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. 2024 Nov 15;10(46):eadp5689. doi: 10.1126/sciadv.adp5689

Fig. 1. In vitro FRET system for detecting m6A modifications on aRNAs.

Fig. 1.

(A) Schematic diagram of the FRET system based on m6A aRNAs. (B) Fluorescence spectrum after MCP-ECFP, YTH-YPet, or YTH-YPet-3A was mixed with aRNA or m6A aRNA. (C) Gel red staining of native polyacrylamide gel electrophoresis (PAGE) after MCP-ECFP, YTH-YPet, or YTH-YPet-3A was mixed with aRNA or m6A aRNA; (i) m6A aRNA, (ii) aRNA (iii) MCP-ECFP + YTH-YPet + m6A aRNA; (iv) MCP-ECFP + YTH-YPet-3A + m6A aRNA, and (v) MCP-ECFP + YTH-YPet + aRNA; blue arrow: the complex of YTH, MCP, and m6A aRNA; red arrow: the complex of MCP and RNA. (D) Ratio of 528/478-nm emission after MCP-ECFP and YTH-YPet were mixed; the red arrow indicates the addition of aRNA or m6A aRNA, and the blue arrow indicates the addition of METTL3/14 at different concentrations; n = 3. (E) Gel red staining of native PAGE for (D) (i) m6A aRNA + YTH-YPet + MCP-ECFP, (ii) aRNA + YTH-YPet + MCP-ECFP + METTL3 (2 μM), (iii) aRNA + YTH-YPet + MCP-ECFP + METTL3 (1 μM), (iv) aRNA + YTH-YPet + MCP-ECFP + METTL3 (0.5 μM), (v) aRNA + YTH-YPet + MCP-ECFP + METTL3 (0 μM); the blue arrow indicates the complex of YTH, MCP, and m6A aRNA; the red arrow indicates the complex of MCP and RNA.