In a study published in Proceedings of the National Academy of Sciences (PNAS), a research team led by Prof. TAN Weihong, Prof. HAN Da, and Prof. GUO Pei from Hangzhou Institute of Medicine (HIM) of the Chinese Academy of Sciences (CAS) reports the tertiary structure of a DNA aptamer-ATP 1:1 binding complex, and elucidates the recognition mechanism. Leveraging structural insights, the team engineers an optimized DNA aptamer with a submicromolar K_D for ATP binding. The optimized aptamer exhibits the highest affinity reported for ATP-binding DNA aptamers to date.

DNA aptamers are powerful molecular tools in biosensing, bioimaging and therapeutics. However, a limited understanding on 3D structures and binding mechanisms hinders their further optimizations and applications. ATP, a central metabolite in cellular energy metabolism, is a key target for aptamer development. A DNA aptamer 1301b is recently reported, binding to a single ATP molecule with a K_D ≈ 2.5 µM. However, its structural basis for recognition remains unclear, lacking guiding principles for rational optimization.

The researchers determined the solution NMR structures of a shortened variant, 1301b_v1, in complex with ATP. The structure reveals an "L"-shaped architecture, where ATP intercalates into the binding pocket formed by two internal loops, as stabilized by hydrogen bonding with guanine and stacking interactions with neighboring bases. Additionally, the researchers showed that Mg²⁺ ions facilitate 1301b_v1 to form a semi-folded structure and further stabilize the binding complex by neutralizing the negatively charged phosphate groups of DNA and ATP. This demonstrated that the aptamer recognizes ATP through an adaptive recognition mechanism.

Guided by these structural insights, the researchers introduced 2'-O-methyl modifications to key residues in the central junction, significantly improving binding affinity and reducing reliance on Mg²⁺ ions. The optimized variant achieved a submicromolar K_D ≈ 0.7 μM and retains specificity for ATP.

This work demonstrates DNA's untapped potential to form intricate 3D folds. The structural insights and engineered aptamer open new avenues for designing high-performance DNA molecular tools toward downstream applications such as diagnostics and targeted therapeutics.