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: Five sgRNAs were selected to generate single-strand nicks at positions 97, 289, 417, 601, and 799 nucleotides from the non-template strand 3′ end. sgRNAs were synthesized in vitro using the EnGen® sgRNA Synthesis Kit (New England Biolabs, #E3322V), and their target sequences are listed in .

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