Phosphodiesterase 1

Three-microgram to 100-ng portions of RNA were digested to single nucleosides with AP (0.2 U; Sigma-Aldrich, St. Louis, MO, USA), phosphodiesterase I (0.02 U; VWR, Radnor, PA, USA), and Benzonase (0.2 U) in tris (pH 8, 5 mM)– and MgCl₂ (1 mM)–containing buffer. Furthermore, tetrahydrouridine (0.5 μg from Merck), butylated hydroxytoluene (1 μM), and pentostatin (0.1 μg) were added to protect modification (45 (link)). The mixture was incubated with the RNA for 2 hours at 37°C. Afterward, samples were filtered through 96-well filter plates (AcroPrep Advance 350 10K Omega, Pall Corporation, NY, USA) for longer than 10 min at 3000g to remove digestive enzymes.

tRNA was digested using 0.6 U nuclease P1 (Sigma-Aldrich) in the presence of ZnCl₂, ammonium acetate pH 5.3, THU, pentostatine and BHT from²¹ (link) at 50°C for one hour followed by overnight incubation at room temperature. The next day, 10 U benzonase (Sigma-Aldrich, Munich, Germany), 0.1 U phosphodiesterase I (VWR, Ismaning, Germany) and MgCl₂ in a final concentration of 1 mM were added and incubated at 37°C for one hour. Then 20 U alkaline phosphatase (Sigma-Aldrich, Munich, Germany) and the proper amount to reach a final concentration of 0.1 M Tris-HCl pH 8 were added. After 2 hours at 37 °C the digestion mix was filtered through a 10 kDa MWCO filter (AcroPrep™ Advance, 350 µl, Omega™ 10K MWCO, Pall, Dreieich, Germany) at 3000 xg for 30 minutes. 18 µL of filtrate was mixed with 2 µL of the 10x SILIS and subjected to LC-MS/MS analysis.

Total tRNA (300 ng), 18S or 28S rRNA (500 ng each) or Poly-A RNA (100 ng) were digested in aqueous digestion mix (30 μL) to single nucleosides by using 2 U alkaline phosphatase, 0.2 U phosphodiesterase I (VWR, Radnor, Pennsylvania, USA), and 2 U benzonase in Tris (pH 8, 5 mM) and MgCl₂ (1 mM) containing buffer. Furthermore, 0.5 μg tetrahydrouridine (Merck, Darmstadt, Germany), 1 μM butylated hydroxytoluene, and 0.1 μg pentostatin were added. After incubation for 2 h at 37 °C, 20 μL of LC-MS buffer A (QQQ) was added to the mixture and then filtered through 96-well filter plates (AcroPrep Advance 350 10 K Omega, PALL Corporation, New York, USA) at 3000 × g at 4 °C for 30 min. A stable isotope labelled SILIS (gen²,^{27 (link)}) was added to each filtrate and calibration solution of synthetic standards before injection into the QQQ MS.

Three-microgram to 100-ng portions of RNA were digested for 2 h at 37°C to single nucleosides with 0.2 U of alkaline phosphatase (Sigma-Aldrich), 0.02 U of phosphodiesterase I (VWR), and 0.2 U of benzonase in 5 mM Tris (pH 8) and 1 mM MgCl₂ containing buffer. Tetrahydrouridine (THU; 0.5 µg; Merck), 1 µM butylated hydroxytoluene (BHT), and 0.1 µg of pentostatin were added to protect modification (Cai et al. 2015 (link)). Afterward, samples were filtered through 96-well filter plates (AcroPrep Advance 350 10K Omega, Pall Corporation) at 3000g for >10 min to remove digestive enzymes.