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<p>RNA’s regulatory and catalytic roles affect gene expression, with circular RNAs (circRNAs) emerging as a unique subclass with broad therapeutic potential. Among circRNA production methods, ribozyme-mediated circularization, especially through group I intron-based systems such as the T4 <i>td-</i>PIE (where PIE indicates permuted intron–exon), offers efficient in vitro synthesis. However, detailed structural insights of the T4 <i>td</i> intron are limited, particularly regarding circularization mechanisms. Here we use cryo-electron microscopy to resolve high-resolution structures of both linear and circular T4 <i>td</i> intron forms. Comparative structural analysis reveals key conformational shifts in the catalytic core, including P1ext domain loss and realignment of critical base pairs in the circular form. Additionally, we identify critical sites and interactions optimizing RNA circularization. Structure-guided mutations enhance circularization efficiency, as validated in the T4 <i>td</i>-PIE system and benchmarked against alternative platforms. These findings enhance our understanding of RNA circularization mechanisms and inform optimizations for large-scale circRNA production, with important implications for RNA-based therapeutics and synthetic biology.</p><p></p>

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Structural insights and engineering of the T4 td intron for improved RNA circularization

  • 期刊论文

  • 发表日期：

    2025年11月18日

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  • Linfeng An,
  • Shanshan Li,
  • Kaiming Zhang

摘要

RNA’s regulatory and catalytic roles affect gene expression, with circular RNAs (circRNAs) emerging as a unique subclass with broad therapeutic potential. Among circRNA production methods, ribozyme-mediated circularization, especially through group I intron-based systems such as the T4 td-PIE (where PIE indicates permuted intron–exon), offers efficient in vitro synthesis. However, detailed structural insights of the T4 td intron are limited, particularly regarding circularization mechanisms. Here we use cryo-electron microscopy to resolve high-resolution structures of both linear and circular T4 td intron forms. Comparative structural analysis reveals key conformational shifts in the catalytic core, including P1ext domain loss and realignment of critical base pairs in the circular form. Additionally, we identify critical sites and interactions optimizing RNA circularization. Structure-guided mutations enhance circularization efficiency, as validated in the T4 td-PIE system and benchmarked against alternative platforms. These findings enhance our understanding of RNA circularization mechanisms and inform optimizations for large-scale circRNA production, with important implications for RNA-based therapeutics and synthetic biology.