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High-Speed Atomic Force Microscopy Reveals Aptamer-Mediated Conformational Trapping of METTL3-METTL14 for m6A
Madhu Biyani1, Chihiro Ueda2, Leonardo Puppulin1
1Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa 920-1192, Japan.
Abstract:
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.
Insights
Researchers visualized the METTL3-METTL14 (M3/M14) complex dynamics using HS-AFM. They discovered RNA binding induces conformational changes, leading to the development of aptamer inhibitors that block cancer cell growth.
Area of Science:
- Epitranscriptomics
- Molecular Biology
- Structural Biology
Background:
- The METTL3-METTL14 (M3/M14) heterodimer is the core component responsible for N6-methyladenosine (m6A) deposition.
- Understanding the conformational dynamics of M3/M14 is crucial for insights into catalytic regulation and cancer therapeutics.
Purpose of the Study:
- To visualize the single-molecule dynamics of the M3/M14 heterodimer.
- To identify novel inhibitors targeting the dynamic states of M3/M14.
- To explore the therapeutic potential of conformationally targeted inhibitors in cancer.
Main Methods:
- High-speed atomic force microscopy (HS-AFM) for single-molecule visualization.
- Competitive in vitro selection for aptamer inhibitor discovery.
- Molecular docking to elucidate aptamer-target interactions.
Main Results:
- Substrate RNA binding induces a conformational transition in M3/M14 from a rigid apo state to a compact, active state.
- Two DNA aptamer inhibitors, M3B and M3L, were identified that bind to the M3/M14 interface.
- Aptamer binding stabilizes a distorted, open M3/M14 conformation, inhibiting activity and reducing cancer cell growth.
Conclusions:
- The study defines a dynamically regulated, targetable state of the m6A writer complex.
- HS-AFM is a powerful tool for uncovering dynamic regulatory mechanisms in epitranscriptomics.
- Conformationally targeted aptamers show therapeutic potential for cancer treatment by inhibiting m6A deposition.
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