High-Speed Atomic Force Microscopy Reveals Aptamer-Mediated Conformational Trapping of METTL3–METTL14 for m ⁶ A Inhibition

The conformational dynamics of METTL3-METTL14 (M3/M14) heterodimer, the catalytic core for N6-methyladenosine (m⁶A) deposition, remain largely unexplored, limiting insight into dynamic regulation of catalysis and opportunities for therapeutic targeting in cancer. Here, we report the first single-molecule visualization of M3/M14 dynamics by high-speed atomic force microscopy (HS-AFM). Our measurements show that substrate RNA binding induces a conformational transition from a rigid apo heterodimer with an open interlobe groove to a compact, catalytically competent state. Guided by these dynamic insights, we identify two potent DNA aptamer inhibitors of M3/M14, M3B, and M3L, using a competitive in vitro selection strategy. HS-AFM integrated with molecular docking reveals that both aptamers insert into the M3/M14 interface, forming a sandwich-like complex that stabilizes a distorted, open conformation and prevents RNA-induced compaction. This conformational trapping inhibits methyltransferase activity, reduces global m⁶A levels, and suppresses A549 lung cancer cell growth. These findings define a dynamically regulated, interlobe targetable state of the m⁶A writer complex and demonstrate the utility of HS-AFM for uncovering dynamic regulatory mechanisms and guiding the development of conformationally targeted therapeutics in epitranscriptomic biology.