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

Abstract

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.

Fig. 1. DNA chemical ligation reactions reported and developed in this study.

A phosphate condensation reaction, B click ligation, C EPT-based ligation, and D this work: cross-activation of phosphorothioate- and sulfonamide-DNA.

Scheme 1

Synthetic route for 5’-DNBSA Thymidine phosphoramidite 6 for the 5’-DNBSA DNA synthesis.

Fig. 2. Screening of cleavage/deprotection conditions for 5′-DNBSA DNA.

A model 14-mer poly(T) oligonucleotide bearing a DNBSA group was released from CPG and deprotected under the four conditions listed. Product distributions were analysed by RP-HPLC, and the yield of intact 5′-DNBSA-DNA (a) is reported. N.D., not detected; rt, room temperature.

Fig. 3. DNA chemical ligation between 3’-PS DNA and 5’-DNBSA DNA on the DNA splint.

A Sequence and structure of the DNA fragments and the splint DNA. The reaction was performed with 8 μM of 3’-PS DNA, 5’-DNBSA DNA and splint DNA at different pH 6 to 8 for 1 to 12 hr at 37 °C. B dPAGE analysis of the crude mixture of the ligation reaction, C Ligation yield of the reaction at each time point and pH.

Fig. 4. Optimization of ligation conditions and mechanistic insight.

A Chemical ligation yield under modified reaction conditions; (a) 20 μM of 3’-PS DNA, 5’-DNBSA DNA and splint DNA without annealing, (b) 20 μM of 3’-PS DNA, 5’-DNBSA DNA and 40 μM of splint DNA without annealing, (c) 20 μM of 3’-PS DNA, 5’-DNBSA DNA and splint DNA with annealing before the reaction (90 °C, 3 min), B Possible reaction mechanism; (a) concerted mechanism, (b) stepwise mechanism.