In-vitro and in-silico selection of DNA-based aptamer towards pork detection using SELEX

Meat authentication is a growing global concern in the food industry, necessitating accurate and efficient methods for source identification. Established techniques such as PCR and mass spectrometry are sensitive and specific, but require high-quality DNA or protein for testing, and are time-consuming. In contrast, aptamer-based detection, a newer approach in food authentication shows promise as a more accessible alternative. Hence, this study attempted to address the need for porcine-specific aptamer capable of binding to porcine proteins. The study aimed to develop and characterize aptamers that bound to any pork protein through SELEX process, combined with Next Generation Sequencing (NGS) analysis, Liquid Chromatography Mass Spectrometry (LC-MS) analysis, and molecular docking simulation. Fourteen rounds of selection using the centrifugal-ultrafiltration separation technique against four negative controls (chicken, duck, beef and lamb) led to the identification of potential pork-binding aptamers. The final pool library was subjected to Sanger sequencing and NGS. Sequence analysis yielded 67 sequences, with the most frequently occurring aptamer, APT#A1, exhibiting the highest binding affinity (27.61 ±1.92 nM) as determined by ELONA. Clustering analysis, combined with motif and network analysis also resulted in five aptamers (APT#A1, APT#A2, APT#A4, APT#A8, APT#A17), to be used for further analysis. However, protein blotting revealed cross-reactivity with multiple proteins from negative samples, necessitating further specificity enhancement. LC-MS analysis consistently identified troponin subunits (TnI and TnT) as potential target markers that bound to the aptamers. Aptamer truncation analysis showed that the removal of the flanking region could affect the stability and binding efficacy of APT#A1. On the other hand, the forward primer binding site was identified as crucial to aptamer binding, as its retention in the truncated sequences of APT#A2 and APT#A4 resulted in improved binding affinity. Molecular docking simulations demonstrated more stable and stronger interactions between aptamers and troponin, notably with specific chains of troponin (TnT and TnI), as compared to that of myosin complex structure. Amino acids that were responsible for the interactions were glutamine (GLN), valine (VAL), glycine (GLY), serine (SER), isoleucine (ILE), methionine (MET), and glutamic acid (GLU) with interactions occurring at a distance of < 3.0 Å by hydrogen bonding. As a conclusion, through combinations of approaches, this study has successfully developed and characterized a high affinity porcine-binding aptamer. These aptamers are a promising element that can use for porcine detection and thus will improve the authentication limit.

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