Rational design of bispecific aptamers with high stability and affinity.
Bispecific aptamers (BsApts) enable dual-target recognition, which is essential for rapid antibiotic detection. However, the practical application of BsApts is often hindered by structural instability, which compromises their binding affinity. To address this, we employed a "sequence-structure-dynamic simulation" framework to design BsApts targeting sulfadiazine and sulfamethoxazole. We introduced a novel C-type configuration utilizing poly-T linkers with complementary terminal poly-A sequences, facilitating self-assembly via A-T base-pairing. Molecular dynamics simulations revealed that this C-type design possesses superior stability, with an RMSD of ∼0.41 nm, significantly lower than traditional L-type constructs (>0.83 nm). Fluorescence assays confirmed that the optimized variant (C10_SME-SDZ) achieved nanomolar affinity (Kd = 52.81 nM for SDZ and 63.18 nM for SME), representing a 23-fold and 19-fold enhancement for SDZ and SME, respectively, compared to parental aptamers. Conversely, L-type aptamers exhibited weak or lost recognition. This study provides a robust computational strategy for engineering high-affinity, stable bispecific aptamers for enhanced food safety monitoring.