Structural optimization, characterization, and evaluation of binding mechanism of aptamers against bovine pregnancy-associated glycoproteins and their application in establishment of a colorimetric aptasensor using Fe-based metal-organic framework as peroxidase mimic tags.

A truncated aptamer (designated A24-3) was identified that specifically binds to bovine pregnancy-associated glycoproteins (bPAG9) with a low dissociation constant (2.04 nM) through two truncation approaches. Circular dichroism spectroscopy indicated that A24-3 formed parallel G-quadruplexes, which was subsequently confirmed using nuclear magnetic resonance (NMR) spectroscopy. Furthermore, a molecular dynamics simulation was employed to investigate the recognition mechanism of A24-3 and bPAG9. Interaction analysis showed that A24-3 folded into a parallel G-quadruplex structure with three G-tetrads, primarily through numerous hydrogen bonds and hydrophobic and π-π interactions. Finally, a novel colorimetric aptasensor was developed for detecting bPAG9 using A24-3 and an Fe-based metal-organic framework as target recognition elements and enzyme mimics, respectively. The method demonstrated a broad detection range from 0.5 to 50 ng/mL, with a low detection limit of 0.03 ng mL, and exhibited a good recovery (91.0-102%) for bPAG9-spiked serum samples. Additionally, the aptasensor was successfully applied to detecting the pregnancy-specific biomarker bPAGs in serum samples.