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.
Shuang Jiang, Yue-Xiang Ren, Xue Wang et al.· Analytica Chimica Acta· 0 citations
Aptamers are valuable recognition elements for biosensing and therapeutic applications; however, an accessible strategy to link structure with function during postselection engineering remains challenging. Here, we introduce the concept of a structure-function landscape for aptamers, constructed by systematic single-point mutagenesis that maps the contribution of each nucleotide to target binding. Using OTC2, an aptamer that recognizes multiple tetracycline antibiotics, as a model, we identified critical binding sites and variable regions within the landscape. Guided by this landscape, we engineered OTC2 through combinatorial mutagenesis of only five nucleotides, yielding an evolved aptamer (OTC2M1) with enhanced affinity and substantially improved binding kinetics. The improved performance translated directly into sensing applications: an aptamer switch based on OTC2M1 achieved a 40-fold improvement in detection sensitivity for tetracycline antibiotics compared to the original aptamer. This work establishes the structure-function landscape as an informative and accessible platform for rational aptamer engineering and highlights its potential to guide the development of high-performance biosensors.
Zhuoer Chen, Qi Sun, Xianlu Lei et al.· Analytical Chemistry· 0 citations
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