Ribonuclease a

Flow cytometry analysis was performed at the Immunophenotyping Platform of the Research Institute of the McGill University Health Centre (MUHC‐RI). Early apoptosis and necrosis were evaluated in cells under propranolol treatment (0, 50, 100, and 200 mmol/L) using Annexin V‐FITC kit (Cell Signaling Technology) following manufacturer's instructions. Unstained cells were used as a negative control. For cell cycle analysis, 8 × 10⁵ to 1.5 × 10⁶ cells were seeded per well in a six‐well plate. Plated cells were then starved during 12 hours in a SF RPMI 1640 media and afterward incubated with 0‐200 µmol/L propranolol. Following the 12‐hour treatment, single cell suspensions were prepared and fixed with ice‐cold 70% ethanol during 1 hour at 4°C. Subsequently, cells were washed with phosphate buffered saline and suspended in 1 µL/mL Ribonuclease A (Thermofisher) for 30 minutes at room temperature. Cells were then transferred into 5 mL polystyrene round‐bottom tubes (Corning), and stained with Propidium iodide (PI) solution (Cat P4170; Sigma) for 30 minutes at 37°C protected from light. Cell cycle was analyzed with BD FACSCanto II system and the PI solution (Sigma) was detected in the blue 488 nm wavelength, 585/42 nm filter.

Synthetic RNA analogs (1.0–5.0 µg) were digested with 0.65 mg/mL of ribonuclease A (Thermo Fisher Scientific), 4.0 µg/mL nucleases P1 (Sigma-Aldrich, St. Louis, MO, USA), 0.2 mg/mL phosphodiesterase I (Sigma-Aldrich) and 2 U alkaline phosphatase (Thermo Fisher Scientific) per 1 µg RNA in 10 mM Tris-HCl (pH 8.0), 5.0 mM MgCl2, 0.1 M KCl, and 0.02% Triton X-100, 0.1 mg/mL BSA at 37 °C for 5–20 h. The deactivation of enzymes was carried out by heating at 75 °C for 10 min and chloroform extraction. The aqueous phase containing nucleosides was collected and was dried in Eppendorf vacuum concentrator 5301 (Eppendorf, Hamburg, Germany). The pellet was dissolved in DEPC-treated water. Samples were analyzed with HPLC-UV (0.15 mL/min flow rate, 40 °C, eluent A—0.05 M aqueous solution of TEAA; eluent B—0.05 M solution of TEAA in a 20% acetonitrile: Linear gradient from 100% A to 100% B in 15 min) on a Milichrome A-02 liquid chromatograph (Econova) or HPLC-MS/MS (as described below). Control nucleosides were obtained from corresponding nucleoside triphosphates (Jena Bioscience) under the same conditions. Using HPLC-UV, it was found that synthetic RNAs obtained in the mixture with 20% m⁵CTP and 30% ΨTP contained 13–15% m⁵C and 25–28% Ψ; analogs obtained in the mixture with 50% m⁵CTP and 50% ΨTP contained 50–52% m⁵C and 52–54% Ψ.

The total genomic DNA was extracted from overnight cultures of the LAB isolates grown in MRS broth (Oxoid Ltd., Hampshire, UK). Biomass was washed twice and resuspended in 100 μL of DNase-free deionized water. DNA extraction was conducted using HigherPurity™ Bacterial Genomic DNA Isolation Kit (Canvax Biotech, S.L., Cordoba, Spain) according to the manufacturer’s instructions. Purification was performed by adding 20 µL of egg white lysozyme (Sigma Aldrich, St. Louis, MO, USA), 10 µL of proteinase K and 5 µL of ribonuclease A 10 mg/mL (Thermo Fisher Scientific Inc., Waltham, MA, USA) to the bacterial suspensions. The quality and concentration of the obtained DNA extracts were assessed through determination of absorbance at the wavelengths of 260 and 280 nm (Shimadzu UV-VIS, Shimadzu Corporation, Kyoto, Japan). The concentration of the obtained DNA extracts was calculated at 30–70 ng/mL. For the quality assessment, genomic DNA from the isolated LAB strains was mixed with a DNA loading buffer and the samples were placed in the wells of the 0.8% agarose gel. The gel electrophoresis system (VWR, Darmstadt, Germany) was powered with a DC voltage of 100 V and the separation process lasted for 40 min.