Screening and characterization of nucleic acid aptamers targeting Brucella surface antigens: identification and analysis of their corresponding binding sites

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Abstract
Brucellosis is a zoonotic infectious disease caused by Brucella species, posing serious threats to public health and livestock industries worldwide. According to the World Health Organization (WHO), over one million new cases are reported annually. However, the actual number may be underestimated due to the bacteria’s intracellular persistence and subclinical bacteremia. These features and Brucella’s genetic variability and immune evasion strategies hinder accurate diagnosis and effective treatment. Thus, there is an urgent need for sensitive and specific molecular probes. This study used whole Brucella bacteria as the target antigen for aptamer selection via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, combined with a microplate-based screening approach. To enhance specificity, a multi-organism counter-selection strategy was implemented using Rhizobium fischeri, Escherichia coli, and Salmonella. After 13 rounds of selection, a high-affinity aptamer library was obtained. Candidate aptamers were cloned, sequenced using T-vector cloning, and analyzed phylogenetically. Five candidates (WS-6, WS-17, WS-44, WS-26, and WS-32) were selected for further characterization. Binding affinity and specificity assays identified WS-6 as the top-performing aptamer (Kd = 16.23 ± 5.84 nM). WS-6 also demonstrated thermal and pH stability. Its target protein was identified as the outer membrane protein BamA (β-barrel assembly machinery A) using Co-immunoprecipitation (Co-IP), mass spectrometry, and molecular docking. Western blotting and pull-down assays confirmed this interaction. Functional studies showed that WS-6-loaded liposomes selectively recognized Brucella and significantly reduced bacterial invasion in host cells. qPCR analysis demonstrated a notable decrease in bacterial load in WS-6-treated cells (Ct values from 22.34 to 20.64, P < 0.05). Mutational analysis of WS-6 confirmed the importance of GC-rich binding sites. Moreover, a truncated version of BamA (BamAK) retained binding capability, validating it as the key antigenic domain. In summary, this study demonstrates the potential of SELEX-derived aptamers as diagnostic and therapeutic tools for Brucella infection. Identifying BamA as a target provides a molecular basis for the future development of precise detection methods and targeted therapies against brucellosis. Graphical abstract

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