t7 rna polymerase  (TaKaRa)

Journal: Nucleic Acids Research

Article Title: Template nicking suppresses promoter-independent antisense transcription in IVT via R-loop-mediated strand displacement

doi: 10.1093/nar/gkaf1536

Figure Lengend Snippet: NiLoT reduces dsRNA without compromising RNA yield or integrity. ( A ) 1% agarose gel and J2 antibody-based dot blot analysis of RNA transcribed from three DNA templates: (i) partially duplexed DNA with 80 bp surrounding the T7 promoter and a single-stranded downstream region (no nick), (ii) nicked DNA (NiLoT) with a site-specific nick at 211 nt upstream of the 3′ end in the non-template strand, and (iii) fully duplexed DNA. 200 ng of each IVT RNA was analyzed. For reference, 200 ng of ssRNA and 20 ng of dsRNA markers were loaded on the gel, and 200 ng of ssRNA and 10 ng of dsRNA controls were included on the dot blot to validate antibody specificity. Total RNA was quantified using the RiboGreen assay. Values for total RNA and dsRNA were normalized to the yield obtained from the fully duplexed DNA template. Statistical comparisons were performed using one-way ANOVA with Šídák’s multiple comparisons test; **** P < .0001 and ns = not significant. ( B ) 1% agarose gel and J2 antibody-based dot blot analysis of RNA transcribed using NiLoT with nicks positioned at various distances (97, 211, 289, 417, 601, and 799 nt) from the 3′ end of the non-template strand (conditions 1–6). 200 ng of each IVT RNA was loaded for both analyses. For reference, 200 ng of ssRNA marker and 20 ng of dsRNA marker were loaded on the agarose gel. Total RNA was quantified using the RiboGreen assay and normalized to the yield obtained from the fully duplexed dsDNA template. ( C ) Comparison of NiLoT with MgCl₂-mediated dsRNA suppression. 1% agarose gel and J2 antibody-based dot blot analysis of RNA transcribed using standard dsDNA templates under two conditions: low MgCl₂ (5 mM) and NiLoT. For both analyses, 200 ng of each IVT RNA was loaded to allow direct comparison of RNA integrity and dsRNA content. For reference, 200 ng of ssRNA marker and 20 ng of dsRNA marker were loaded on the agarose gel, and 200 ng of ssRNA and 10 ng of dsRNA controls were included on the dot blot. Total RNA yield was independently quantified by RiboGreen assay, and bar graph values reflect total RNA recovered per IVT reaction. RNA and dsRNA levels were normalized to the yield obtained from the dsDNA template under 8 mM MgCl₂ conditions. Statistical comparisons were performed using one-way ANOVA with Šídák’s multiple comparisons test; **** P < .0001.

Article Snippet: For polymerase comparison experiments, three T7 RNA polymerases were used under identical reaction conditions; WT #1: Takara wild-type T7 RNA polymerase (Takara, #2540A), used throughout the study.

Techniques: Agarose Gel Electrophoresis, Dot Blot, Marker, Comparison

Journal: Nucleic Acids Research

Article Title: Template nicking suppresses promoter-independent antisense transcription in IVT via R-loop-mediated strand displacement

doi: 10.1093/nar/gkaf1536

Figure Lengend Snippet: Promoter-independent antisense transcription contributes to dsRNA. ( A ) 1% agarose gel electrophoresis analysis of eGFP RNA synthesized by T7 RNA polymerase and treated with increasing concentrations of S1 nuclease (0.04, 0.08, 0.16, and 0.24 U) or RNase III (0.002, 0.004, 0.008, and 0.016 U). ( B ) 1% agarose gel and J2 antibody-based dot blot analysis of RNA transcribed from four DNA templates: (i) ssDNA containing only the T7 promoter region (80 nt), (ii) single-stranded template strand (904 nt), (iii) partially duplexed DNA with 80 bp encompassing the T7 promoter and a single-stranded downstream region, and (iv) fully duplexed DNA. For both analyses, 200 ng of each IVT RNA was loaded. As controls, 200 ng of ssRNA and 20 ng of dsRNA markers were included on the agarose gel, and 200 ng of ssRNA and 10 ng of dsRNA were spotted on the dot blot to confirm J2 antibody specificity. Total RNA was quantified using the RiboGreen assay. Values for total RNA and dsRNA were normalized to the yield obtained from the fully duplexed dsDNA template. Statistical comparisons were performed using one-way ANOVA with Šídák’s multiple comparisons test; **** P < .0001.

Article Snippet: For polymerase comparison experiments, three T7 RNA polymerases were used under identical reaction conditions; WT #1: Takara wild-type T7 RNA polymerase (Takara, #2540A), used throughout the study.

Techniques: Agarose Gel Electrophoresis, Synthesized, Dot Blot

Journal: Nucleic Acids Research

Article Title: Template nicking suppresses promoter-independent antisense transcription in IVT via R-loop-mediated strand displacement

doi: 10.1093/nar/gkaf1536

Figure Lengend Snippet: NiLoT induces strand displacement via R-loop formation. ( A ) Schematic illustration of the proposed mechanism: T7 RNAP transcription induces R-loop formation at the nick site, resulting in displacement of the non-template strand and inhibition of antisense transcription initiation. ( B ) Dot blot analysis using the S9.6 antibody to detect RNA:DNA hybrids over a time-course IVT reaction using either dsDNA or NiLoT. Nonlinear regression was performed using a one-phase association model (least-squares method, GraphPad Prism version 8.0.2). The fitting equation was \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $Y = \ {{Y}_0} + ( {\mathrm{Plateau} - \ {{Y}_0}} ) \times ( {1 - {{e}^{ - kx}}} )$\end{document} . Detailed fitting parameters and 95% confidence intervals are provided in . ( C ) Schematic of the S1 nuclease protection assay designed to detect strand displacement by hybridization of a FAM-labeled probe to the displaced non-template strand. ( D ) 10% denaturing PAGE analysis of time-course IVT reactions using either dsDNA or NiLoT templates, following FAM-probe hybridization and S1 digestion. P/C: Positive control, generated by pre-annealing the 30 nt FAM-labeled probe with a fully complementary 30 nt oligonucleotide before S1 nuclease digestion. Band intensities of protected FAM-probe were quantified and normalized to the positive control. Statistical comparisons were performed using one-way ANOVA with Šídák’s multiple comparisons test.

Article Snippet: For polymerase comparison experiments, three T7 RNA polymerases were used under identical reaction conditions; WT #1: Takara wild-type T7 RNA polymerase (Takara, #2540A), used throughout the study.

Techniques: Inhibition, Dot Blot, Hybridization, Labeling, Positive Control, Generated

Journal: Nucleic Acids Research

Article Title: Template nicking suppresses promoter-independent antisense transcription in IVT via R-loop-mediated strand displacement

doi: 10.1093/nar/gkaf1536

Figure Lengend Snippet: NiLoT suppresses dsRNA across different template formats, transcript sizes, T7 RNAP variants, and modified NTPs. ( A ) 1% agarose gel and J2 antibody-based dot blot analysis of RNA transcribed using either PCR-amplified or linearized plasmid DNA templates, prepared as dsDNA or NiLoT. 200 ng of each IVT RNA was loaded for both analyses. For reference, 200 ng of ssRNA marker and 20 ng of dsRNA marker were loaded on the agarose gel. Total RNA was quantified using the RiboGreen assay. Values for total RNA and dsRNA were normalized to the yield obtained from the PCR-based dsDNA template with unmodified NTPs. Statistical comparisons were performed using two-tailed, unpaired t -test; ** P < .01. ( B ) Agarose gel electrophoresis and J2 dot blot analysis of IVT products from 1.0 kb and 3.9 kb templates. RNAs were synthesized using either dsDNA or NiLoT templates encoding 1.0 kb (eGFP) or 3.9 kb (SARS-CoV-2 spike) transcripts. For each condition, 200 ng of IVT RNA was analyzed by 1% agarose gel electrophoresis and by dot blot using the J2 antibody. For reference, 200 ng of ssRNA and 20 ng of dsRNA were loaded on the gel as controls. Total RNA concentrations were measured using the RiboGreen assay, and dsRNA levels were quantified from dot blots. All values were normalized to the total RNA and dsRNA yield obtained from the corresponding dsDNA-derived transcripts. ( C ) Representative 1% agarose gel and J2 antibody-based dot blot analyses of IVT RNA transcribed from either conventional dsDNA or NiLoT templates using three T7 RNA polymerases: WT #1 (vendor 1, wild-type), WT #2 (vendor 2, wild-type), and Mutant #1 (vendor 1, engineered low-dsRNA variant). For each condition, 200 ng of RNA was loaded per assay. Reference lanes include 200 ng ssRNA and 20 ng dsRNA markers (gel), and 200 ng ssRNA and 10 ng dsRNA controls (dot blot). Total RNA was quantified using the RiboGreen assay. RNA yield and dsRNA content were normalized to the output from the dsDNA template transcribed with WT #1. Statistical comparisons were performed using a two-tailed, unpaired t -test; ** P < .01 and **** P < .0001. ( D ) Chemical structures and abbreviations of modified nucleotides used for IVT: Ψ (pseudouridine), m 1 Ψ (N1-methylpseudouridine), m 5 C (5-methylcytidine), and m 6 A (N6-methyladenosine). ( E ) 1% agarose gel and J2 antibody–based dot blot analysis of eGFP mRNA transcribed using either dsDNA or NiLoT templates, with or without modified NTPs shown in panel ( D ). 200 ng of IVT mRNA was loaded per lane/spot. For reference, 200 ng of ssRNA marker and 20 ng of dsRNA marker were loaded on the agarose gel, and 200 ng of ssRNA and 10 ng of dsRNA controls were included on the dot blot. The J2 blot confirms that NiLoT-derived mRNAs contain markedly lower levels of dsRNA contaminants than dsDNA-derived counterparts. Total RNA was quantified using the RiboGreen assay. Values for total RNA and dsRNA were normalized to the yield obtained from the dsDNA template with unmodified NTPs.

Article Snippet: For polymerase comparison experiments, three T7 RNA polymerases were used under identical reaction conditions; WT #1: Takara wild-type T7 RNA polymerase (Takara, #2540A), used throughout the study.

Techniques: Modification, Agarose Gel Electrophoresis, Dot Blot, Amplification, Plasmid Preparation, Marker, Two Tailed Test, Synthesized, Derivative Assay, Mutagenesis, Variant Assay

Journal: Nucleic Acids Research

Article Title: Template nicking suppresses promoter-independent antisense transcription in IVT via R-loop-mediated strand displacement

doi: 10.1093/nar/gkaf1536

Figure Lengend Snippet: NiLoT reduces dsRNA without compromising RNA yield or integrity. ( A ) 1% agarose gel and J2 antibody-based dot blot analysis of RNA transcribed from three DNA templates: (i) partially duplexed DNA with 80 bp surrounding the T7 promoter and a single-stranded downstream region (no nick), (ii) nicked DNA (NiLoT) with a site-specific nick at 211 nt upstream of the 3′ end in the non-template strand, and (iii) fully duplexed DNA. 200 ng of each IVT RNA was analyzed. For reference, 200 ng of ssRNA and 20 ng of dsRNA markers were loaded on the gel, and 200 ng of ssRNA and 10 ng of dsRNA controls were included on the dot blot to validate antibody specificity. Total RNA was quantified using the RiboGreen assay. Values for total RNA and dsRNA were normalized to the yield obtained from the fully duplexed DNA template. Statistical comparisons were performed using one-way ANOVA with Šídák’s multiple comparisons test; **** P < .0001 and ns = not significant. ( B ) 1% agarose gel and J2 antibody-based dot blot analysis of RNA transcribed using NiLoT with nicks positioned at various distances (97, 211, 289, 417, 601, and 799 nt) from the 3′ end of the non-template strand (conditions 1–6). 200 ng of each IVT RNA was loaded for both analyses. For reference, 200 ng of ssRNA marker and 20 ng of dsRNA marker were loaded on the agarose gel. Total RNA was quantified using the RiboGreen assay and normalized to the yield obtained from the fully duplexed dsDNA template. ( C ) Comparison of NiLoT with MgCl₂-mediated dsRNA suppression. 1% agarose gel and J2 antibody-based dot blot analysis of RNA transcribed using standard dsDNA templates under two conditions: low MgCl₂ (5 mM) and NiLoT. For both analyses, 200 ng of each IVT RNA was loaded to allow direct comparison of RNA integrity and dsRNA content. For reference, 200 ng of ssRNA marker and 20 ng of dsRNA marker were loaded on the agarose gel, and 200 ng of ssRNA and 10 ng of dsRNA controls were included on the dot blot. Total RNA yield was independently quantified by RiboGreen assay, and bar graph values reflect total RNA recovered per IVT reaction. RNA and dsRNA levels were normalized to the yield obtained from the dsDNA template under 8 mM MgCl₂ conditions. Statistical comparisons were performed using one-way ANOVA with Šídák’s multiple comparisons test; **** P < .0001.

Article Snippet: For polymerase comparison experiments, three T7 RNA polymerases were used under identical reaction conditions; WT #1: Takara wild-type T7 RNA polymerase (Takara, #2540A), used throughout the study.

Techniques: Agarose Gel Electrophoresis, Dot Blot, Marker, Comparison

Journal: Nucleic Acids Research

Article Title: Template nicking suppresses promoter-independent antisense transcription in IVT via R-loop-mediated strand displacement

doi: 10.1093/nar/gkaf1536

Figure Lengend Snippet: Promoter-independent antisense transcription contributes to dsRNA. ( A ) 1% agarose gel electrophoresis analysis of eGFP RNA synthesized by T7 RNA polymerase and treated with increasing concentrations of S1 nuclease (0.04, 0.08, 0.16, and 0.24 U) or RNase III (0.002, 0.004, 0.008, and 0.016 U). ( B ) 1% agarose gel and J2 antibody-based dot blot analysis of RNA transcribed from four DNA templates: (i) ssDNA containing only the T7 promoter region (80 nt), (ii) single-stranded template strand (904 nt), (iii) partially duplexed DNA with 80 bp encompassing the T7 promoter and a single-stranded downstream region, and (iv) fully duplexed DNA. For both analyses, 200 ng of each IVT RNA was loaded. As controls, 200 ng of ssRNA and 20 ng of dsRNA markers were included on the agarose gel, and 200 ng of ssRNA and 10 ng of dsRNA were spotted on the dot blot to confirm J2 antibody specificity. Total RNA was quantified using the RiboGreen assay. Values for total RNA and dsRNA were normalized to the yield obtained from the fully duplexed dsDNA template. Statistical comparisons were performed using one-way ANOVA with Šídák’s multiple comparisons test; **** P < .0001.

Article Snippet: For polymerase comparison experiments, three T7 RNA polymerases were used under identical reaction conditions; WT #1: Takara wild-type T7 RNA polymerase (Takara, #2540A), used throughout the study.

Techniques: Agarose Gel Electrophoresis, Synthesized, Dot Blot

Journal: Nucleic Acids Research

Article Title: Template nicking suppresses promoter-independent antisense transcription in IVT via R-loop-mediated strand displacement

doi: 10.1093/nar/gkaf1536

Figure Lengend Snippet: NiLoT induces strand displacement via R-loop formation. ( A ) Schematic illustration of the proposed mechanism: T7 RNAP transcription induces R-loop formation at the nick site, resulting in displacement of the non-template strand and inhibition of antisense transcription initiation. ( B ) Dot blot analysis using the S9.6 antibody to detect RNA:DNA hybrids over a time-course IVT reaction using either dsDNA or NiLoT. Nonlinear regression was performed using a one-phase association model (least-squares method, GraphPad Prism version 8.0.2). The fitting equation was \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $Y = \ {{Y}_0} + ( {\mathrm{Plateau} - \ {{Y}_0}} ) \times ( {1 - {{e}^{ - kx}}} )$\end{document} . Detailed fitting parameters and 95% confidence intervals are provided in . ( C ) Schematic of the S1 nuclease protection assay designed to detect strand displacement by hybridization of a FAM-labeled probe to the displaced non-template strand. ( D ) 10% denaturing PAGE analysis of time-course IVT reactions using either dsDNA or NiLoT templates, following FAM-probe hybridization and S1 digestion. P/C: Positive control, generated by pre-annealing the 30 nt FAM-labeled probe with a fully complementary 30 nt oligonucleotide before S1 nuclease digestion. Band intensities of protected FAM-probe were quantified and normalized to the positive control. Statistical comparisons were performed using one-way ANOVA with Šídák’s multiple comparisons test.

Article Snippet: For polymerase comparison experiments, three T7 RNA polymerases were used under identical reaction conditions; WT #1: Takara wild-type T7 RNA polymerase (Takara, #2540A), used throughout the study.

Techniques: Inhibition, Dot Blot, Hybridization, Labeling, Positive Control, Generated

Journal: Nucleic Acids Research

Article Title: Template nicking suppresses promoter-independent antisense transcription in IVT via R-loop-mediated strand displacement

doi: 10.1093/nar/gkaf1536

Figure Lengend Snippet: NiLoT suppresses dsRNA across different template formats, transcript sizes, T7 RNAP variants, and modified NTPs. ( A ) 1% agarose gel and J2 antibody-based dot blot analysis of RNA transcribed using either PCR-amplified or linearized plasmid DNA templates, prepared as dsDNA or NiLoT. 200 ng of each IVT RNA was loaded for both analyses. For reference, 200 ng of ssRNA marker and 20 ng of dsRNA marker were loaded on the agarose gel. Total RNA was quantified using the RiboGreen assay. Values for total RNA and dsRNA were normalized to the yield obtained from the PCR-based dsDNA template with unmodified NTPs. Statistical comparisons were performed using two-tailed, unpaired t -test; ** P < .01. ( B ) Agarose gel electrophoresis and J2 dot blot analysis of IVT products from 1.0 kb and 3.9 kb templates. RNAs were synthesized using either dsDNA or NiLoT templates encoding 1.0 kb (eGFP) or 3.9 kb (SARS-CoV-2 spike) transcripts. For each condition, 200 ng of IVT RNA was analyzed by 1% agarose gel electrophoresis and by dot blot using the J2 antibody. For reference, 200 ng of ssRNA and 20 ng of dsRNA were loaded on the gel as controls. Total RNA concentrations were measured using the RiboGreen assay, and dsRNA levels were quantified from dot blots. All values were normalized to the total RNA and dsRNA yield obtained from the corresponding dsDNA-derived transcripts. ( C ) Representative 1% agarose gel and J2 antibody-based dot blot analyses of IVT RNA transcribed from either conventional dsDNA or NiLoT templates using three T7 RNA polymerases: WT #1 (vendor 1, wild-type), WT #2 (vendor 2, wild-type), and Mutant #1 (vendor 1, engineered low-dsRNA variant). For each condition, 200 ng of RNA was loaded per assay. Reference lanes include 200 ng ssRNA and 20 ng dsRNA markers (gel), and 200 ng ssRNA and 10 ng dsRNA controls (dot blot). Total RNA was quantified using the RiboGreen assay. RNA yield and dsRNA content were normalized to the output from the dsDNA template transcribed with WT #1. Statistical comparisons were performed using a two-tailed, unpaired t -test; ** P < .01 and **** P < .0001. ( D ) Chemical structures and abbreviations of modified nucleotides used for IVT: Ψ (pseudouridine), m 1 Ψ (N1-methylpseudouridine), m 5 C (5-methylcytidine), and m 6 A (N6-methyladenosine). ( E ) 1% agarose gel and J2 antibody–based dot blot analysis of eGFP mRNA transcribed using either dsDNA or NiLoT templates, with or without modified NTPs shown in panel ( D ). 200 ng of IVT mRNA was loaded per lane/spot. For reference, 200 ng of ssRNA marker and 20 ng of dsRNA marker were loaded on the agarose gel, and 200 ng of ssRNA and 10 ng of dsRNA controls were included on the dot blot. The J2 blot confirms that NiLoT-derived mRNAs contain markedly lower levels of dsRNA contaminants than dsDNA-derived counterparts. Total RNA was quantified using the RiboGreen assay. Values for total RNA and dsRNA were normalized to the yield obtained from the dsDNA template with unmodified NTPs.

Article Snippet: For polymerase comparison experiments, three T7 RNA polymerases were used under identical reaction conditions; WT #1: Takara wild-type T7 RNA polymerase (Takara, #2540A), used throughout the study.

Techniques: Modification, Agarose Gel Electrophoresis, Dot Blot, Amplification, Plasmid Preparation, Marker, Two Tailed Test, Synthesized, Derivative Assay, Mutagenesis, Variant Assay