Incorporation of a Hydrazone Base-Pair Analog as a Nucleic Acid Staple: Structure, Stability, Triggered Release, and Ligase Studies.

Abstract

Hydrogen bonding and hydrophobic stacking are the primary interactions that control the structure and functionality of DNA systems, including those of most synthetic variants. These interactions are weak and are highly dynamic. Tunable Orthogonal Reversible Covalent (TORC) bonding utilizes dynamic covalent interactions that can be tuned both kinetically and thermodynamically. Here, we report the implementation of dynamic hydrazone bonding into a DNA duplex to greatly enhance its thermostability. The hydrazone-containing DNA duplex can only be denatured with a chemical trigger. Additionally, we generated different geometric configurations of the hydrazone within the DNA duplex using different constitutional isomers of the precursor aldehyde and hydrazide nucleotides. Through combined experimental and simulation analyses, we determined the optimal geometric configurations that most closely resemble canonical DNA structures. Finally, we demonstrated the susceptibility of our synthetic DNA system to enzymatic ligation by joining cohesive nicked-end fragments. Overall, the work demonstrates the utility of exchangeable covalent hydrazone chemistry to create a chemically addressable genetic system that incorporates novel chemical functionalities for advanced applications in DNA nanotechnology, therapeutics, and computing.