Dialysis unit

Loading efficiency (LE) and loading capacity (LC) were analyzed by high-performance liquid chromatography (HPLC; Agilent 1100 [Beijing, China]; flow rate: 1.0 mL/min; mobile phase: acetonitrile/water 53/47; column: Diamonsil™ C18, 4.6×200 mm, 5 µm; detection wavelength: 230 nm, and temperature: 25°C). The in vitro release of Doc was carried out using a dialysis unit (molecular weight cutoff [MWCO] 20,000 Da; Thermo Fisher Scientific, Waltham, MA, USA) at 37°C. Briefly, 1 mL of GNRs/DocL-R (concentration of GNRs 0.4 nM) was sealed to a dialysis unit. The dialysis unit was submerged into 50 mL PBS that contained 0.5% (w/w) Tween 80 (pH 7.4 or pH 5.0). The dialysis unit with GNRs/DocL-R (concentration of GNRs 0.4 nM) + laser was exposed to laser radiation (808 nm) for 10 min. At set time points (0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 h), 1 mL of release media was withdrawn and 1 mL of fresh 0.5% (w/w) Tween 80 PBS solution was refilled. The obtained release samples were centrifuged at 10,000 rpm for 10 min. Supernatants were analyzed by HPLC to calculate the cumulative release (%) of Doc.
To test thermal response, GNRs/DocL-R was exposed to laser radiation at 808 nm for 10 min. Temperature increase was recorded at set time points. Curves of thermal response to laser radiation were plotted.

The purified DBD and the annealed DNA fragment were dialyzed separately overnight against the SEC buffer using a dialysis unit (Thermo Fisher Scientific) with a 10 kDa cutoff membrane. Samples were diluted to the working concentration and degassed prior to the experiments. ITC data were obtained on a Malvern Panalytical PEAQ-ITC microcalorimeter. Measurements were performed by titrating the protein (cell) with DNA (syringe). The heat of dilution for the protein and DNA were obtained by titrating them separately against the buffer. Experiments were performed at 10, 15, 20, 25 and 30°C for the embERRE/IR3 DNA in order to evaluate the temperature-dependency of the protein/DNA interaction. Data were processed with the MicroCal PEAQ-ITC Analysis software and with the AFFINImeter v 1.2.3 software using a two binding site model (50 (link)). ITC data were also collected for the binding of the ERRα DBD to IR3/ERE DNA and to the ERRE extended half-site DNA (at 15 and 20°C, respectively, in order to optimize the signal/noise ratio for these DNA sequence) and to the ERRE extended half-site DNA (at 20°C).