Probing the Formation Kinetics and Thermodynamics with Rationally Designed Analytical Tools Enables One-Pot Synthesis and Purification of a Tetrahedral DNA Nanostructure

The development of robust analytical tools capable of probing the formation kinetics and thermodynamics of DNA nanostructures is a crucial step toward better understanding and manufacturing of diverse DNA-based materials. Herein, we introduce a real-time fluorescence anisotropy assay and rationally designed DNA reaction termination probes (DRTPs) as a set of new tools for exploring the formation mechanisms of DNA nanostructures. We deployed these tools for probing the formation of a classic tetrahedral DNA nanostructure (TDN) as a model system. Our tools revealed that the formation of TDN was dominated by simultaneous hybridization, whereas its undesired side products were caused mainly through step-wise hybridization. An optimal reaction temperature exists that favors the formation of TDN over side products. With insight into the TDN formation mechanism, we further engineered magnetic DRTPs to achieve single-step purification of TDN, enabling 10-fold improvement in the ratio between the targeted TDN and undesired side products without tedious procedures or bulky instruments. Combining the optimal reaction and purification conditions, we finally demonstrated the one-pot synthesis and purification of TDN. The analytical techniques offered in this work may hold potential to find wide applications and inspire new analytical methods for structural DNA nanotechnology.