Prostar 325

A total ion chromatogram (TIC) was obtained using an Agilent Varian ProStar HPLC system equipped with a ProStar 325 UV-Vis detector (ProStar 325, Varian, Palo Alto, USA) and a C18 column (Pursuit, Agilent Technologies, Inc., Santa Clara, CA, USA), which was protected with a guard column. For the mobile phase, pump A utilized Milli-Q water (Millipore-Merck, Darmstadt, Germany) acidified with 0.1% acetic acid; Pump C used HPLC-grade acetonitrile (Merck, Darmstadt, Germany) and pump B used methanol (HPLC grade, Merck, Darmstadt, Germany) to clean the column. The program used for elution was carried out from 0 to 15 min with gradient ranged from 95% A and 0.5% C to 72% A and 28% C. Then the gradient ranged from 72% A and 28% C to 32% A to 68% C in the interval between 16 and 55 min, reaching the concentration of 100% for C in the interval of 55–60 min. The detection of compounds of interest was performed at a wavelength (λ) of 254 nm, and the volume injected was 20 μl, with the mobile phase flow set at 0.6 ml/min.

The four glucuronides (Figure 1) were obtained by hemisynthesis as previously described [50 (link)] with some modifications. One molar equivalent εVin was dissolved in ethanol and then mixed with 20 molar equivalents of K₂CO₃. Six molar equivalents of acetobromo-glucuronic acid methyl-ester dissolved in ethanol were added, and the reaction mixture was stirred at 50 °C for 24 h under agitation. The reaction was then stopped by lowering the pH below 5 with 1% formic acid. Solvents were evaporated with a vacuum rotatory evaporator before redissolving sample in methanol:water (50:50, v:v). The mixture was injected into semi-preparative high-performance liquid chromatography equipped with a binary pump and UV-VIS detector used at 320 nm (Prostar 325, Varian, Palo Alto, CA, USA). Solvents A and B were ultra-pure water and acetonitrile:water (50:50, v:v), respectively, both acidified with 0.025% of TFA. The gradient, used with a 2 mL/min flow rate, was as follows: 0 min, 38% B; 0–55 min, 38–48% B; 55–57 min, 48–100% B; 57–60 min, 100% B; 60–62 min, 100–38% B; 62–64 min; 38% B. Metabolites were individually collected and then injected in UPLC-DAD-MS to ensure their purity before being freeze-dried. For method validation and a calibration curve, V2G (Figure 1) was used as it is the most predominant isomer found in rats [33 (link)].

The unmodified RNA oligomers were synthesized in Division of Bioorganic Chemistry of CMMS PAS Łódź by dr Anna Maciaszek using Gene World automated DNA/RNA synthesizer at a 0.2 μmol scale, with commercially available LCA–CPG supports (Biosearch Technologies, Inc., Petaluma, CA) and the standard phosphoramidite monomers (Glen Research, Sterling, VA, USA). The phosphoramidites were dissolved in anhydrous CH3CN at concentration of 0.07 M, and a 0.25 M solution of 5-Benzylmercapto-1H-tetrazole in anhydrous CH3CN was used as an activator. Oligonucleotides were deprotected and isolated using a binary Varian HPLC system, consisting of two PrepStar 218 pumps and a ProStar 325 UV/VIS detector set at 260 nm. A reverse phase HPLC column (PRP-1, C18, 7 μm, 305 × 7 mm, Hamilton, Reno, NV, USA) was eluted with a 1% min − 1 gradient of CH3CN in 0.1 M TEAB (pH 7.3) at a flow rate 2.5 ml min − 1. The miR494 and miR146a mimics sequences: miR494-5p 5ʹ-AGGUUGUCCGUGUUGUCUUCUC-3ʹ; miR494-3p 5ʹ-UGAAACAUACACGGGAAACCUC-3ʹ; miR146a-5p 5ʹ-UGAGAACUGAAUUCCAUGGGUU-3ʹ; miR146a-3p 5ʹ-CCUCUGAAAUUCAGUUCUUCAG-3’.