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. 2022 Sep 22;23(19):11139. doi: 10.3390/ijms231911139

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© 2022 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Methylation by 2′-O-ribose prevents IFN-α but not IL-6 secretion, independent of the position. (a) Sequences of unmethylated RNA63 (U) or methylated derivatives (M1, M2, M3, and M4): 2′-O-ribose-methylated guanosines are indicated with an X. (b) RNA63 derivatives were either treated with 0.005 units of RNase T2/µg RNA (+) or mock treated (−), and RNA fragmentation was visualized in polyacrylamide gel electrophoresis. Position of the RNA fragments of interest are indicated with an arrow () and a line (). (c,d) PBMCs were stimulated with RNA63 derivatives at a final concentration of 10 µg/mL: 1 µM CpG ODN 2216 or 5 µg/mL RNA40. Supernatants were harvested 20 h post-stimulation (h p. s.) and both (c) IFN-α and (d) IL-6 concentrations were measured in ELISA. Graphs depict six independent experiments with PBMCs obtained from six individual donors, each in biological duplicates (twelve measurements per data point, mean + S.D). Data were analyzed using paired t-tests. **** p < 0.0001, *** p < 0.001.

Inline graphic — Methylation by 2′-O-ribose prevents IFN-α but not IL-6 secretion, independent of the position. (a) Sequences of unmethylated RNA63 (U) or methylated derivatives (M1, M2, M3, and M4): 2′-O-ribose-methylated guanosines are indicated with an X. (b) RNA63 derivatives were either treated with 0.005 units of RNase T2/µg RNA (+) or mock treated (−), and RNA fragmentation was visualized in polyacrylamide gel electrophoresis. Position of the RNA fragments of interest are indicated with an arrow () and a line (). (c,d) PBMCs were stimulated with RNA63 derivatives at a final concentration of 10 µg/mL: 1 µM CpG ODN 2216 or 5 µg/mL RNA40. Supernatants were harvested 20 h post-stimulation (h p. s.) and both (c) IFN-α and (d) IL-6 concentrations were measured in ELISA. Graphs depict six independent experiments with PBMCs obtained from six individual donors, each in biological duplicates (twelve measurements per data point, mean + S.D). Data were analyzed using paired t-tests. **** p < 0.0001, *** p < 0.001.