Global Quantitative Analysis of Ligation Reactions in Self‐Assembled DNA Nanostructures at the Single‐Nick Level

<jats:title>ABSTRACT</jats:title> <jats:p>Ligation of staple strands in DNA origami nanostructures (DONs) can yield enhanced structural stability in critical environments. This process can be viewed as performing hundreds of parallel reactions programmed on a self‐assembled nanoscale platform. While previous studies have focused on investigating the collective results of the chemical or enzymatic ligation reactions, herein, the global quantitative analysis of individual ligation reactions is achieved using quantitative PCR (qPCR). By mapping enzymatic ligation efficiency on a trapezoidal substructure representing one‐third of a triangular DON, ligation is shown to preferentially occur at the trapezoid edges rather than at inner sites. Excellent agreement between the experimental ligation yields and docking simulations suggests that this is a result of variations in the ligase docking probability. Ligation products involving more than two consecutive sequences can be generated with each enzyme‐catalyzed reaction as an independent event. Interestingly, the sharp contrast between the edges vs. the inner sites has been abolished by changing the reaction conditions and performing the ligation in a DMSO co‐solvent system. This analytic method provides unprecedented insight into the multiple ligation reactions occurring in parallel within complex DONs and will be an invaluable tool in the translation of DONs from the lab to real‐world applications.</jats:p>