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A) Schematic of DNA construct preparation for single-molecule force spectroscopy. A ∼6.8 kb plasmid was linearized with the restriction enzyme NotI and the 5’-overhangs were biotinylated using Klenow fragment. The CpG methyltransferase (MTase) M.SssI was used to methylate all cytosines at the C-5 position (5mC) within the double-stranded CG dinucleotide sequence. B) Interpolation of force-extension data shows that the averaged force-distance (F,d) curves for unmethylated (−5mC) and methylated (+5mC) DNA follow different trajectories, reflecting variation in the mechanical properties upon CpG methylation. C ) CpG methylation significantly increases the DNA contour length (L C ) (Student’s t-test, p < 0.001, n = 34 and n = 26 for -5mC and +5mC, respectively). Box plots show quartiles; whiskers show the distribution excluding outliers. D ) CpG methylation significantly increases the DNA persistence length (L p ) from 47 ± 8 nm (unmethylated) to 52 ± 7 nm (methylated) (Student’s T-test, p < 0.05, n = 34 and n = 26 for -5mC and +5mC, respectively).

Journal: bioRxiv

Article Title: Multiscale Physical Effects of CpG Methylation on DNA Mechanics, Nucleosome Wrapping, and Chromatin Condensates

doi: 10.64898/2026.04.13.715649

Figure Lengend Snippet: A) Schematic of DNA construct preparation for single-molecule force spectroscopy. A ∼6.8 kb plasmid was linearized with the restriction enzyme NotI and the 5’-overhangs were biotinylated using Klenow fragment. The CpG methyltransferase (MTase) M.SssI was used to methylate all cytosines at the C-5 position (5mC) within the double-stranded CG dinucleotide sequence. B) Interpolation of force-extension data shows that the averaged force-distance (F,d) curves for unmethylated (−5mC) and methylated (+5mC) DNA follow different trajectories, reflecting variation in the mechanical properties upon CpG methylation. C ) CpG methylation significantly increases the DNA contour length (L C ) (Student’s t-test, p < 0.001, n = 34 and n = 26 for -5mC and +5mC, respectively). Box plots show quartiles; whiskers show the distribution excluding outliers. D ) CpG methylation significantly increases the DNA persistence length (L p ) from 47 ± 8 nm (unmethylated) to 52 ± 7 nm (methylated) (Student’s T-test, p < 0.05, n = 34 and n = 26 for -5mC and +5mC, respectively).

Article Snippet: 5’oOverhangs were filled with dGTP and biotin-16-dCTP using Klenow fragment (Jena Bioscience).

Techniques: Construct, Force Spectroscopy, Plasmid Preparation, Sequencing, Methylation, CpG Methylation Assay