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

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.