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<p>Chemical ligation of oligonucleotides enables assembly of long DNA constructs essential for genome engineering, DNA nanotechnology, and molecular diagnostics, but current methods often require external activators and suffer from reactive intermediate instability. Here we show a reagent-free DNA autoligation strategy based on intramolecular cross-activation between 3′-phosphorothioate (PS) and 5′-dinitrobenzenesulfonamide (DNBSA) termini on a splint DNA, yielding a P3′ → N5′ phosphoramidate linkage under near-physiological conditions. Ligation proceeds with over 80% yield at 37 °C and pH 8 without external reagents. The DNBSA group exhibits exceptional aqueous stability, and in situ formation of reactive intermediates contributes to high efficiency. This strategy expands the current toolkit for assembling DNA constructs and may facilitate future biotechnological and therapeutic studies.</p>

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Chemical autoligation with phosphorothioate- and sulfonamide-terminated DNA via intramolecular cross-activation

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年8月6日

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  • Hayato Yokoe,
  • Kengo Kokubo,
  • Kazuki Yamaoka,
  • Ryota Oikawa,
  • Fumiaki Tomoike,
  • Naoko Abe,
  • Yasuaki Kimura,
  • Hiroshi Abe

摘要

Chemical ligation of oligonucleotides enables assembly of long DNA constructs essential for genome engineering, DNA nanotechnology, and molecular diagnostics, but current methods often require external activators and suffer from reactive intermediate instability. Here we show a reagent-free DNA autoligation strategy based on intramolecular cross-activation between 3′-phosphorothioate (PS) and 5′-dinitrobenzenesulfonamide (DNBSA) termini on a splint DNA, yielding a P3′ → N5′ phosphoramidate linkage under near-physiological conditions. Ligation proceeds with over 80% yield at 37 °C and pH 8 without external reagents. The DNBSA group exhibits exceptional aqueous stability, and in situ formation of reactive intermediates contributes to high efficiency. This strategy expands the current toolkit for assembling DNA constructs and may facilitate future biotechnological and therapeutic studies.