Mechanism of pH influence on aptamer binding with Cd²⁺ revealed by molecular dynamics simulation

Aptamers can bind specifically to heavy metal ions, and aptamer-based biosensors for heavy metal detection with high sensitivity have been extensively developed. However, their detection sensitivity is quite limited to specific conditions due to unknown mechanisms of environmental influence, such as the pH factor, which restrict the development and application of biosensors. In this work, the intrinsic mechanism of pH influence on CD-2-1 aptamer binding with Cd2+ in aqueous solution was investigated with molecular dynamics (MD) simulation. The calculated data demonstrated that as the pH value increased in the range of 2.40-7.90, the capability of aptamer binding to Cd2+ was enhanced, which was consistent with experimental results. The intrinsic reason lies in the protonation of three key bases: 7G, 12C, and 16C. With a decrease in pH value, several bases were protonated, causing a rearrangement of hydrogen bonds and structural change to the aptamer. Finally, the stem-loop structure of the aptamer was totally destroyed, and it became unlikely to bind Cd2+. The decrease in the absolute value of binding free energy also indicated weakened binding interactions. It has been emphasized that the stem-loop structure and the C-shape loop character are the specific spatial structures for Cd2+ binding. This work reveals the mechanism behind the influence of pH on aptamer binding to Cd2+ in aqueous solution, providing a theoretical reference for heavy metal detection in complicated environments, which is helpful for the development and application of aptamer-based biosensors.