Dynabeads m 280 streptavidin magnetic beads

The biotin-labeled miR-155-5p mimic, biotin-labeled mutated miR-155-5p and negative control were transfected into HSC3 and HSC4 cells. After 48 h, the cells were harvested and lysed, and the lysate was added to the Dynabeads™ M-280 streptavidin magnetic beads (Invitrogen, Carlsbad, CA, USA). The mixture was incubated at room temperature for 15-30 min. The enrichment of the co-deposited TP53INP1 RNA was determined using RT-PCR.

MCM loading, MCM phosphorylation, and MCM-DDK binding assays were performed on a linear 3 kbp ARS305-containing DNA covalently linked on one end to HpaII methyltransferase (M.HpaII) and immobilized on paramagnetic beads via a 5’ photocleavable biotin on the other end. The template was PCR-amplified from p470 using oligo DR772, which contains a photocleavable 5’ biotin moiety, and oligo DR2417, which contains a M.HpaII-binding sequence modified with 5-fluoro-2′-deoxycytidine (BioSynthesis). The purified PCR product was coupled to Dynabeads M280 streptavidin magnetic beads (Invitrogen). M.HpaII (NEB) was conjugated to bead-bound DNA in 50 mM Tris-HCl pH 7.5, 10 mM EDTA, 100 μM SAM at a ratio of 4 units M.HpaII per 90 fmol of DNA for 16 hr at 37°C with agitation. M.HpaII-conjugated bead-bound DNA was washed and stored in 10 mM HEPES-KOH pH 7.6/50 mM KOAc/1 mM DTT at 4°C.

The linear ssDNA was also used for the DNA binding assay. The preparation of linear ssDNA followed the protocol described in Marimuthu et al. [30 (link)] using the biotin-streptavidin separation method. First, a biotinylated DNA fragment containing the whole CAV genome was amplified by PCR using the EmeraldAmp Max PCR Master kit (Takara, Japan) from pCAV with a designed primer set, including the reverse biotinylated primer Biotin-CAV-r: biotin-labelled-GATTGT GCGGTGAACGAATTAG, and the forward regular primer CAV-f: GAATTCCGAGTGGTTACTATTC. After PCR amplification, the biotinylated PCR product was then immobilized on 40 μl Dynabeads M-280 Streptavidin magnetic beads (Invitrogen, USA) and incubated at 4 °C overnight. After washing the DNA-bonded beads twice with B/W buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 M NaCl), the washed beads were incubated in 150 μl elution buffer (0.1 M NaOH, 1 mM EDTA, pH 13.0) to perform alkaline denaturation. Under the high alkaline environment, the desired non-biotinylated strand can be separated from the biotinylated strand and suspended in the supernatant. After magnet adsorption, the supernatants were collected, and the linear ssDNA was further purified by a PCR clean-up kit (Geneaid, Taiwan). The linear ssDNA was diluted to 50 ng/ml with DNA-binding buffer and store at − 20 °C until required.