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<p>Salt stress significantly impairs rice productivity by disrupting ion homeostasis and generating oxidative damage that undermines protein synthesis. In rice, the translation elongation factor eEF1 plays a critical role in the accurate, GTP-dependent delivery of aminoacyl-tRNAs to the ribosome, a process that becomes compromised under stress conditions. Here, we report the design and comprehensive characterization of a nucleic acid aptamer (S2-A) that binds rice eEF1 with nanomolar affinity. Using iterative SELEX from both a fully randomized (N40) and a stem-enriched (Stem2) library, we enriched aptamers that converge on a conserved stem–bulge architecture. Binding analyses via EMSA revealed an apparent dissociation constant of 5.3 nM for S2-A, while structural predictions using RNAstructure and AlphaFold-based modeling, together with MDockPP docking, indicated that S2-A targets the GTP-binding domain of eEF1. Site-directed mutagenesis and fluorescence polarization assays identified Ile585, Lys621, and Arg625 as critical for the aptamer–eEF1 interaction, with the K621A mutation causing the most pronounced loss of binding. Functionally, rice seedlings transfected with S2-A aptamer under 150 mM NaCl stress exhibited improved growth, enhanced chlorophyll content, reduced lipid peroxidation, and a coordinated upregulation of key salt stress-responsive genes (<i>OsSOS1</i>, <i>OsHKT1</i>, <i>OsDREB2A</i>). These findings demonstrate that aptamer-mediated stabilization of eEF1 preserves translational efficiency and contributes to enhanced salt tolerance in rice, providing a proof-of-concept that aptamer-mediated stabilization of eEF1A can enhance salt tolerance in rice.</p>

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Aptamer‑mediated modulation of eEF1 enhances salt stress tolerance in rice

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月2日

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  • Yongxiang Huang,
  • Haomin Chen,
  • Zhihao Xie,
  • Daming Chen,
  • Mingming Chen

摘要

Salt stress significantly impairs rice productivity by disrupting ion homeostasis and generating oxidative damage that undermines protein synthesis. In rice, the translation elongation factor eEF1 plays a critical role in the accurate, GTP-dependent delivery of aminoacyl-tRNAs to the ribosome, a process that becomes compromised under stress conditions. Here, we report the design and comprehensive characterization of a nucleic acid aptamer (S2-A) that binds rice eEF1 with nanomolar affinity. Using iterative SELEX from both a fully randomized (N40) and a stem-enriched (Stem2) library, we enriched aptamers that converge on a conserved stem–bulge architecture. Binding analyses via EMSA revealed an apparent dissociation constant of 5.3 nM for S2-A, while structural predictions using RNAstructure and AlphaFold-based modeling, together with MDockPP docking, indicated that S2-A targets the GTP-binding domain of eEF1. Site-directed mutagenesis and fluorescence polarization assays identified Ile585, Lys621, and Arg625 as critical for the aptamer–eEF1 interaction, with the K621A mutation causing the most pronounced loss of binding. Functionally, rice seedlings transfected with S2-A aptamer under 150 mM NaCl stress exhibited improved growth, enhanced chlorophyll content, reduced lipid peroxidation, and a coordinated upregulation of key salt stress-responsive genes (OsSOS1, OsHKT1, OsDREB2A). These findings demonstrate that aptamer-mediated stabilization of eEF1 preserves translational efficiency and contributes to enhanced salt tolerance in rice, providing a proof-of-concept that aptamer-mediated stabilization of eEF1A can enhance salt tolerance in rice.