DHMs were fabricated by the step-wise spotting of DNA and histone droplets into wells of standard 96-well microplates. To increase the binding efficiency of DHMs, untreated 96-well microplates (CLS3370; Corning, Corning, NY, USA) were coated with 0.001% poly-L-lysine (P4707; Sigma-Aldrich, St. Louis, MO, USA) for 10 min and rinsed once with water. Once dry, the DNA layer was prepared by combining a solution of 0.1 mg/mL lambda-phage methylated DNA (D9768; Sigma-Aldrich, St. Louis, MO, USA) with a 400 mM trehalose solution (T-104-4; Pfanstiehl, Waukegan, IL, USA) at a 1:1 ratio. Suspending the soluble DNA within a trehalose phase added stability, localization, and uniformity to the final structures. The trehalose phase could be washed off prior to testing, leaving only the chromatin fibers in each well. As a note, histones could not be pre-mixed with the DNA solution as they instantly formed fibers and therefore could not be dispensed with liquid-handling techniques.
A sub-microliter droplet, 0.6 μL, of the DNA–trehalose solution was dispensed into each well. The droplets were vitrified for 24 h in a vacuum desiccator. A 0.6 μL droplet containing a 0.5 mg/mL histone solution (H9250; Sigma-Aldrich, St. Louis, MO, USA) was then deposited over the vitrified DNA spot and dried for another 24 h in a vacuum desiccator. The concentration of DNA and histone solutions, as well as the volume, can be modified to vary the resulting morphology and compaction of the DHM chromatin structure. The binding of histones to DNA strands resulted in a defined sub-millimeter mesh of condensed chromatin fibers within the boundaries of the initial droplet area. Once fabricated, the resulting vitrified fibrous DNA structures were stable at room temperature until use.
A sub-microliter droplet, 0.6 μL, of the DNA–trehalose solution was dispensed into each well. The droplets were vitrified for 24 h in a vacuum desiccator. A 0.6 μL droplet containing a 0.5 mg/mL histone solution (H9250; Sigma-Aldrich, St. Louis, MO, USA) was then deposited over the vitrified DNA spot and dried for another 24 h in a vacuum desiccator. The concentration of DNA and histone solutions, as well as the volume, can be modified to vary the resulting morphology and compaction of the DHM chromatin structure. The binding of histones to DNA strands resulted in a defined sub-millimeter mesh of condensed chromatin fibers within the boundaries of the initial droplet area. Once fabricated, the resulting vitrified fibrous DNA structures were stable at room temperature until use.
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