Journal: bioRxiv
Article Title: Single-Molecule Imaging Reveals Transcription-Driven Supercoiling in Unconstrained DNA
doi: 10.1101/2025.11.08.687369
Figure Lengend Snippet: (a) Schematic of the experimental setup. A 20 kb DNA molecule is asymmetrically labeled with digoxigenin (DIG) at the 5′ end of one strand and biotin at the 5′ end of the complementary strand. The DIG-labeled end is tethered to the surface of a µ-Slide that coated with anti-DIG, while the biotin-labeled end is attached to a streptavidin-coated paramagnetic bead. The DNA molecule is stretched horizontally by a stack of magnets positioned atop and alongside of the flow channel. DNA conformations are monitored in real time using an inverted fluorescence microscope. (b) Schematic of the 20 kb DNA construct, mapped to a normalized [0, 1] scale for visualization. The sequence is derived from the E. coli genome and includes three synthetic promoters (all in the same orientation) driving fluorescent reporters near the 0.6 position. mB: tmBroccoli RNA aptamer. (c) Representative composite image of a tethered DNA molecule under a magnetic field, showing bright-field (gray) and Cy5-labeled DNA (red). The observation was performed at 37°C in transcription buffer. Scale bar: 5 µm. (d) Visualization of DNA and synthesized tmBroccoli mRNA in transcription buffer supplemented with RNAP and DFHBI fluorophore, after a 45-minute incubation at 37°C. Images from left to right: bright-field, tmBroccoli fluorescence (mRNA), Cy5 fluorescence (DNA), and a merged composite. Scale bar: 2 µm. (e) Real-time analysis of DNA conformational changes in the presence of transcription buffer and RNAP at 37°C, presented as a time-series image marked with fluorescence intensity peaks. The illustration figures (a and b) were created in BioRender ( https://BioRender.com ).
Article Snippet: 80161, 80601, or 80621) were coated with 30 μg/mL anti-digoxigenin antibody (Bio-Rad Laboratories Inc., USA, Cat. No. 3210-0488) by incubating overnight at 4 °C.
Techniques: Labeling, Fluorescence, Microscopy, Construct, Sequencing, Derivative Assay, Synthesized, Incubation
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![(a) Schematic of the experimental setup. A 20 kb DNA molecule is asymmetrically labeled with <t>digoxigenin</t> (DIG) at the 5′ end of one strand and biotin at the 5′ end of the complementary strand. The DIG-labeled end is tethered to the surface of a µ-Slide that coated with <t>anti-DIG,</t> while the biotin-labeled end is attached to a streptavidin-coated paramagnetic bead. The DNA molecule is stretched horizontally by a stack of magnets positioned atop and alongside of the flow channel. DNA conformations are monitored in real time using an inverted fluorescence microscope. (b) Schematic of the 20 kb DNA construct, mapped to a normalized [0, 1] scale for visualization. The sequence is derived from the E. coli genome and includes three synthetic promoters (all in the same orientation) driving fluorescent reporters near the 0.6 position. mB: tmBroccoli RNA aptamer. (c) Representative composite image of a tethered DNA molecule under a magnetic field, showing bright-field (gray) and Cy5-labeled DNA (red). The observation was performed at 37°C in transcription buffer. Scale bar: 5 µm. (d) Visualization of DNA and synthesized tmBroccoli mRNA in transcription buffer supplemented with RNAP and DFHBI fluorophore, after a 45-minute incubation at 37°C. Images from left to right: bright-field, tmBroccoli fluorescence (mRNA), Cy5 fluorescence (DNA), and a merged composite. Scale bar: 2 µm. (e) Real-time analysis of DNA conformational changes in the presence of transcription buffer and RNAP at 37°C, presented as a time-series image marked with fluorescence intensity peaks. The illustration figures (a and b) were created in BioRender ( https://BioRender.com ).](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_69/10__1101_slash_2025__11__08__687369/10__1101_slash_2025__11__08__687369___F1.large.jpg)