Fluorescein 5 thiosemicarbazide

Chemicals and reagents were obtained from the following commercial sources:
2',7'-dichlorodihydrofluorescein diacetate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), RPMI 1600 and an antibiotic solution containing 10,000 units of penicillin, 10 mg of streptomycin and 25 μg of amphotericin B per mL were purchased from Thermo Fisher Scientific, Gibco (Grand Island, NY, USA). BlockAce was purchased from DS Pharma Biomedical (Japan), Biotin-HABP (biotinylated linker protein hyaluronan, 0.25mg/mL) was purchased from Hokudo Co. (Japan), Anti-Human Collagen type 1 antibody was purchased from Rockland Immunochemicals (US), Anti-human MMP-1, anti-IgG/HRP, and Streptavidin-HRP were purchased from R&D Systems (Japan), Can Get Signal was purchased from Toyobo Co. (Japan), EZ West blue dye was purchased from Atto Corp. (Japan), Chlorogenic acid (CGA) was purchased from Cayman Chemical (US). Fluorescein-5-thiosemicarbazide and sodium hyaluronate, 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The bicinchoninic acid (BCA) protein assay reagent kit was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

5 nmol tRNA were incubated 50 min in 400 µL volume, containing 100 mM NaOAc pH5.5, 2.5 mM KIO₄ at 4 °C. After ethanol precipitation the labelling reaction was carried out in 100 mM NaOAc pH 5.5 with 1 mM fluorescein-5-thiosemicarbazide (Sigma Aldrich). RNA was again ethanol precipitated and dissolved in H₂O. Unreacted fluorophore was removed using Zeba spin desalting columns (ThermoFisher Scientific). spDnmt2 was desalted into assay buffer 20 mM Tris/HCl, 50 mM NaCl prior to the experiment. Anisotropy measurements were performed using a Fluoromax III (Horiba Jobin Yvon) fluorimeter and data was evaluated with FluorEssence software (Horiba Jobin Yvon).