S. islandicus Cmr-α complex was used as the positive reference.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    Related Articles

    Northern Blot:

    Article Title: Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase
    Article Snippet: .. For northern blotting of crRNA, 100 ng of unlabeled crRNA was mixed with equal volume of 2× RNA loading dye (New England Biolabs) and fractionated in the 12% denaturing polyacrylamide gel. .. Northern blo tting analysis was conducted as described previously , using radio-labeled RNA S1–40 (Supplementary Table ).

    Article Title: Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase
    Article Snippet: .. For northern blotting of crRNA, 100 ng of unlabeled crRNA was mixed with equal volume of 2 × RNA loading dye (New England Biolabs) and fractionated in the 12% denaturing polyacrylamide gel. .. Northern blotting analysis was conducted as described previously , using radiolabeled RNA S1-40 (Supplementary Table S1).

    Clear Native PAGE:

    Article Title: Effect of UV Radiation on Fluorescent RNA Aptamers’ Functional and Templating Ability
    Article Snippet: .. For native PAGE, the annealed RNA was mixed with 6× gel loading dye (NEB) before being loaded onto a 10 % native gel (acrylamide: bisacrylamide (29:1)). .. Gels were run at 325 V for 1 h in 1× TBE buffer and stained by SYBR gold.

    Purification:

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription
    Article Snippet: .. Typically, purified IVT RNA was denatured in 2× RNA loading dye (New England Biolabs) and heated at 70°C for 2 min before analyzing on TBE-Urea polyacrylamide gels. .. SybrGold staining (ThermoFisher Scientific) was performed for RNA visualization and gels were scanned with an Amersham Typhoon Biomolecular Imager (GE Healthcare).

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    UV cross-link analysis of the interaction between SisCsx1 and different <t>RNA</t> oligos. ( A ) SisCsx1 (3 μM) was incubated with increasing concentrations of S 3 A 7 (0, 0.1, 1, 10, 50 μM) at <t>70°C</t> for 10 min and exposed to UV irradiation for 30 min. Then, the samples were resolved by SDS-loading buffer, analyzed by SDS-PAGE and visualized by Coomassie staining. ( B and C ) Labeled indicated RNA oligos ( Supplementary Table S2 ) were incubated with 3 μM SisCsx1 at 70°C for 10 min and exposed to UV irradiation for 30 min. Then the samples were loaded onto a SDS-polyacrylamide gel and analyzed by autoradiography (upper panel) and Coomassie staining (lower panel). The right panels show the relative amount of the indicated RNA oligos bound by SisCsx1. Only the signal from the main band was calculated and the amount of Csx1-associated S 3 A 7 and CA 4 was set as ‘1’ in (B) and (C), respectively.
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    UV cross-link analysis of the interaction between SisCsx1 and different RNA oligos. ( A ) SisCsx1 (3 μM) was incubated with increasing concentrations of S 3 A 7 (0, 0.1, 1, 10, 50 μM) at 70°C for 10 min and exposed to UV irradiation for 30 min. Then, the samples were resolved by SDS-loading buffer, analyzed by SDS-PAGE and visualized by Coomassie staining. ( B and C ) Labeled indicated RNA oligos ( Supplementary Table S2 ) were incubated with 3 μM SisCsx1 at 70°C for 10 min and exposed to UV irradiation for 30 min. Then the samples were loaded onto a SDS-polyacrylamide gel and analyzed by autoradiography (upper panel) and Coomassie staining (lower panel). The right panels show the relative amount of the indicated RNA oligos bound by SisCsx1. Only the signal from the main band was calculated and the amount of Csx1-associated S 3 A 7 and CA 4 was set as ‘1’ in (B) and (C), respectively.

    Journal: Nucleic Acids Research

    Article Title: Allosteric regulation of Csx1, a type IIIB-associated CARF domain ribonuclease by RNAs carrying a tetraadenylate tail

    doi: 10.1093/nar/gkx726

    Figure Lengend Snippet: UV cross-link analysis of the interaction between SisCsx1 and different RNA oligos. ( A ) SisCsx1 (3 μM) was incubated with increasing concentrations of S 3 A 7 (0, 0.1, 1, 10, 50 μM) at 70°C for 10 min and exposed to UV irradiation for 30 min. Then, the samples were resolved by SDS-loading buffer, analyzed by SDS-PAGE and visualized by Coomassie staining. ( B and C ) Labeled indicated RNA oligos ( Supplementary Table S2 ) were incubated with 3 μM SisCsx1 at 70°C for 10 min and exposed to UV irradiation for 30 min. Then the samples were loaded onto a SDS-polyacrylamide gel and analyzed by autoradiography (upper panel) and Coomassie staining (lower panel). The right panels show the relative amount of the indicated RNA oligos bound by SisCsx1. Only the signal from the main band was calculated and the amount of Csx1-associated S 3 A 7 and CA 4 was set as ‘1’ in (B) and (C), respectively.

    Article Snippet: All the reactions were performed at 70°C and stopped at the indicated time point by supplementing 2 × RNA loading dye (New England Biolabs) and cooling on ice.

    Techniques: Incubation, Irradiation, SDS Page, Staining, Labeling, Autoradiography

    E rns is a nicking endoribonuclease. ( a ) Schematic representation of the RNase activity assay with the modified hybrids. A single-stranded RNA of positive polarity (ssRNA+) labeled in red together with either a single-stranded methylated RNA (metRNA) or a ssDNA of negative polarity, both labeled in green, were boiled and cooled down at room temperature for hybridization (RNA/metRNA or RNA/DNA). Strep-tag purified wild-type (C171), monomeric (R171) and RNase-inactive mutant (H30F) of E rns were incubated at the concentrations indicated with 625 nM single-stranded metRNA or DNA ( b ), double-stranded RNA/DNA− ( c ) or double-stranded RNA/metRNA-hybrids ( d ). Samples were separated by 14% SDS-PAGE and fluorescence was analysed with a Li-Cor Odyssey system. Due to the known, defined length of the directly labeled fragments (30 b for ssRNA, 30 bp for dsRNA), no size ladder was applied. Non-cropped gels as representative experiment out of three ( b ) or four ( c , d ) are shown.

    Journal: Scientific Reports

    Article Title: Homodimerisation-independent cleavage of dsRNA by a pestiviral nicking endoribonuclease

    doi: 10.1038/s41598-018-26557-4

    Figure Lengend Snippet: E rns is a nicking endoribonuclease. ( a ) Schematic representation of the RNase activity assay with the modified hybrids. A single-stranded RNA of positive polarity (ssRNA+) labeled in red together with either a single-stranded methylated RNA (metRNA) or a ssDNA of negative polarity, both labeled in green, were boiled and cooled down at room temperature for hybridization (RNA/metRNA or RNA/DNA). Strep-tag purified wild-type (C171), monomeric (R171) and RNase-inactive mutant (H30F) of E rns were incubated at the concentrations indicated with 625 nM single-stranded metRNA or DNA ( b ), double-stranded RNA/DNA− ( c ) or double-stranded RNA/metRNA-hybrids ( d ). Samples were separated by 14% SDS-PAGE and fluorescence was analysed with a Li-Cor Odyssey system. Due to the known, defined length of the directly labeled fragments (30 b for ssRNA, 30 bp for dsRNA), no size ladder was applied. Non-cropped gels as representative experiment out of three ( b ) or four ( c , d ) are shown.

    Article Snippet: GoTaq DNA Polymerase, BenchTop 100 bp DNA ladder and RNasin were from Promega, whereas the RNA loading dye was obtained from New England Biolabs (Ipswich, MA).

    Techniques: Activity Assay, Modification, Labeling, Methylation, Hybridization, Strep-tag, Purification, Mutagenesis, Incubation, SDS Page, Fluorescence

    3′-extended RNA by-products are formed during IVT reactions. ( A ) dsRNA immunoblot assay using a dsRNA-specific antibody (J2) on three different mRNA sequences (CLuc, RFP, and GFP). Poly(I:C) and RNase III treatment is used as a control to detect and validate dsRNA, respectively. For the immunoblots, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses, 0.05 µg of RNA was loaded in each well. ( B , C ) IVT reactions using short templates (30 bp) run on denaturing gels under standard conditions (four rNTPs), and under conditions where one rNTP was eliminated to prevent formation of the dsRNA products (−rUTP for B and −rCTP for C ). RNase III digestion control to confirm the dsRNA nature of the extended by-product. Chemically synthesized RNA was used as a control. ( D , E ) Intact mass spectrometry of IVT products when all four rNTPs are present (+rUTP for D and +rCTP for E ), and when one rNTP is eliminated to prevent formation of the dsRNA products (−rUTP for D and −rCTP for E ). Expected mass of extended products is calculated based on average rNTP values.

    Journal: RNA

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    doi: 10.1261/rna.073858.119

    Figure Lengend Snippet: 3′-extended RNA by-products are formed during IVT reactions. ( A ) dsRNA immunoblot assay using a dsRNA-specific antibody (J2) on three different mRNA sequences (CLuc, RFP, and GFP). Poly(I:C) and RNase III treatment is used as a control to detect and validate dsRNA, respectively. For the immunoblots, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses, 0.05 µg of RNA was loaded in each well. ( B , C ) IVT reactions using short templates (30 bp) run on denaturing gels under standard conditions (four rNTPs), and under conditions where one rNTP was eliminated to prevent formation of the dsRNA products (−rUTP for B and −rCTP for C ). RNase III digestion control to confirm the dsRNA nature of the extended by-product. Chemically synthesized RNA was used as a control. ( D , E ) Intact mass spectrometry of IVT products when all four rNTPs are present (+rUTP for D and +rCTP for E ), and when one rNTP is eliminated to prevent formation of the dsRNA products (−rUTP for D and −rCTP for E ). Expected mass of extended products is calculated based on average rNTP values.

    Article Snippet: Typically, purified IVT RNA was denatured in 2× RNA loading dye (New England Biolabs) and heated at 70°C for 2 min before analyzing on TBE-Urea polyacrylamide gels.

    Techniques: Western Blot, Nucleic Acid Electrophoresis, Synthesized, Mass Spectrometry

    High-temperature IVT with TsT7-1 leads to a reduction in 3′-extended by-products. ( A ) Denaturing gel analysis of IVT products synthesized from short IVT template-1 using TsT7-1 performed at a temperature range of 37°C to 55°C. Chemically synthesized short IVT template 1 RNA was used as a control. Equal amounts were loaded in each lane. ( B ) Intact mass spectrometry analysis of Template IVT short-1 and Template IVT short-2 using TsT7-1 under standard conditions (all four rNTPs; +rUTP or rCTP) or lacking one rNTP (−rUTP or −rCTP) performed at either 37°C or 50°C. Expected run-off transcript (including n + 1 and n + 2 products) is highlighted in orange, and products longer than the run-off transcript are highlighted in gray. ( C ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized with TsT7-1 or wild-type RNAP at a temperature range from 37°C to 55°C. For the immunoblots with TsT7-1, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses with TsT7-1, 0.05 µg of RNA was loaded in each well. ( D ) Intact mass spectrometry analysis of IVT reactions in the presence or absence of TsT7-1 on chemically synthesized RNA ( n , n + 1, n + 2) performed at either 37°C or 50°C. Products longer than the run-off transcript are highlighted in gray.

    Journal: RNA

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    doi: 10.1261/rna.073858.119

    Figure Lengend Snippet: High-temperature IVT with TsT7-1 leads to a reduction in 3′-extended by-products. ( A ) Denaturing gel analysis of IVT products synthesized from short IVT template-1 using TsT7-1 performed at a temperature range of 37°C to 55°C. Chemically synthesized short IVT template 1 RNA was used as a control. Equal amounts were loaded in each lane. ( B ) Intact mass spectrometry analysis of Template IVT short-1 and Template IVT short-2 using TsT7-1 under standard conditions (all four rNTPs; +rUTP or rCTP) or lacking one rNTP (−rUTP or −rCTP) performed at either 37°C or 50°C. Expected run-off transcript (including n + 1 and n + 2 products) is highlighted in orange, and products longer than the run-off transcript are highlighted in gray. ( C ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized with TsT7-1 or wild-type RNAP at a temperature range from 37°C to 55°C. For the immunoblots with TsT7-1, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses with TsT7-1, 0.05 µg of RNA was loaded in each well. ( D ) Intact mass spectrometry analysis of IVT reactions in the presence or absence of TsT7-1 on chemically synthesized RNA ( n , n + 1, n + 2) performed at either 37°C or 50°C. Products longer than the run-off transcript are highlighted in gray.

    Article Snippet: Typically, purified IVT RNA was denatured in 2× RNA loading dye (New England Biolabs) and heated at 70°C for 2 min before analyzing on TBE-Urea polyacrylamide gels.

    Techniques: Synthesized, Mass Spectrometry, Nucleic Acid Electrophoresis, Western Blot

    Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.

    Journal: RNA

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    doi: 10.1261/rna.073858.119

    Figure Lengend Snippet: Template-encoded poly(A) tailing reduces antisense by-product formation. ( A ) dsRNA immunoblot with J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from CLuc templates with varying length (30, 60, 120 bp) of poly(T) sequence at 3′ end under standard conditions. ( B ) Immunoblot and native gel electrophoresis analysis of IVT reactions on 512B::CLuc chimeric template with poly(T) (60 and 120 bp) sequence at the 3′ end. IVT reactions were performed at 37°C or 50°C.

    Article Snippet: Typically, purified IVT RNA was denatured in 2× RNA loading dye (New England Biolabs) and heated at 70°C for 2 min before analyzing on TBE-Urea polyacrylamide gels.

    Techniques: Nucleic Acid Electrophoresis, Synthesized, Sequencing

    Thermostable (Ts) RNAPs are active at high temperatures and do not affect 3′-extended RNA by-product formation. ( A ) Melting temperatures of wild-type T7 RNAP (orange), TsT7-1 (blue), and TsT7-2 (red) determined by nano-differential scanning fluorimetry. ( B ) Molecular beacon assay for the efficiency of IVT with TsT7-1 or TsT7-2 compared to wild-type RNAP at temperatures ranging from 37°C to 62°C. ( C ) Gel electrophoreses analyses of CLuc RNA synthesized with either wild-type RNAP, TsT7-1, or TsT7-2 RNAPs at temperatures ranging from 37°C to 62°C. Equal volume of IVT reactions were loaded in each well to reflect the differences in RNA yield at each temperature. ( D ) dsRNA immunoblot using J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from IVT reactions performed at 37°C using wild-type T7, TsT7-1, and TsT7-2 RNAPs. For the immunoblots, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses, 0.05 µg of RNA was loaded in each well. ( E ) Denaturing gel analysis of IVT reactions using Template IVT short-1 with Ts RNAPs compared to wild-type RNAP. Reactions were performed with either four NTPs (+rUTP) or with three NTPs (−rUTP). ( F ) Intact mass spectrometry of short IVT products from TsT7-1 under standard conditions (37°C) with products longer than the run-off transcript highlighted in gray and the expected run-off transcripts in orange (including n + 1, n + 2 products from nontemplated additions).

    Journal: RNA

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    doi: 10.1261/rna.073858.119

    Figure Lengend Snippet: Thermostable (Ts) RNAPs are active at high temperatures and do not affect 3′-extended RNA by-product formation. ( A ) Melting temperatures of wild-type T7 RNAP (orange), TsT7-1 (blue), and TsT7-2 (red) determined by nano-differential scanning fluorimetry. ( B ) Molecular beacon assay for the efficiency of IVT with TsT7-1 or TsT7-2 compared to wild-type RNAP at temperatures ranging from 37°C to 62°C. ( C ) Gel electrophoreses analyses of CLuc RNA synthesized with either wild-type RNAP, TsT7-1, or TsT7-2 RNAPs at temperatures ranging from 37°C to 62°C. Equal volume of IVT reactions were loaded in each well to reflect the differences in RNA yield at each temperature. ( D ) dsRNA immunoblot using J2 antibody and gel electrophoresis analysis of CLuc RNA synthesized from IVT reactions performed at 37°C using wild-type T7, TsT7-1, and TsT7-2 RNAPs. For the immunoblots, 1.0 µg of RNA was loaded for each mRNA. For the gel electrophoresis analyses, 0.05 µg of RNA was loaded in each well. ( E ) Denaturing gel analysis of IVT reactions using Template IVT short-1 with Ts RNAPs compared to wild-type RNAP. Reactions were performed with either four NTPs (+rUTP) or with three NTPs (−rUTP). ( F ) Intact mass spectrometry of short IVT products from TsT7-1 under standard conditions (37°C) with products longer than the run-off transcript highlighted in gray and the expected run-off transcripts in orange (including n + 1, n + 2 products from nontemplated additions).

    Article Snippet: Typically, purified IVT RNA was denatured in 2× RNA loading dye (New England Biolabs) and heated at 70°C for 2 min before analyzing on TBE-Urea polyacrylamide gels.

    Techniques: Nano Differential Scanning Fluorimetry, Synthesized, Nucleic Acid Electrophoresis, Western Blot, Mass Spectrometry

    mRNAs generated by high-temperature IVT with TsT7-1 are functional and have reduced immunogenicity in vivo. ( A ) Luciferase activity from HEK293 cells transfected with CLuc mRNA (unmodified or modified with pseudouridine) synthesized with wild-type T7 or TsT7-1 RNAP at 37°C or 50°C. ( B ) Representative chromatogram for CLuc mRNA separated on an HPLC column. Absorbance at 260 nm representing the RNA products in the IVT reaction. dsRNA immunoblot with J2 antibody on fractions collected from the HPLC purification. ( C ) IFN-α levels in supernatant collected from DCs transfected with either crude IVT CLuc mRNA or CLuc mRNA purified with HPLC. IFN-α levels in the supernatant were measured 24 h post-transfection. Error bars are standard error of the mean. Poly(I:C), a synthetic analog of dsRNA and Resiquimod (R848), an activator of Toll-like receptors were used as controls for interferon activation.

    Journal: RNA

    Article Title: Synthesis of low immunogenicity RNA with high-temperature in vitro transcription

    doi: 10.1261/rna.073858.119

    Figure Lengend Snippet: mRNAs generated by high-temperature IVT with TsT7-1 are functional and have reduced immunogenicity in vivo. ( A ) Luciferase activity from HEK293 cells transfected with CLuc mRNA (unmodified or modified with pseudouridine) synthesized with wild-type T7 or TsT7-1 RNAP at 37°C or 50°C. ( B ) Representative chromatogram for CLuc mRNA separated on an HPLC column. Absorbance at 260 nm representing the RNA products in the IVT reaction. dsRNA immunoblot with J2 antibody on fractions collected from the HPLC purification. ( C ) IFN-α levels in supernatant collected from DCs transfected with either crude IVT CLuc mRNA or CLuc mRNA purified with HPLC. IFN-α levels in the supernatant were measured 24 h post-transfection. Error bars are standard error of the mean. Poly(I:C), a synthetic analog of dsRNA and Resiquimod (R848), an activator of Toll-like receptors were used as controls for interferon activation.

    Article Snippet: Typically, purified IVT RNA was denatured in 2× RNA loading dye (New England Biolabs) and heated at 70°C for 2 min before analyzing on TBE-Urea polyacrylamide gels.

    Techniques: Generated, Functional Assay, In Vivo, Luciferase, Activity Assay, Transfection, Modification, Synthesized, High Performance Liquid Chromatography, Purification, Activation Assay