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( A ) Illustration of the single molecule imaging assay, which combines microfluidics, surface immobilization and <t>TIRF</t> microscopy. ( B ) Typical field-of-view showing individual flow-aligned DNA strands (green) with MpARF2 nanoclusters (magenta) bound to the DNA templates. [MpARF2] = 1 nM. Scale bar: 5 µm. Inset: filtered Region Of Interest (ROI). ( C ) Kymograph of a nanocluster binding to DNA, illustrating the high temporal stability of a nanocluster once bound, with virtually no lateral movement. ( D ) Binding isotherm of nanoclusters, determined from the area fraction of protein covering the DNA, ϕ . Inset: binary-filtered ROIs used in coverage analysis. ( E ) Spatial distribution of binding events along individual DNA strands (solid) compared to the predicted IR7 positions (dashed). Inset: enrichment of protein on nonspecific (green) and IR7-containing (orange) DNA templates, measured as the ratio of protein intensity on DNA compared to the surrounding background. *** P <0.001; Welch’s t-test.
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( A ) Illustration of the single molecule imaging assay, which combines microfluidics, surface immobilization and TIRF microscopy. ( B ) Typical field-of-view showing individual flow-aligned DNA strands (green) with MpARF2 nanoclusters (magenta) bound to the DNA templates. [MpARF2] = 1 nM. Scale bar: 5 µm. Inset: filtered Region Of Interest (ROI). ( C ) Kymograph of a nanocluster binding to DNA, illustrating the high temporal stability of a nanocluster once bound, with virtually no lateral movement. ( D ) Binding isotherm of nanoclusters, determined from the area fraction of protein covering the DNA, ϕ . Inset: binary-filtered ROIs used in coverage analysis. ( E ) Spatial distribution of binding events along individual DNA strands (solid) compared to the predicted IR7 positions (dashed). Inset: enrichment of protein on nonspecific (green) and IR7-containing (orange) DNA templates, measured as the ratio of protein intensity on DNA compared to the surrounding background. *** P <0.001; Welch’s t-test.

Journal: bioRxiv

Article Title: Nanoclustering of a plant transcription factor enables strong yet specific DNA binding

doi: 10.1101/2025.11.05.686732

Figure Lengend Snippet: ( A ) Illustration of the single molecule imaging assay, which combines microfluidics, surface immobilization and TIRF microscopy. ( B ) Typical field-of-view showing individual flow-aligned DNA strands (green) with MpARF2 nanoclusters (magenta) bound to the DNA templates. [MpARF2] = 1 nM. Scale bar: 5 µm. Inset: filtered Region Of Interest (ROI). ( C ) Kymograph of a nanocluster binding to DNA, illustrating the high temporal stability of a nanocluster once bound, with virtually no lateral movement. ( D ) Binding isotherm of nanoclusters, determined from the area fraction of protein covering the DNA, ϕ . Inset: binary-filtered ROIs used in coverage analysis. ( E ) Spatial distribution of binding events along individual DNA strands (solid) compared to the predicted IR7 positions (dashed). Inset: enrichment of protein on nonspecific (green) and IR7-containing (orange) DNA templates, measured as the ratio of protein intensity on DNA compared to the surrounding background. *** P <0.001; Welch’s t-test.

Article Snippet: Microfluidic chips were mounted on a custom built TIRF microscope ( ) and connected to a distributor valve (MUX-D-12, Elveflow) using Tygon tubing.

Techniques: Imaging, Microscopy, Binding Assay