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    Name:
    E coli Poly A Polymerase
    Description:
    E coli Poly A Polymerase 500 units
    Catalog Number:
    m0276l
    Price:
    292
    Size:
    500 units
    Category:
    DNA Polymerases
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    Structured Review

    New England Biolabs mirnas
    E coli Poly A Polymerase
    E coli Poly A Polymerase 500 units
    https://www.bioz.com/result/mirnas/product/New England Biolabs
    Average 93 stars, based on 328 article reviews
    Price from $9.99 to $1999.99
    mirnas - by Bioz Stars, 2020-10
    93/100 stars

    Images

    1) Product Images from "Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions"

    Article Title: Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211739

    Relationship between target mRNAs of XmiRs and generation of ΔXmiRs mice. (A) A dendrogram of hierarchical clustering analysis of target mRNAs of XmiRs and their neighboring miRNAs. (B) Venn diagram showing the relationship among putative target mRNAs of miR-741-3p, miR-871-3p, and miR-880-3p. Corresponding gene lists are shown in S3 Table . (C) A schematic presentation of the WT and ΔXmiRs locus. gR-741 and gR-871 represent positions of guide RNAs used for genome editing. (D) Representative PCR for genotyping of WT and ΔXmiRs (OT84) mice. Arrows in the right panel represent primers used for PCR. (E) Expression of XmiRs in WT and a ΔXmiRs testes (F2 of OT84) determined with semi-quantitative RT-PCR analysis. U6 snRNA was used as an internal control. (F) HE-stained sections of seminiferous tubules in WT (left) and ΔXmiR (F2 of OT100) (right) testes at 8, 12, 16, and 30 weeks of age. The second and fourth panels for 30 weeks show higher magnification views corresponding to the rectangular area in the first and third panels. Lower two panels show mildly affected seminiferous tubules. Arrowheads show abnormal seminiferous tubules. Scale bar = 50 μm (8, 12, 16 weeks), 200 μm (30 weeks, lower magnification), 100 μm (30 weeks, higher magnification).
    Figure Legend Snippet: Relationship between target mRNAs of XmiRs and generation of ΔXmiRs mice. (A) A dendrogram of hierarchical clustering analysis of target mRNAs of XmiRs and their neighboring miRNAs. (B) Venn diagram showing the relationship among putative target mRNAs of miR-741-3p, miR-871-3p, and miR-880-3p. Corresponding gene lists are shown in S3 Table . (C) A schematic presentation of the WT and ΔXmiRs locus. gR-741 and gR-871 represent positions of guide RNAs used for genome editing. (D) Representative PCR for genotyping of WT and ΔXmiRs (OT84) mice. Arrows in the right panel represent primers used for PCR. (E) Expression of XmiRs in WT and a ΔXmiRs testes (F2 of OT84) determined with semi-quantitative RT-PCR analysis. U6 snRNA was used as an internal control. (F) HE-stained sections of seminiferous tubules in WT (left) and ΔXmiR (F2 of OT100) (right) testes at 8, 12, 16, and 30 weeks of age. The second and fourth panels for 30 weeks show higher magnification views corresponding to the rectangular area in the first and third panels. Lower two panels show mildly affected seminiferous tubules. Arrowheads show abnormal seminiferous tubules. Scale bar = 50 μm (8, 12, 16 weeks), 200 μm (30 weeks, lower magnification), 100 μm (30 weeks, higher magnification).

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Staining

    The expression profile of miRNAs in various tissues and cell lines. (A) A heat map of hierarchical clustering of miRNAs detected in small RNA-seq data used in this study. (B) A heat map of 20 miRNAs highly expressed in PGCs. Relative miRNA expression is described according to the color scale. Red and green indicate high and low expression, respectively. Mouse embryonic fibroblasts (MEFs), embryonic stem (ES) cells, primordial germ cells (PGCs), spermatogonia (SPG), spermatozoa (SPZ). (C) The locus of XmiR genes on the X chromosome. (D) The expression of XmiRs in testes, ES cells, and MEFs determined by quantitative RT-PCR. Each expression level was normalized to the expression of U6 snRNA. The expression in ES cells was set as 1.0. Error bars show standard errors of three biological replicates. **P
    Figure Legend Snippet: The expression profile of miRNAs in various tissues and cell lines. (A) A heat map of hierarchical clustering of miRNAs detected in small RNA-seq data used in this study. (B) A heat map of 20 miRNAs highly expressed in PGCs. Relative miRNA expression is described according to the color scale. Red and green indicate high and low expression, respectively. Mouse embryonic fibroblasts (MEFs), embryonic stem (ES) cells, primordial germ cells (PGCs), spermatogonia (SPG), spermatozoa (SPZ). (C) The locus of XmiR genes on the X chromosome. (D) The expression of XmiRs in testes, ES cells, and MEFs determined by quantitative RT-PCR. Each expression level was normalized to the expression of U6 snRNA. The expression in ES cells was set as 1.0. Error bars show standard errors of three biological replicates. **P

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR

    2) Product Images from "An origin of the immunogenicity of in vitro transcribed RNA"

    Article Title: An origin of the immunogenicity of in vitro transcribed RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky177

    The dsRNA byproduct is formed by sense and antisense RNAs generated in promoter-dependent and -independent manners, respectively. (A and B) Transcriptional start sites and end sites for the intended 512B product ( A ) and its complementary RNA byproduct (c512B) ( B ), as examined by 5′- and 3′-RACE. Transcriptional start and end sites are shown upstream of the poly A tail in the 5′- and 3′-RACE sequences, respectively. Cyan underscores in the sequence chromatograms indicate sequences matching those in the template. The location of the matching sequence in the template is shown in the schematic on the right. The red box in (B) indicates the reverse complement sequence of the T7 promoter. ( C ) Schematic illustrating the results in (A and B). Transcription using a template with a single T7 promoter results in the production of both sense and antisense transcripts, which differ in length by the size of the T7 promoter. Solid and dotted lines indicate DNA and RNA, respectively. ( D ) Native PAGE analysis of T7 transcripts generated using DNA template with a single T7 promoter (1), DNA template without the T7 promoter (2), and gel-purified 512B ssRNA as a template (3). RNA template alone (4) was compared with (3). ( E ) Native PAGE analysis of T7 transcripts generated using fully duplexed DNA template (1), template strand ssDNA alone (512B-3end in Supplementary Table S1 ) (2), and partially duplexed DNA template (generated by annealing 512B-3end with T7promoter_top_strand, Supplementary Table S1 ) (3). * indicates unknown ssRNA byproduct from transcription.
    Figure Legend Snippet: The dsRNA byproduct is formed by sense and antisense RNAs generated in promoter-dependent and -independent manners, respectively. (A and B) Transcriptional start sites and end sites for the intended 512B product ( A ) and its complementary RNA byproduct (c512B) ( B ), as examined by 5′- and 3′-RACE. Transcriptional start and end sites are shown upstream of the poly A tail in the 5′- and 3′-RACE sequences, respectively. Cyan underscores in the sequence chromatograms indicate sequences matching those in the template. The location of the matching sequence in the template is shown in the schematic on the right. The red box in (B) indicates the reverse complement sequence of the T7 promoter. ( C ) Schematic illustrating the results in (A and B). Transcription using a template with a single T7 promoter results in the production of both sense and antisense transcripts, which differ in length by the size of the T7 promoter. Solid and dotted lines indicate DNA and RNA, respectively. ( D ) Native PAGE analysis of T7 transcripts generated using DNA template with a single T7 promoter (1), DNA template without the T7 promoter (2), and gel-purified 512B ssRNA as a template (3). RNA template alone (4) was compared with (3). ( E ) Native PAGE analysis of T7 transcripts generated using fully duplexed DNA template (1), template strand ssDNA alone (512B-3end in Supplementary Table S1 ) (2), and partially duplexed DNA template (generated by annealing 512B-3end with T7promoter_top_strand, Supplementary Table S1 ) (3). * indicates unknown ssRNA byproduct from transcription.

    Techniques Used: Generated, Sequencing, Clear Native PAGE, Purification

    3) Product Images from "RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation †Electronic supplementary information (ESI) available: Fig. S1–S7. See DOI: 10.1039/c5sc03946k"

    Article Title: RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation †Electronic supplementary information (ESI) available: Fig. S1–S7. See DOI: 10.1039/c5sc03946k

    Journal: Chemical Science

    doi: 10.1039/c5sc03946k

    Dependence of BiFC fluorescence enhancement on target RNA expression in E. coli . (A) Time course of the difference in mean fluorescence between cells expressing RNA REX(A) – TERC(Q) and REX(A) – TERC(Qm) in the presence and absence of RNA induction by ATc. (B) RNA level in cells before and after induction with ATc. Data are given in mean ± SD of three experiments. IPTG was added at 0 h with or without ATc. Representative distribution of eGFP fluorescence of corresponding cells is given in Fig. S4B. †
    Figure Legend Snippet: Dependence of BiFC fluorescence enhancement on target RNA expression in E. coli . (A) Time course of the difference in mean fluorescence between cells expressing RNA REX(A) – TERC(Q) and REX(A) – TERC(Qm) in the presence and absence of RNA induction by ATc. (B) RNA level in cells before and after induction with ATc. Data are given in mean ± SD of three experiments. IPTG was added at 0 h with or without ATc. Representative distribution of eGFP fluorescence of corresponding cells is given in Fig. S4B. †

    Techniques Used: Bimolecular Fluorescence Complementation Assay, Fluorescence, RNA Expression, Expressing

    Pull-down of G-quadruplex-recognizing proteins by RNA from E. coli . Cells were lysed in the presence of RNase inhibitor or RNase A. Probe proteins associated with the indicated target RNAs were pulled down with an immobilized DNA oligomer complementary to the 5′ end of the RNA and detected by an antibody against eGFP. “NS” indicates a non-specifically stained band that can serve as a loading control.
    Figure Legend Snippet: Pull-down of G-quadruplex-recognizing proteins by RNA from E. coli . Cells were lysed in the presence of RNase inhibitor or RNase A. Probe proteins associated with the indicated target RNAs were pulled down with an immobilized DNA oligomer complementary to the 5′ end of the RNA and detected by an antibody against eGFP. “NS” indicates a non-specifically stained band that can serve as a loading control.

    Techniques Used: Staining

    Dependence of BiFC fluorescence on the number of G 3 tracts in the RNA target expressed in E. coli . Distribution of (A) eGFP fluorescence or (B) forward scattering intensity or (C) relative RNA expression (mean ± SD of three experiments) was assayed and processed as in Fig. 4 .
    Figure Legend Snippet: Dependence of BiFC fluorescence on the number of G 3 tracts in the RNA target expressed in E. coli . Distribution of (A) eGFP fluorescence or (B) forward scattering intensity or (C) relative RNA expression (mean ± SD of three experiments) was assayed and processed as in Fig. 4 .

    Techniques Used: Bimolecular Fluorescence Complementation Assay, Fluorescence, RNA Expression

    Detection of BiFC fluorescence in E. coli cells by flow cytometry. Distribution of (A–C) eGFP fluorescence and (D–F) forward scattering intensity was collected from cells expressing G N -REX(A) and RHAU(Q)-G C probe proteins plus the indicated RNA. (G–I) Relative RNA expression (mean ± SD of three experiments) assayed by qPCR.
    Figure Legend Snippet: Detection of BiFC fluorescence in E. coli cells by flow cytometry. Distribution of (A–C) eGFP fluorescence and (D–F) forward scattering intensity was collected from cells expressing G N -REX(A) and RHAU(Q)-G C probe proteins plus the indicated RNA. (G–I) Relative RNA expression (mean ± SD of three experiments) assayed by qPCR.

    Techniques Used: Bimolecular Fluorescence Complementation Assay, Fluorescence, Flow Cytometry, Cytometry, Expressing, RNA Expression, Real-time Polymerase Chain Reaction

    4) Product Images from "Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions"

    Article Title: Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211739

    Relationship between target mRNAs of XmiRs and generation of ΔXmiRs mice. (A) A dendrogram of hierarchical clustering analysis of target mRNAs of XmiRs and their neighboring miRNAs. (B) Venn diagram showing the relationship among putative target mRNAs of miR-741-3p, miR-871-3p, and miR-880-3p. Corresponding gene lists are shown in S3 Table . (C) A schematic presentation of the WT and ΔXmiRs locus. gR-741 and gR-871 represent positions of guide RNAs used for genome editing. (D) Representative PCR for genotyping of WT and ΔXmiRs (OT84) mice. Arrows in the right panel represent primers used for PCR. (E) Expression of XmiRs in WT and a ΔXmiRs testes (F2 of OT84) determined with semi-quantitative RT-PCR analysis. U6 snRNA was used as an internal control. (F) HE-stained sections of seminiferous tubules in WT (left) and ΔXmiR (F2 of OT100) (right) testes at 8, 12, 16, and 30 weeks of age. The second and fourth panels for 30 weeks show higher magnification views corresponding to the rectangular area in the first and third panels. Lower two panels show mildly affected seminiferous tubules. Arrowheads show abnormal seminiferous tubules. Scale bar = 50 μm (8, 12, 16 weeks), 200 μm (30 weeks, lower magnification), 100 μm (30 weeks, higher magnification).
    Figure Legend Snippet: Relationship between target mRNAs of XmiRs and generation of ΔXmiRs mice. (A) A dendrogram of hierarchical clustering analysis of target mRNAs of XmiRs and their neighboring miRNAs. (B) Venn diagram showing the relationship among putative target mRNAs of miR-741-3p, miR-871-3p, and miR-880-3p. Corresponding gene lists are shown in S3 Table . (C) A schematic presentation of the WT and ΔXmiRs locus. gR-741 and gR-871 represent positions of guide RNAs used for genome editing. (D) Representative PCR for genotyping of WT and ΔXmiRs (OT84) mice. Arrows in the right panel represent primers used for PCR. (E) Expression of XmiRs in WT and a ΔXmiRs testes (F2 of OT84) determined with semi-quantitative RT-PCR analysis. U6 snRNA was used as an internal control. (F) HE-stained sections of seminiferous tubules in WT (left) and ΔXmiR (F2 of OT100) (right) testes at 8, 12, 16, and 30 weeks of age. The second and fourth panels for 30 weeks show higher magnification views corresponding to the rectangular area in the first and third panels. Lower two panels show mildly affected seminiferous tubules. Arrowheads show abnormal seminiferous tubules. Scale bar = 50 μm (8, 12, 16 weeks), 200 μm (30 weeks, lower magnification), 100 μm (30 weeks, higher magnification).

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Staining

    The expression profile of miRNAs in various tissues and cell lines. (A) A heat map of hierarchical clustering of miRNAs detected in small RNA-seq data used in this study. (B) A heat map of 20 miRNAs highly expressed in PGCs. Relative miRNA expression is described according to the color scale. Red and green indicate high and low expression, respectively. Mouse embryonic fibroblasts (MEFs), embryonic stem (ES) cells, primordial germ cells (PGCs), spermatogonia (SPG), spermatozoa (SPZ). (C) The locus of XmiR genes on the X chromosome. (D) The expression of XmiRs in testes, ES cells, and MEFs determined by quantitative RT-PCR. Each expression level was normalized to the expression of U6 snRNA. The expression in ES cells was set as 1.0. Error bars show standard errors of three biological replicates. **P
    Figure Legend Snippet: The expression profile of miRNAs in various tissues and cell lines. (A) A heat map of hierarchical clustering of miRNAs detected in small RNA-seq data used in this study. (B) A heat map of 20 miRNAs highly expressed in PGCs. Relative miRNA expression is described according to the color scale. Red and green indicate high and low expression, respectively. Mouse embryonic fibroblasts (MEFs), embryonic stem (ES) cells, primordial germ cells (PGCs), spermatogonia (SPG), spermatozoa (SPZ). (C) The locus of XmiR genes on the X chromosome. (D) The expression of XmiRs in testes, ES cells, and MEFs determined by quantitative RT-PCR. Each expression level was normalized to the expression of U6 snRNA. The expression in ES cells was set as 1.0. Error bars show standard errors of three biological replicates. **P

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR

    5) Product Images from "A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation"

    Article Title: A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.011132

    QL47 inhibits eukaryotic but not prokaryotic protein synthesis. A , E. coli cells carrying the pUA66- rrnB plasmid that constitutively expresses GFP ( 24 ) were treated with DMSO, 250 μg/ml G418, or 50 μ m QL47. The intracellular GFP fluorescence signal was then measured continuously for 14 h at 37 °C. The signal obtained from growth medium was subtracted, and data are presented as means ± S.D. of 12 experimental replicates. One representative experiment is shown from two independent experiments. B , analysis of in vitro translation assays performed in rabbit reticulocyte lysates, yeast cell lysates, or a reconstituted E. coli cell-free synthesis system (PURExpress®). Translation in rabbit reticulocyte lysates was performed in the presence of DMSO, 30 μg/ml CHX, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed reporter DV subgenomic RNA was used as a template, and the luciferase signal was measured after 90-min incubation at 30 °C. Data are presented as means normalized to DMSO ± S.D. of four experimental replicates. Translation in yeast cell lysates was performed in the presence of DMSO, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed vesicular stomatitis virus (VSV) RNA bearing a luciferase reporter gene ( 44 ) was used as a template, and the luciferase signal was measured after 2-h incubation at 25 °C. Data are presented as means normalized to DMSO ± S.D. of three experimental replicates. Translation in a reconstituted E. coli cell-free synthesis system (PURExpress®) was performed in the presence of DMSO, 250 μg/ml G418, 100 μ m QL47, or 100 μ m compound 14. A plasmid expressing GFP under control of a T7 promoter was used as a template. After 1-h incubation at 37 °C, the total protein content was analyzed by Western blotting. The reporter protein was detected using a GFP antibody, and its abundance was normalized to the loading control (histidine tag). Data are presented as means normalized to DMSO ± S.D. of two technical replicates. One representative experiment is shown from four (rabbit reticulocyte lysates) or two (yeast cell lysates and E. coli cell-free synthesis system) independent experiments. Asterisks indicate that the differences between experimental samples and the DMSO-treated control samples are statistically significant when compared using unpaired t test: ***, p
    Figure Legend Snippet: QL47 inhibits eukaryotic but not prokaryotic protein synthesis. A , E. coli cells carrying the pUA66- rrnB plasmid that constitutively expresses GFP ( 24 ) were treated with DMSO, 250 μg/ml G418, or 50 μ m QL47. The intracellular GFP fluorescence signal was then measured continuously for 14 h at 37 °C. The signal obtained from growth medium was subtracted, and data are presented as means ± S.D. of 12 experimental replicates. One representative experiment is shown from two independent experiments. B , analysis of in vitro translation assays performed in rabbit reticulocyte lysates, yeast cell lysates, or a reconstituted E. coli cell-free synthesis system (PURExpress®). Translation in rabbit reticulocyte lysates was performed in the presence of DMSO, 30 μg/ml CHX, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed reporter DV subgenomic RNA was used as a template, and the luciferase signal was measured after 90-min incubation at 30 °C. Data are presented as means normalized to DMSO ± S.D. of four experimental replicates. Translation in yeast cell lysates was performed in the presence of DMSO, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed vesicular stomatitis virus (VSV) RNA bearing a luciferase reporter gene ( 44 ) was used as a template, and the luciferase signal was measured after 2-h incubation at 25 °C. Data are presented as means normalized to DMSO ± S.D. of three experimental replicates. Translation in a reconstituted E. coli cell-free synthesis system (PURExpress®) was performed in the presence of DMSO, 250 μg/ml G418, 100 μ m QL47, or 100 μ m compound 14. A plasmid expressing GFP under control of a T7 promoter was used as a template. After 1-h incubation at 37 °C, the total protein content was analyzed by Western blotting. The reporter protein was detected using a GFP antibody, and its abundance was normalized to the loading control (histidine tag). Data are presented as means normalized to DMSO ± S.D. of two technical replicates. One representative experiment is shown from four (rabbit reticulocyte lysates) or two (yeast cell lysates and E. coli cell-free synthesis system) independent experiments. Asterisks indicate that the differences between experimental samples and the DMSO-treated control samples are statistically significant when compared using unpaired t test: ***, p

    Techniques Used: Plasmid Preparation, Fluorescence, In Vitro, Luciferase, Incubation, Expressing, Western Blot

    6) Product Images from "A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation"

    Article Title: A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.011132

    QL47 inhibits protein synthesis in vitro . A , SDS-PAGE analysis of in vitro translations performed in rabbit reticulocyte lysates for 90 min at 30 °C in the presence of precharged FluoroTect TM lysine tRNA and DMSO, 40 μ m QL47, or 30 μg/ml CHX. An in vitro transcribed reporter RNA bearing the EMCV IRES and a luciferase ( Luc ) reporter gene ( 42 ) was used as a template. One representative experiment is shown from three independent experiments. The abundance of neosynthesized fluorescent proteins was measured and is graphically presented on the right . Data are presented as means normalized to DMSO ± S.D. of two independent experiments. B , rabbit reticulocyte lysates were pretreated with DMSO or 25 μ m proteasome inhibitor lactacystin for 15 min at room temperature. Subsequently, an in vitro transcribed reporter DV subgenomic RNA ( 40 ) was added, and lysates were incubated in the presence of DMSO or 40 μ m of the indicated small molecules for 90 min at 30 °C. The luciferase signal was measured, and data are presented as means ± S.D. of two technical replicates. One representative experiment is shown from two independent experiments. RLU , relative light units. C , translation of in vitro transcribed reporter DV subgenomic RNA in rabbit reticulocyte lysates was performed for 90 min at 30 °C in the presence of DMSO, 30 μg/ml CHX, or 40 μ m of QL47 and the indicated analogs. The luciferase signal was measured, and data are presented as means ± S.D. of two technical replicates. One representative experiment is shown from three independent experiments.
    Figure Legend Snippet: QL47 inhibits protein synthesis in vitro . A , SDS-PAGE analysis of in vitro translations performed in rabbit reticulocyte lysates for 90 min at 30 °C in the presence of precharged FluoroTect TM lysine tRNA and DMSO, 40 μ m QL47, or 30 μg/ml CHX. An in vitro transcribed reporter RNA bearing the EMCV IRES and a luciferase ( Luc ) reporter gene ( 42 ) was used as a template. One representative experiment is shown from three independent experiments. The abundance of neosynthesized fluorescent proteins was measured and is graphically presented on the right . Data are presented as means normalized to DMSO ± S.D. of two independent experiments. B , rabbit reticulocyte lysates were pretreated with DMSO or 25 μ m proteasome inhibitor lactacystin for 15 min at room temperature. Subsequently, an in vitro transcribed reporter DV subgenomic RNA ( 40 ) was added, and lysates were incubated in the presence of DMSO or 40 μ m of the indicated small molecules for 90 min at 30 °C. The luciferase signal was measured, and data are presented as means ± S.D. of two technical replicates. One representative experiment is shown from two independent experiments. RLU , relative light units. C , translation of in vitro transcribed reporter DV subgenomic RNA in rabbit reticulocyte lysates was performed for 90 min at 30 °C in the presence of DMSO, 30 μg/ml CHX, or 40 μ m of QL47 and the indicated analogs. The luciferase signal was measured, and data are presented as means ± S.D. of two technical replicates. One representative experiment is shown from three independent experiments.

    Techniques Used: In Vitro, SDS Page, Luciferase, Incubation

    QL47 inhibits protein synthesis in live cells. A , chemical structures of QL47 and the derivatives YKL-04-085, QL47R, and YKL-03-109 (compound 14) ( 6 , 9 ). QL47R is an inactive analog due to replacement of the cysteine-reactive acrylamide moiety with a nonreactive propyl amide. Compound 14 is a negative control compound with significantly diminished antiviral activity. B , HEK293T cells were transfected with an in vitro transcribed reporter DV subgenomic RNA bearing the virus's seven nonstructural genes ( NS1–NS5 ) as well as a NanoLuc®-proline, glutamate, serine, threonine ( NlucP ) luciferase reporter gene. The intracellular reporter activity was immediately measured for 1 h at 37 °C. Cells were treated with DMSO or 2 μ m QL47 at 1 h post-transfection, and the intracellular luciferase signal was then measured continuously for 3 h at 37 °C. The luciferase signal obtained from mock-transfected cells was subtracted, and data are presented as means ± S.D. of three experimental replicates. One representative experiment is shown from two independent experiments. RLU , relative light units. C , measurement of puromycin incorporation in nascent cellular proteins. Huh7 cells were treated with DMSO, 50 μg/ml CHX, or 2 μ m QL47 for 1 h. The cells were next pulse-labeled with 1 μ m puromycin for 30 min, and their cellular contents were analyzed by Western blotting. Prematurely terminated polypeptides were detected using a puromycin-specific antibody. Their abundance was normalized to the loading control (tubulin) and is presented as a percentage of the DMSO-treated control samples. One representative experiment is shown from two independent experiments. D , metabolic labeling of cellular proteins with radiolabeled amino acids. Huh7 cells were starved for 30 min in methionine/cysteine-free medium and concomitantly treated with DMSO, 30 μg/ml CHX, 2 μ m QL47, or 2 μ m compound 14. The medium was next supplemented with a mixture of 35 S-Met and 35 S-Cys for 30 min. Total cell lysates were analyzed by SDS-PAGE, followed by autoradiography to measure bulk protein synthesis. Neosynthesized protein abundance is presented as a percentage of the DMSO-treated control samples. One representative experiment is shown from three independent experiments. E , Huh7 cells were treated with DMSO or 2 μ m QL47, and metabolic labeling ( red boxes ) was performed as indicated in D 1 h and 24 h post-treatment. Analysis of the neosynthesized proteins was performed as in D , and their abundance is presented as a percentage of the DMSO-treated control samples at each time point. One representative experiment is shown from two independent experiments.
    Figure Legend Snippet: QL47 inhibits protein synthesis in live cells. A , chemical structures of QL47 and the derivatives YKL-04-085, QL47R, and YKL-03-109 (compound 14) ( 6 , 9 ). QL47R is an inactive analog due to replacement of the cysteine-reactive acrylamide moiety with a nonreactive propyl amide. Compound 14 is a negative control compound with significantly diminished antiviral activity. B , HEK293T cells were transfected with an in vitro transcribed reporter DV subgenomic RNA bearing the virus's seven nonstructural genes ( NS1–NS5 ) as well as a NanoLuc®-proline, glutamate, serine, threonine ( NlucP ) luciferase reporter gene. The intracellular reporter activity was immediately measured for 1 h at 37 °C. Cells were treated with DMSO or 2 μ m QL47 at 1 h post-transfection, and the intracellular luciferase signal was then measured continuously for 3 h at 37 °C. The luciferase signal obtained from mock-transfected cells was subtracted, and data are presented as means ± S.D. of three experimental replicates. One representative experiment is shown from two independent experiments. RLU , relative light units. C , measurement of puromycin incorporation in nascent cellular proteins. Huh7 cells were treated with DMSO, 50 μg/ml CHX, or 2 μ m QL47 for 1 h. The cells were next pulse-labeled with 1 μ m puromycin for 30 min, and their cellular contents were analyzed by Western blotting. Prematurely terminated polypeptides were detected using a puromycin-specific antibody. Their abundance was normalized to the loading control (tubulin) and is presented as a percentage of the DMSO-treated control samples. One representative experiment is shown from two independent experiments. D , metabolic labeling of cellular proteins with radiolabeled amino acids. Huh7 cells were starved for 30 min in methionine/cysteine-free medium and concomitantly treated with DMSO, 30 μg/ml CHX, 2 μ m QL47, or 2 μ m compound 14. The medium was next supplemented with a mixture of 35 S-Met and 35 S-Cys for 30 min. Total cell lysates were analyzed by SDS-PAGE, followed by autoradiography to measure bulk protein synthesis. Neosynthesized protein abundance is presented as a percentage of the DMSO-treated control samples. One representative experiment is shown from three independent experiments. E , Huh7 cells were treated with DMSO or 2 μ m QL47, and metabolic labeling ( red boxes ) was performed as indicated in D 1 h and 24 h post-treatment. Analysis of the neosynthesized proteins was performed as in D , and their abundance is presented as a percentage of the DMSO-treated control samples at each time point. One representative experiment is shown from two independent experiments.

    Techniques Used: Negative Control, Activity Assay, Transfection, In Vitro, Luciferase, Labeling, Western Blot, SDS Page, Autoradiography

    QL47 inhibits eukaryotic but not prokaryotic protein synthesis. A , E. coli cells carrying the pUA66- rrnB plasmid that constitutively expresses GFP ( 24 ) were treated with DMSO, 250 μg/ml G418, or 50 μ m QL47. The intracellular GFP fluorescence signal was then measured continuously for 14 h at 37 °C. The signal obtained from growth medium was subtracted, and data are presented as means ± S.D. of 12 experimental replicates. One representative experiment is shown from two independent experiments. B , analysis of in vitro translation assays performed in rabbit reticulocyte lysates, yeast cell lysates, or a reconstituted E. coli cell-free synthesis system (PURExpress®). Translation in rabbit reticulocyte lysates was performed in the presence of DMSO, 30 μg/ml CHX, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed reporter DV subgenomic RNA was used as a template, and the luciferase signal was measured after 90-min incubation at 30 °C. Data are presented as means normalized to DMSO ± S.D. of four experimental replicates. Translation in yeast cell lysates was performed in the presence of DMSO, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed vesicular stomatitis virus (VSV) RNA bearing a luciferase reporter gene ( 44 ) was used as a template, and the luciferase signal was measured after 2-h incubation at 25 °C. Data are presented as means normalized to DMSO ± S.D. of three experimental replicates. Translation in a reconstituted E. coli cell-free synthesis system (PURExpress®) was performed in the presence of DMSO, 250 μg/ml G418, 100 μ m QL47, or 100 μ m compound 14. A plasmid expressing GFP under control of a T7 promoter was used as a template. After 1-h incubation at 37 °C, the total protein content was analyzed by Western blotting. The reporter protein was detected using a GFP antibody, and its abundance was normalized to the loading control (histidine tag). Data are presented as means normalized to DMSO ± S.D. of two technical replicates. One representative experiment is shown from four (rabbit reticulocyte lysates) or two (yeast cell lysates and E. coli cell-free synthesis system) independent experiments. Asterisks indicate that the differences between experimental samples and the DMSO-treated control samples are statistically significant when compared using unpaired t test: ***, p
    Figure Legend Snippet: QL47 inhibits eukaryotic but not prokaryotic protein synthesis. A , E. coli cells carrying the pUA66- rrnB plasmid that constitutively expresses GFP ( 24 ) were treated with DMSO, 250 μg/ml G418, or 50 μ m QL47. The intracellular GFP fluorescence signal was then measured continuously for 14 h at 37 °C. The signal obtained from growth medium was subtracted, and data are presented as means ± S.D. of 12 experimental replicates. One representative experiment is shown from two independent experiments. B , analysis of in vitro translation assays performed in rabbit reticulocyte lysates, yeast cell lysates, or a reconstituted E. coli cell-free synthesis system (PURExpress®). Translation in rabbit reticulocyte lysates was performed in the presence of DMSO, 30 μg/ml CHX, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed reporter DV subgenomic RNA was used as a template, and the luciferase signal was measured after 90-min incubation at 30 °C. Data are presented as means normalized to DMSO ± S.D. of four experimental replicates. Translation in yeast cell lysates was performed in the presence of DMSO, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed vesicular stomatitis virus (VSV) RNA bearing a luciferase reporter gene ( 44 ) was used as a template, and the luciferase signal was measured after 2-h incubation at 25 °C. Data are presented as means normalized to DMSO ± S.D. of three experimental replicates. Translation in a reconstituted E. coli cell-free synthesis system (PURExpress®) was performed in the presence of DMSO, 250 μg/ml G418, 100 μ m QL47, or 100 μ m compound 14. A plasmid expressing GFP under control of a T7 promoter was used as a template. After 1-h incubation at 37 °C, the total protein content was analyzed by Western blotting. The reporter protein was detected using a GFP antibody, and its abundance was normalized to the loading control (histidine tag). Data are presented as means normalized to DMSO ± S.D. of two technical replicates. One representative experiment is shown from four (rabbit reticulocyte lysates) or two (yeast cell lysates and E. coli cell-free synthesis system) independent experiments. Asterisks indicate that the differences between experimental samples and the DMSO-treated control samples are statistically significant when compared using unpaired t test: ***, p

    Techniques Used: Plasmid Preparation, Fluorescence, In Vitro, Luciferase, Incubation, Expressing, Western Blot

    7) Product Images from "Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions"

    Article Title: Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211739

    The expression profile of miRNAs in various tissues and cell lines. (A) A heat map of hierarchical clustering of miRNAs detected in small RNA-seq data used in this study. (B) A heat map of 20 miRNAs highly expressed in PGCs. Relative miRNA expression is described according to the color scale. Red and green indicate high and low expression, respectively. Mouse embryonic fibroblasts (MEFs), embryonic stem (ES) cells, primordial germ cells (PGCs), spermatogonia (SPG), spermatozoa (SPZ). (C) The locus of XmiR genes on the X chromosome. (D) The expression of XmiRs in testes, ES cells, and MEFs determined by quantitative RT-PCR. Each expression level was normalized to the expression of U6 snRNA. The expression in ES cells was set as 1.0. Error bars show standard errors of three biological replicates. **P
    Figure Legend Snippet: The expression profile of miRNAs in various tissues and cell lines. (A) A heat map of hierarchical clustering of miRNAs detected in small RNA-seq data used in this study. (B) A heat map of 20 miRNAs highly expressed in PGCs. Relative miRNA expression is described according to the color scale. Red and green indicate high and low expression, respectively. Mouse embryonic fibroblasts (MEFs), embryonic stem (ES) cells, primordial germ cells (PGCs), spermatogonia (SPG), spermatozoa (SPZ). (C) The locus of XmiR genes on the X chromosome. (D) The expression of XmiRs in testes, ES cells, and MEFs determined by quantitative RT-PCR. Each expression level was normalized to the expression of U6 snRNA. The expression in ES cells was set as 1.0. Error bars show standard errors of three biological replicates. **P

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR

    8) Product Images from "An origin of the immunogenicity of in vitro transcribed RNA"

    Article Title: An origin of the immunogenicity of in vitro transcribed RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky177

    The dsRNA byproduct is formed by sense and antisense RNAs generated in promoter-dependent and -independent manners, respectively. (A and B) Transcriptional start sites and end sites for the intended 512B product ( A ) and its complementary RNA byproduct (c512B) ( B ), as examined by 5′- and 3′-RACE. Transcriptional start and end sites are shown upstream of the poly A tail in the 5′- and 3′-RACE sequences, respectively. Cyan underscores in the sequence chromatograms indicate sequences matching those in the template. The location of the matching sequence in the template is shown in the schematic on the right. The red box in (B) indicates the reverse complement sequence of the T7 promoter. ( C ) Schematic illustrating the results in (A and B). Transcription using a template with a single T7 promoter results in the production of both sense and antisense transcripts, which differ in length by the size of the T7 promoter. Solid and dotted lines indicate DNA and RNA, respectively. ( D ) Native PAGE analysis of T7 transcripts generated using DNA template with a single T7 promoter (1), DNA template without the T7 promoter (2), and gel-purified 512B ssRNA as a template (3). RNA template alone (4) was compared with (3). ( E ) (3). * indicates unknown ssRNA byproduct from transcription.
    Figure Legend Snippet: The dsRNA byproduct is formed by sense and antisense RNAs generated in promoter-dependent and -independent manners, respectively. (A and B) Transcriptional start sites and end sites for the intended 512B product ( A ) and its complementary RNA byproduct (c512B) ( B ), as examined by 5′- and 3′-RACE. Transcriptional start and end sites are shown upstream of the poly A tail in the 5′- and 3′-RACE sequences, respectively. Cyan underscores in the sequence chromatograms indicate sequences matching those in the template. The location of the matching sequence in the template is shown in the schematic on the right. The red box in (B) indicates the reverse complement sequence of the T7 promoter. ( C ) Schematic illustrating the results in (A and B). Transcription using a template with a single T7 promoter results in the production of both sense and antisense transcripts, which differ in length by the size of the T7 promoter. Solid and dotted lines indicate DNA and RNA, respectively. ( D ) Native PAGE analysis of T7 transcripts generated using DNA template with a single T7 promoter (1), DNA template without the T7 promoter (2), and gel-purified 512B ssRNA as a template (3). RNA template alone (4) was compared with (3). ( E ) (3). * indicates unknown ssRNA byproduct from transcription.

    Techniques Used: Generated, Sequencing, Clear Native PAGE, Purification

    9) Product Images from "Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing"

    Article Title: Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing

    Journal: bioRxiv

    doi: 10.1101/2019.12.18.880849

    Detection of transcript boundaries. a , Left panel: Separation between uncorrected Nanopore-predicted TSS and comparison to Illumina d(ifferential) RNA-Seq data from published data sets for E. coli 28 , P. furiosus 53 and H. volcanii 60 . Right panel: The translocation speed of the last 12 nucleotides (nt) is not controlled, as the motor protein is falling off. Therefore, native RNA reads are shortened by ∼12 nt. b , Position of TSS is corrected for 12 nucleotides to calculate the length of 5’ untranslated regions (UTR) in the Nanopore data sets (purple). 5’ UTRs are compared to d(ifferential) RNA-Seq Illumina data sets (light-green). Median values are indicated by a black bar inside the distribution (compare Supplementary Fig. 6). c , Length of 3’ UTRs is based on the prediction of transcription termination sites (TTS) and the comparison to annotated gene ends. Distribution of lengths is shown for Nanopore data sets (purple) and compared to Term-Seq Illumina data from E. coli and H. volcanii l (light-green) 28 . d , MEME analysis 53 of extracted sequences upstream of Nanopore-predicted TSS reveals bacterial (position −10) and archaeal-specific promoter elements (BRE: B-recognition element, TATA: TATA-box recognized by transcription factor B), therefore validating the positions of predicted TSS. e , Nucleotide enrichment meta analysis was carried out by comparing the genomic sequences surrounding the TTS (−45 to +45) to randomly selected intergenic positions of the respective organism (n: 10000) (Terminator motifs in Supplementary Fig. 7).
    Figure Legend Snippet: Detection of transcript boundaries. a , Left panel: Separation between uncorrected Nanopore-predicted TSS and comparison to Illumina d(ifferential) RNA-Seq data from published data sets for E. coli 28 , P. furiosus 53 and H. volcanii 60 . Right panel: The translocation speed of the last 12 nucleotides (nt) is not controlled, as the motor protein is falling off. Therefore, native RNA reads are shortened by ∼12 nt. b , Position of TSS is corrected for 12 nucleotides to calculate the length of 5’ untranslated regions (UTR) in the Nanopore data sets (purple). 5’ UTRs are compared to d(ifferential) RNA-Seq Illumina data sets (light-green). Median values are indicated by a black bar inside the distribution (compare Supplementary Fig. 6). c , Length of 3’ UTRs is based on the prediction of transcription termination sites (TTS) and the comparison to annotated gene ends. Distribution of lengths is shown for Nanopore data sets (purple) and compared to Term-Seq Illumina data from E. coli and H. volcanii l (light-green) 28 . d , MEME analysis 53 of extracted sequences upstream of Nanopore-predicted TSS reveals bacterial (position −10) and archaeal-specific promoter elements (BRE: B-recognition element, TATA: TATA-box recognized by transcription factor B), therefore validating the positions of predicted TSS. e , Nucleotide enrichment meta analysis was carried out by comparing the genomic sequences surrounding the TTS (−45 to +45) to randomly selected intergenic positions of the respective organism (n: 10000) (Terminator motifs in Supplementary Fig. 7).

    Techniques Used: RNA Sequencing Assay, Translocation Assay, Genomic Sequencing

    Nanopore-based native RNA sequencing of prokaryotes. a , Key steps of library preparation: (1) native RNA is polyadenylated, which allows library preparation using the direct RNA kit from Oxford Nanopore and sequencing on a MinION device. (2) 3’ ligation is performed to add an adapter carrying the motor-protein (red square), which unzips the RNA-cDNA hybrid and pulls the RNA through the Nanopore (detailed description see Supplementary Fig. 1a). b , Data sets for three prokaryotic model organisms ( Ecoli : Escherichia coli, Pfu : Pyrococcus furiosus, Hvo : Haloferax volcanii ) were collected and mapped to their respective reference genome. Transcript abundances of genomic features (protein coding genes (CDS): red, 5S rRNA: green, 16S rRNA: purple, 23S rRNA: light-purple) were estimated using featurecounts 50 (TEX-treated samples are shown as example in Fig. 1 ). c , Aligned read lengths across different genomic features. d , Comparison of read identities between CDS (red) and rRNA (grey)-mapping reads.
    Figure Legend Snippet: Nanopore-based native RNA sequencing of prokaryotes. a , Key steps of library preparation: (1) native RNA is polyadenylated, which allows library preparation using the direct RNA kit from Oxford Nanopore and sequencing on a MinION device. (2) 3’ ligation is performed to add an adapter carrying the motor-protein (red square), which unzips the RNA-cDNA hybrid and pulls the RNA through the Nanopore (detailed description see Supplementary Fig. 1a). b , Data sets for three prokaryotic model organisms ( Ecoli : Escherichia coli, Pfu : Pyrococcus furiosus, Hvo : Haloferax volcanii ) were collected and mapped to their respective reference genome. Transcript abundances of genomic features (protein coding genes (CDS): red, 5S rRNA: green, 16S rRNA: purple, 23S rRNA: light-purple) were estimated using featurecounts 50 (TEX-treated samples are shown as example in Fig. 1 ). c , Aligned read lengths across different genomic features. d , Comparison of read identities between CDS (red) and rRNA (grey)-mapping reads.

    Techniques Used: RNA Sequencing Assay, Sequencing, Ligation

    Related Articles

    Functional Assay:

    Article Title: Mouse Embryonic Stem Cells Have Underdeveloped Antiviral Mechanisms That Can Be Exploited for the Development of mRNA-Mediated Gene Expression Strategy
    Article Snippet: .. The purified RNA transcripts were polyadenylated by Escherichia coli Poly(A) polymerase (New England Biolabs), resulting in functional mRNA, m7 GpppA-EGFP-polyA, and m7 GpppA-Etv2-polyA. .. ESCs (D3 cell line, ATCC) were cultured in the standard mESC medium that contains leukemia inhibitory factor as previously described [ ].

    In Vitro:

    Article Title: A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation
    Article Snippet: .. For pcDNA3-RLUC-POLIRES-FLUC and pcDNA3-RLUC-CrPV-IRES-FLUC, in vitro transcripts were synthesized from Xho I-linearized plasmids using the mMessage mMachine T7 transcription kit (Thermo Fisher Scientific, AM1344) and then polyadenylated using E. coli poly(A) polymerase (New England Biolabs, M0276). .. In vitro transcripts were synthesized from Xho I-linearized pBS-EMCV-Fluci and Xba I-linearized mung bean nuclease–treated pSGR-JFH1/Luc using the AmpliScribe T7-Flash transcription kit (Lucigen, ASF3257).

    Synthesized:

    Article Title: A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation
    Article Snippet: .. For pcDNA3-RLUC-POLIRES-FLUC and pcDNA3-RLUC-CrPV-IRES-FLUC, in vitro transcripts were synthesized from Xho I-linearized plasmids using the mMessage mMachine T7 transcription kit (Thermo Fisher Scientific, AM1344) and then polyadenylated using E. coli poly(A) polymerase (New England Biolabs, M0276). .. In vitro transcripts were synthesized from Xho I-linearized pBS-EMCV-Fluci and Xba I-linearized mung bean nuclease–treated pSGR-JFH1/Luc using the AmpliScribe T7-Flash transcription kit (Lucigen, ASF3257).

    Incubation:

    Article Title: Exploring prokaryotic transcription, operon structures, rRNA maturation and modifications using Nanopore-based native RNA sequencing
    Article Snippet: .. Briefly, 5 µg RNA, 20 units poly(A) polymerase, 2 µl reaction buffer and 1 mM ATP were incubated for 15 min at 37°C in a total reaction volume of 50 µl. .. To stop the reaction and to remove the enzyme, the poly(A)-tailed RNA was purified with the RNeasy MinElute Cleanup Kit (Qiagen).

    Purification:

    Article Title: RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation †Electronic supplementary information (ESI) available: Fig. S1–S7. See DOI: 10.1039/c5sc
    Article Snippet: .. RNA was then polyadenylated by E. coli poly(A) polymerase (M0276S, NEB) in a 50 μl volume containing 50 mM Tris–HCl (pH 7.9), 250 mM NaCl, 10 mM MgCl2 , 1.5 μg RNA, 2.5 U poly(A) polymerase, and 1 mM ATP. cDNA was generated from the purified tailed RNA using M-MLV Reverse Transcriptase (M368B, Promega). ..

    Article Title: Mouse Embryonic Stem Cells Have Underdeveloped Antiviral Mechanisms That Can Be Exploited for the Development of mRNA-Mediated Gene Expression Strategy
    Article Snippet: .. The purified RNA transcripts were polyadenylated by Escherichia coli Poly(A) polymerase (New England Biolabs), resulting in functional mRNA, m7 GpppA-EGFP-polyA, and m7 GpppA-Etv2-polyA. .. ESCs (D3 cell line, ATCC) were cultured in the standard mESC medium that contains leukemia inhibitory factor as previously described [ ].

    Generated:

    Article Title: RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation †Electronic supplementary information (ESI) available: Fig. S1–S7. See DOI: 10.1039/c5sc
    Article Snippet: .. RNA was then polyadenylated by E. coli poly(A) polymerase (M0276S, NEB) in a 50 μl volume containing 50 mM Tris–HCl (pH 7.9), 250 mM NaCl, 10 mM MgCl2 , 1.5 μg RNA, 2.5 U poly(A) polymerase, and 1 mM ATP. cDNA was generated from the purified tailed RNA using M-MLV Reverse Transcriptase (M368B, Promega). ..

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    New England Biolabs poly a polymerase
    The dsRNA byproduct is formed by sense and antisense RNAs generated in promoter-dependent and -independent manners, respectively. (A and B) Transcriptional start sites and end sites for the intended 512B product ( A ) and its complementary <t>RNA</t> byproduct (c512B) ( B ), as examined by 5′- and 3′-RACE. Transcriptional start and end sites are shown upstream of the <t>poly</t> A tail in the 5′- and 3′-RACE sequences, respectively. Cyan underscores in the sequence chromatograms indicate sequences matching those in the template. The location of the matching sequence in the template is shown in the schematic on the right. The red box in (B) indicates the reverse complement sequence of the T7 promoter. ( C ) Schematic illustrating the results in (A and B). Transcription using a template with a single T7 promoter results in the production of both sense and antisense transcripts, which differ in length by the size of the T7 promoter. Solid and dotted lines indicate DNA and RNA, respectively. ( D ) Native PAGE analysis of T7 transcripts generated using DNA template with a single T7 promoter (1), DNA template without the T7 promoter (2), and gel-purified 512B ssRNA as a template (3). RNA template alone (4) was compared with (3). ( E ) Native PAGE analysis of T7 transcripts generated using fully duplexed DNA template (1), template strand ssDNA alone (512B-3end in Supplementary Table S1 ) (2), and partially duplexed DNA template (generated by annealing 512B-3end with T7promoter_top_strand, Supplementary Table S1 ) (3). * indicates unknown ssRNA byproduct from transcription.
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    The dsRNA byproduct is formed by sense and antisense RNAs generated in promoter-dependent and -independent manners, respectively. (A and B) Transcriptional start sites and end sites for the intended 512B product ( A ) and its complementary RNA byproduct (c512B) ( B ), as examined by 5′- and 3′-RACE. Transcriptional start and end sites are shown upstream of the poly A tail in the 5′- and 3′-RACE sequences, respectively. Cyan underscores in the sequence chromatograms indicate sequences matching those in the template. The location of the matching sequence in the template is shown in the schematic on the right. The red box in (B) indicates the reverse complement sequence of the T7 promoter. ( C ) Schematic illustrating the results in (A and B). Transcription using a template with a single T7 promoter results in the production of both sense and antisense transcripts, which differ in length by the size of the T7 promoter. Solid and dotted lines indicate DNA and RNA, respectively. ( D ) Native PAGE analysis of T7 transcripts generated using DNA template with a single T7 promoter (1), DNA template without the T7 promoter (2), and gel-purified 512B ssRNA as a template (3). RNA template alone (4) was compared with (3). ( E ) Native PAGE analysis of T7 transcripts generated using fully duplexed DNA template (1), template strand ssDNA alone (512B-3end in Supplementary Table S1 ) (2), and partially duplexed DNA template (generated by annealing 512B-3end with T7promoter_top_strand, Supplementary Table S1 ) (3). * indicates unknown ssRNA byproduct from transcription.

    Journal: Nucleic Acids Research

    Article Title: An origin of the immunogenicity of in vitro transcribed RNA

    doi: 10.1093/nar/gky177

    Figure Lengend Snippet: The dsRNA byproduct is formed by sense and antisense RNAs generated in promoter-dependent and -independent manners, respectively. (A and B) Transcriptional start sites and end sites for the intended 512B product ( A ) and its complementary RNA byproduct (c512B) ( B ), as examined by 5′- and 3′-RACE. Transcriptional start and end sites are shown upstream of the poly A tail in the 5′- and 3′-RACE sequences, respectively. Cyan underscores in the sequence chromatograms indicate sequences matching those in the template. The location of the matching sequence in the template is shown in the schematic on the right. The red box in (B) indicates the reverse complement sequence of the T7 promoter. ( C ) Schematic illustrating the results in (A and B). Transcription using a template with a single T7 promoter results in the production of both sense and antisense transcripts, which differ in length by the size of the T7 promoter. Solid and dotted lines indicate DNA and RNA, respectively. ( D ) Native PAGE analysis of T7 transcripts generated using DNA template with a single T7 promoter (1), DNA template without the T7 promoter (2), and gel-purified 512B ssRNA as a template (3). RNA template alone (4) was compared with (3). ( E ) Native PAGE analysis of T7 transcripts generated using fully duplexed DNA template (1), template strand ssDNA alone (512B-3end in Supplementary Table S1 ) (2), and partially duplexed DNA template (generated by annealing 512B-3end with T7promoter_top_strand, Supplementary Table S1 ) (3). * indicates unknown ssRNA byproduct from transcription.

    Article Snippet: For 3′-RACE, 3′ end of the RNA was first extended with poly A tails using the poly A polymerase (NEB).

    Techniques: Generated, Sequencing, Clear Native PAGE, Purification

    Dependence of BiFC fluorescence enhancement on target RNA expression in E. coli . (A) Time course of the difference in mean fluorescence between cells expressing RNA REX(A) – TERC(Q) and REX(A) – TERC(Qm) in the presence and absence of RNA induction by ATc. (B) RNA level in cells before and after induction with ATc. Data are given in mean ± SD of three experiments. IPTG was added at 0 h with or without ATc. Representative distribution of eGFP fluorescence of corresponding cells is given in Fig. S4B. †

    Journal: Chemical Science

    Article Title: RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation †Electronic supplementary information (ESI) available: Fig. S1–S7. See DOI: 10.1039/c5sc03946k

    doi: 10.1039/c5sc03946k

    Figure Lengend Snippet: Dependence of BiFC fluorescence enhancement on target RNA expression in E. coli . (A) Time course of the difference in mean fluorescence between cells expressing RNA REX(A) – TERC(Q) and REX(A) – TERC(Qm) in the presence and absence of RNA induction by ATc. (B) RNA level in cells before and after induction with ATc. Data are given in mean ± SD of three experiments. IPTG was added at 0 h with or without ATc. Representative distribution of eGFP fluorescence of corresponding cells is given in Fig. S4B. †

    Article Snippet: RNA was then polyadenylated by E. coli poly(A) polymerase (M0276S, NEB) in a 50 μl volume containing 50 mM Tris–HCl (pH 7.9), 250 mM NaCl, 10 mM MgCl2 , 1.5 μg RNA, 2.5 U poly(A) polymerase, and 1 mM ATP. cDNA was generated from the purified tailed RNA using M-MLV Reverse Transcriptase (M368B, Promega).

    Techniques: Bimolecular Fluorescence Complementation Assay, Fluorescence, RNA Expression, Expressing

    Pull-down of G-quadruplex-recognizing proteins by RNA from E. coli . Cells were lysed in the presence of RNase inhibitor or RNase A. Probe proteins associated with the indicated target RNAs were pulled down with an immobilized DNA oligomer complementary to the 5′ end of the RNA and detected by an antibody against eGFP. “NS” indicates a non-specifically stained band that can serve as a loading control.

    Journal: Chemical Science

    Article Title: RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation †Electronic supplementary information (ESI) available: Fig. S1–S7. See DOI: 10.1039/c5sc03946k

    doi: 10.1039/c5sc03946k

    Figure Lengend Snippet: Pull-down of G-quadruplex-recognizing proteins by RNA from E. coli . Cells were lysed in the presence of RNase inhibitor or RNase A. Probe proteins associated with the indicated target RNAs were pulled down with an immobilized DNA oligomer complementary to the 5′ end of the RNA and detected by an antibody against eGFP. “NS” indicates a non-specifically stained band that can serve as a loading control.

    Article Snippet: RNA was then polyadenylated by E. coli poly(A) polymerase (M0276S, NEB) in a 50 μl volume containing 50 mM Tris–HCl (pH 7.9), 250 mM NaCl, 10 mM MgCl2 , 1.5 μg RNA, 2.5 U poly(A) polymerase, and 1 mM ATP. cDNA was generated from the purified tailed RNA using M-MLV Reverse Transcriptase (M368B, Promega).

    Techniques: Staining

    Dependence of BiFC fluorescence on the number of G 3 tracts in the RNA target expressed in E. coli . Distribution of (A) eGFP fluorescence or (B) forward scattering intensity or (C) relative RNA expression (mean ± SD of three experiments) was assayed and processed as in Fig. 4 .

    Journal: Chemical Science

    Article Title: RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation †Electronic supplementary information (ESI) available: Fig. S1–S7. See DOI: 10.1039/c5sc03946k

    doi: 10.1039/c5sc03946k

    Figure Lengend Snippet: Dependence of BiFC fluorescence on the number of G 3 tracts in the RNA target expressed in E. coli . Distribution of (A) eGFP fluorescence or (B) forward scattering intensity or (C) relative RNA expression (mean ± SD of three experiments) was assayed and processed as in Fig. 4 .

    Article Snippet: RNA was then polyadenylated by E. coli poly(A) polymerase (M0276S, NEB) in a 50 μl volume containing 50 mM Tris–HCl (pH 7.9), 250 mM NaCl, 10 mM MgCl2 , 1.5 μg RNA, 2.5 U poly(A) polymerase, and 1 mM ATP. cDNA was generated from the purified tailed RNA using M-MLV Reverse Transcriptase (M368B, Promega).

    Techniques: Bimolecular Fluorescence Complementation Assay, Fluorescence, RNA Expression

    Detection of BiFC fluorescence in E. coli cells by flow cytometry. Distribution of (A–C) eGFP fluorescence and (D–F) forward scattering intensity was collected from cells expressing G N -REX(A) and RHAU(Q)-G C probe proteins plus the indicated RNA. (G–I) Relative RNA expression (mean ± SD of three experiments) assayed by qPCR.

    Journal: Chemical Science

    Article Title: RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation †Electronic supplementary information (ESI) available: Fig. S1–S7. See DOI: 10.1039/c5sc03946k

    doi: 10.1039/c5sc03946k

    Figure Lengend Snippet: Detection of BiFC fluorescence in E. coli cells by flow cytometry. Distribution of (A–C) eGFP fluorescence and (D–F) forward scattering intensity was collected from cells expressing G N -REX(A) and RHAU(Q)-G C probe proteins plus the indicated RNA. (G–I) Relative RNA expression (mean ± SD of three experiments) assayed by qPCR.

    Article Snippet: RNA was then polyadenylated by E. coli poly(A) polymerase (M0276S, NEB) in a 50 μl volume containing 50 mM Tris–HCl (pH 7.9), 250 mM NaCl, 10 mM MgCl2 , 1.5 μg RNA, 2.5 U poly(A) polymerase, and 1 mM ATP. cDNA was generated from the purified tailed RNA using M-MLV Reverse Transcriptase (M368B, Promega).

    Techniques: Bimolecular Fluorescence Complementation Assay, Fluorescence, Flow Cytometry, Cytometry, Expressing, RNA Expression, Real-time Polymerase Chain Reaction

    QL47 inhibits eukaryotic but not prokaryotic protein synthesis. A , E. coli cells carrying the pUA66- rrnB plasmid that constitutively expresses GFP ( 24 ) were treated with DMSO, 250 μg/ml G418, or 50 μ m QL47. The intracellular GFP fluorescence signal was then measured continuously for 14 h at 37 °C. The signal obtained from growth medium was subtracted, and data are presented as means ± S.D. of 12 experimental replicates. One representative experiment is shown from two independent experiments. B , analysis of in vitro translation assays performed in rabbit reticulocyte lysates, yeast cell lysates, or a reconstituted E. coli cell-free synthesis system (PURExpress®). Translation in rabbit reticulocyte lysates was performed in the presence of DMSO, 30 μg/ml CHX, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed reporter DV subgenomic RNA was used as a template, and the luciferase signal was measured after 90-min incubation at 30 °C. Data are presented as means normalized to DMSO ± S.D. of four experimental replicates. Translation in yeast cell lysates was performed in the presence of DMSO, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed vesicular stomatitis virus (VSV) RNA bearing a luciferase reporter gene ( 44 ) was used as a template, and the luciferase signal was measured after 2-h incubation at 25 °C. Data are presented as means normalized to DMSO ± S.D. of three experimental replicates. Translation in a reconstituted E. coli cell-free synthesis system (PURExpress®) was performed in the presence of DMSO, 250 μg/ml G418, 100 μ m QL47, or 100 μ m compound 14. A plasmid expressing GFP under control of a T7 promoter was used as a template. After 1-h incubation at 37 °C, the total protein content was analyzed by Western blotting. The reporter protein was detected using a GFP antibody, and its abundance was normalized to the loading control (histidine tag). Data are presented as means normalized to DMSO ± S.D. of two technical replicates. One representative experiment is shown from four (rabbit reticulocyte lysates) or two (yeast cell lysates and E. coli cell-free synthesis system) independent experiments. Asterisks indicate that the differences between experimental samples and the DMSO-treated control samples are statistically significant when compared using unpaired t test: ***, p

    Journal: The Journal of Biological Chemistry

    Article Title: A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation

    doi: 10.1074/jbc.RA119.011132

    Figure Lengend Snippet: QL47 inhibits eukaryotic but not prokaryotic protein synthesis. A , E. coli cells carrying the pUA66- rrnB plasmid that constitutively expresses GFP ( 24 ) were treated with DMSO, 250 μg/ml G418, or 50 μ m QL47. The intracellular GFP fluorescence signal was then measured continuously for 14 h at 37 °C. The signal obtained from growth medium was subtracted, and data are presented as means ± S.D. of 12 experimental replicates. One representative experiment is shown from two independent experiments. B , analysis of in vitro translation assays performed in rabbit reticulocyte lysates, yeast cell lysates, or a reconstituted E. coli cell-free synthesis system (PURExpress®). Translation in rabbit reticulocyte lysates was performed in the presence of DMSO, 30 μg/ml CHX, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed reporter DV subgenomic RNA was used as a template, and the luciferase signal was measured after 90-min incubation at 30 °C. Data are presented as means normalized to DMSO ± S.D. of four experimental replicates. Translation in yeast cell lysates was performed in the presence of DMSO, 40 μ m QL47, or 40 μ m compound 14. An in vitro transcribed vesicular stomatitis virus (VSV) RNA bearing a luciferase reporter gene ( 44 ) was used as a template, and the luciferase signal was measured after 2-h incubation at 25 °C. Data are presented as means normalized to DMSO ± S.D. of three experimental replicates. Translation in a reconstituted E. coli cell-free synthesis system (PURExpress®) was performed in the presence of DMSO, 250 μg/ml G418, 100 μ m QL47, or 100 μ m compound 14. A plasmid expressing GFP under control of a T7 promoter was used as a template. After 1-h incubation at 37 °C, the total protein content was analyzed by Western blotting. The reporter protein was detected using a GFP antibody, and its abundance was normalized to the loading control (histidine tag). Data are presented as means normalized to DMSO ± S.D. of two technical replicates. One representative experiment is shown from four (rabbit reticulocyte lysates) or two (yeast cell lysates and E. coli cell-free synthesis system) independent experiments. Asterisks indicate that the differences between experimental samples and the DMSO-treated control samples are statistically significant when compared using unpaired t test: ***, p

    Article Snippet: For pcDNA3-RLUC-POLIRES-FLUC and pcDNA3-RLUC-CrPV-IRES-FLUC, in vitro transcripts were synthesized from Xho I-linearized plasmids using the mMessage mMachine T7 transcription kit (Thermo Fisher Scientific, AM1344) and then polyadenylated using E. coli poly(A) polymerase (New England Biolabs, M0276).

    Techniques: Plasmid Preparation, Fluorescence, In Vitro, Luciferase, Incubation, Expressing, Western Blot

    The dsRNA byproduct is formed by sense and antisense RNAs generated in promoter-dependent and -independent manners, respectively. (A and B) Transcriptional start sites and end sites for the intended 512B product ( A ) and its complementary RNA byproduct (c512B) ( B ), as examined by 5′- and 3′-RACE. Transcriptional start and end sites are shown upstream of the poly A tail in the 5′- and 3′-RACE sequences, respectively. Cyan underscores in the sequence chromatograms indicate sequences matching those in the template. The location of the matching sequence in the template is shown in the schematic on the right. The red box in (B) indicates the reverse complement sequence of the T7 promoter. ( C ) Schematic illustrating the results in (A and B). Transcription using a template with a single T7 promoter results in the production of both sense and antisense transcripts, which differ in length by the size of the T7 promoter. Solid and dotted lines indicate DNA and RNA, respectively. ( D ) Native PAGE analysis of T7 transcripts generated using DNA template with a single T7 promoter (1), DNA template without the T7 promoter (2), and gel-purified 512B ssRNA as a template (3). RNA template alone (4) was compared with (3). ( E ) (3). * indicates unknown ssRNA byproduct from transcription.

    Journal: Nucleic Acids Research

    Article Title: An origin of the immunogenicity of in vitro transcribed RNA

    doi: 10.1093/nar/gky177

    Figure Lengend Snippet: The dsRNA byproduct is formed by sense and antisense RNAs generated in promoter-dependent and -independent manners, respectively. (A and B) Transcriptional start sites and end sites for the intended 512B product ( A ) and its complementary RNA byproduct (c512B) ( B ), as examined by 5′- and 3′-RACE. Transcriptional start and end sites are shown upstream of the poly A tail in the 5′- and 3′-RACE sequences, respectively. Cyan underscores in the sequence chromatograms indicate sequences matching those in the template. The location of the matching sequence in the template is shown in the schematic on the right. The red box in (B) indicates the reverse complement sequence of the T7 promoter. ( C ) Schematic illustrating the results in (A and B). Transcription using a template with a single T7 promoter results in the production of both sense and antisense transcripts, which differ in length by the size of the T7 promoter. Solid and dotted lines indicate DNA and RNA, respectively. ( D ) Native PAGE analysis of T7 transcripts generated using DNA template with a single T7 promoter (1), DNA template without the T7 promoter (2), and gel-purified 512B ssRNA as a template (3). RNA template alone (4) was compared with (3). ( E ) (3). * indicates unknown ssRNA byproduct from transcription.

    Article Snippet: For 3′-RACE, 3′ end of the RNA was first extended with poly A tails using the poly A polymerase (NEB).

    Techniques: Generated, Sequencing, Clear Native PAGE, Purification