Re 206

Total HM carbohydrates were analyzed using GPC. Glycans were separated by the GPC stationary phase and eluted according to size and charge. Neutral mono-, di-, and oligosaccharides, and acidic oligosaccharides with different degrees of polymerization (DP), could be detected. HM carbohydrate solution was prepared by dissolving 0.2 g/mL of total HM carbohydrates in ultrapure water (Sartorius Arium Pro) containing 2% (v/v) 2-propanol at 37 °C. Five milliliters of 0.2 µM filter-sterilized HM carbohydrate solution were injected for each GPC run. The sample loop was cleaned by ultrapure water prior to analysis. Two connected Kronlab ECO50 columns (5 × 110 cm) packed with Toyopearl HW 40 (TOSOH BIOSCIENCE) were used. Milli-Q water was maintained at 50 °C using a heating bath (Lauda, RE 206) for column equilibration. Milli-Q water containing 2% (v/v) 2-propanol was used as the eluent. The flow rate of the eluent was set at 1.65 mL/min. Eluting glycans were monitored by refractive index detection (Shodex, RI-101). The resulting chromatograms were analyzed by using Chromeleon^® software (Thermo Scientific).

Thermal denaturation melting experiments were performed in a UV-Vis spectrophotometer (Shimadzu UV-2401PC). 900 μL of RNA-DNA hybrid nanoshape sample solution at 0.5 μM concentration was prepared with degassed buffer solution, transferred in a 1 cm quartz cuvette and covered with an oil layer to prevent evaporation. The temperature was raised from 15 to 80 °C at a rate of 0.5 °C per minute using a controlled temperature recirculating water system (Lauda RE 206), measuring the absorption every 1 °C at 260 nm.

Cloud point measurements were carried out in a high pressure, variable volume view cell equipped with a sapphire window on the end for visual observations. The cell was equipped with a pressure transducer and an internal thermocouple. It was thermostated by a water/isopropanol alcohol mixture delivered by a Lauda RE206 circulating pump. CO₂ is delivered by an ISCO 260D automatic syringe pump. An amount of 55 mg of polymer was weighed and transferred to the cell along with a clean stirring bar at a starting volume of 6.40 mL. Subsequently, the cell was fed with CO₂ at about 25 °C and 10.9 MPa. Then, the cell was heated to 65 °C (taking care to adjust the volume of the cell in order to stay below a pressure of 35 MPa; safety rupture disk at 50 MPa). Cloud points (one-phase/two-phase transition) were obtained by decreasing the pressure of the cell by increasing the cell volume through a hand-driven piston after 15 min of stirring at a given temperature. The cell was cooled by steps of 5 °C down to 25 °C. The uncertainty of the cloud point pressure was ±0.5 MPa.