Near Quantitative Ligation Results in Resistance of DNA Origami Against Nuclease and Cell Lysate

There have been limited efforts to ligate the staple nicks in DNA origami which is crucial for their stability against thermal and mechanical treatments, and chemical and biological environments. Here, two near quantitative ligation methods are demonstrated for the native backbone linkage at the nicks in origami: i) a cosolvent dimethyl sulfoxide (DMSO)-assisted enzymatic ligation and ii) enzyme-free chemical ligation by CNBr. Both methods achieved over 90% ligation in 2D origami, only CNBr-method resulted in & AP;80% ligation in 3D origami, while the enzyme-alone yielded 31-55% (2D) or 22-36% (3D) ligation. Only CNBr-method worked efficiently for 3D origami. The CNBr-mediated reaction is completed within 5 min, while DMSO-method took overnight. Ligation by these methods improved the structural stability up to 30 & DEG;C, stability during the electrophoresis and subsequent extraction, and against nuclease and cell lysate. These methods are straightforward, non-tedious, and superior in terms of cost, reaction time, and efficiency. Two distinct methods are demonstrated for ligating the staple nicks in 2D and 3D DNA origami that results in near quantitative ligation yields. These methods are successfully tested on eight different origami. Ligation by these methods improves their thermal stability, stability during the electrophoresis and subsequent extraction, and against nuclease and cell lysate.image