Superose 12 10 300

Unmodified, recombinant human histones H2A or H2A(K119C), H2B, H3, H3(K56Q), and H4 were expressed and purified as described [19 (link)]. Histones H3(K115ac, K122ac) and H4(K77ac, K79ac) were produced by expressed protein ligation as described [39 (link), 41 (link), 42 (link)]. The synthetic acetylations were confirmed by mass spectrometry analysis (Fig 1). Octamers were refolded at equimolar histone concentrations and purified by Superose 12 10/300 (GE Healthcare) size exclusion chromatography in 10 mM Tris-HCl pH 7.5, 2 M NaCl, 1 mM EDTA. Nucleosomes were reconstituted with Cy5 labelled 145 bp D02 DNA or 147 bp 601 DNA and histone octamer at a 1:1 molar ratio by double dialysis against 5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 mM benzamidine [43 (link)]. The products were separated by sucrose gradient velocity centrifugation [43 (link)]. Gradient fractions were analyzed by separation on a native polyacrylamide gel electrophoresis (PAGE) and imaged using a Typhoon 9410 variable mode fluorescent imager (GE Healthcare) or a Sapphire Biomolecular Imager (Azure Biosystems) (Fig 2). Fractions with fluorescent NPS DNA bound by histone octamer were combined. The sample buffer was exchanged to 5 mM Tris-HCl pH 7.5, 0.5 mM EDTA and nucleosomes were concentrated with Amicon Ultra centrifugal filters (EMD Millipore). Nucleosomes were stored at 4°C.

Analysis of the aggregation status of labeled and not labeled γ-C and KTI (2 mg/mL) was performed by using the serial system composed by a HPLC pump (515 HPLC pump, Waters), a SEC column (Superose 12, 10/300, GE Healthcare, Milan, Italy), a UV spectrophotometer (Dual λ absorbance detector 2487, Waters), a multi-angle light scattering device (DAWN HELEOS, Wyatt, Santa Barbara, CA, USA), provided with a fast photon counter (QELS), and a differential refractometer (Waters), as previously described in Caparo et al. [21 (link)]. Two hundred µL of samples, previously centrifuged to remove the insoluble fraction, were injected with PBS (pH 7.5) as mobile phase, at a flow rate of 0.5 mL/min. Molar masses were calculated by means of the Astra V software vs. 5.3.4.20 (Wyatt), using a dn/dc value of 0.185 for γ-C samples.

Intasomes were assembled as previously described [23 (link)]. Briefly, 50 mM Bis-tris propane, pH 7.5, 500 mM NaCl, 120 μM PFV IN, and 50 μM vDNA were combined in a total volume of 150 μL and dialyzed overnight at 18 °C against 20 mM Bis-tris propane, pH 7.5, 200 mM NaCl, 2 mM DTT, and 25 μM ZnCl₂. The intasome aggregates were solubilized by increasing the concentration of NaCl from 200 to 320 mM and incubating on ice. The intasomes were purified by size exclusion chromatography using a Superose 12 10/300 (GE Healthcare, Chicago, IL, USA) equilibrated with 20 mM Bis-tris propane, pH 7.5, 320 mM NaCl, and 10% glycerol. Fractions containing intasomes were aliquoted, snap frozen with liquid nitrogen, and stored at −80 °C. PFV intasomes appear to retain activity for one year at −80 °C.