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: To confirm S9.6 specificity, IVT solution (without DNase I treatment) was treated with 10 U RNase H (New England Biolabs, #M0297S) at 37°C for 30 min. RNA was purified with the Monarch® RNA Cleanup Kit and used for dot blot.

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: To confirm S9.6 specificity, IVT solution (without DNase I treatment) was treated with 10 U RNase H (New England Biolabs, #M0297S) at 37°C for 30 min. RNA was purified with the Monarch® RNA Cleanup Kit and used for dot blot.

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