Guanosine

Free adenosine, guanosine, and cytosine standards were purchased from Acros Organics and uridine was purchased from Tokyo Chemical Industry. To generate Ψ and m1Ψ standards, 50 nmol of UTP (NEB), ΨTP (ApexBio), and m1ΨTP (ApexBio) were diluted to a final volume of 25 μL in 100 mM Tris pH 7.5 buffer and incubated with 10 U of calf intestinal alkaline phosphatase (CIP; NEB) at 37°C for 4 h. For analysis of the synthetic RNA, 1 nmol of synthetic RNA was digested by nuclease P1 (Millipore Sigma, 10 Units) at 50°C for 150 min. Afterward, Tris pH 7.5 was added to a final concentration of 100 mM to adjust the pH and 10 U of CIP was added. The reaction was incubated at 37°C for an additional 90 min to dephosphorylate the nucleotide 5′-monophosophates. For all reactions treated with CIP, the resulting nucleosides were filtered using a 0.22 μm pore size syringe filter. To run each sample, 10 μL was loaded onto a Zorbax Eclipse Plus C18 column (2.1 × 50 mm, 1.8 micron) paired with an Agilent 6490 QQQ triple-quadrupole LC mass spectrometer. Runs were analyzed using multiple-reaction monitoring in positive-ion mode. The transitions used were: 268.1→136 (A), 244.1→112 (C), 284.2→152 (G), 245.1→113 (U), 245.1→125 (Ψ), 259.1→139 (m1Ψ).