RNA Nucleoside Digestion and Purification

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.

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Ignatova V.V., Stolz P., Kaiser S., Gustafsson T.H., Lastres P.R., Sanz-Moreno A., Cho Y.L., Amarie O.V., Aguilar-Pimentel A., Klein-Rodewald T., Calzada-Wack J., Becker L., Marschall S., Kraiger M., Garrett L., Seisenberger C., Hölter S.M., Borland K., Van De Logt E., Jansen P.W., Baltissen M.P., Valenta M., Vermeulen M., Wurst W., Gailus-Durner V., Fuchs H., Hrabe de Angelis M., Rando O.J., Kellner S.M., Bultmann S, & Schneider R. (2020). The rRNA m6A methyltransferase METTL5 is involved in pluripotency and developmental programs. Genes & Development, 34(9-10), 715-729.

Publication 2020

alkaline phosphatase phosphodiesterase I Tetrahydrouridine (THU; AcroPrep Advance 350 10K Omega

Alkaline phosphatase Benzonase Buffer Digestive Enzymes Mgcl2 Nucleosides Pentostatin Phosphodiesterase i Tris

Corresponding Organization :

Other organizations : Helmholtz Zentrum München, Ludwig-Maximilians-Universität München, University of Massachusetts Chan Medical School, Oncode Institute, Radboud University Nijmegen, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Technical University of Munich, German Center for Neurodegenerative Diseases, Munich Cluster for Systems Neurology, Heinrich Heine University Düsseldorf, German Center for Diabetes Research, Deutsches Diabetes-Zentrum e.V.

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Variable analysis

independent variables

Amount of RNA (three-microgram to 100-ng portions)

dependent variables

Amount of single nucleosides produced

control variables

Digestion time (2 h)
Digestion temperature (37°C)
Concentration of alkaline phosphatase (0.2 U)
Concentration of phosphodiesterase I (0.02 U)
Concentration of benzonase (0.2 U)
PH of Tris buffer (pH 8)
Concentration of MgCl2 (1 mM)
Concentration of tetrahydrouridine (THU) (0.5 µg)
Concentration of butylated hydroxytoluene (BHT) (1 µM)
Concentration of pentostatin (0.1 µg)

positive controls

Not mentioned

negative controls

Not mentioned

Annotations

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