Isolation and characterization of ssDNA aptamers against BipD antigen of Burkholderia pseudomallei

• Published in: Analytical biochemistry 2024-12, Vol.695, p.115655, Article 115655
• Main Authors: Selvam, Kasturi, Najib, Mohamad Ahmad, Khalid, Muhammad Fazli, Yunus, Muhammad Hafiznur, Wahab, Habibah A., Harun, Azian, Zainulabid, Ummu Afeera, Fadzli Mustaffa, Khairul Mohd, Aziah, Ismail
• Format: Article
• Language: English
• Subjects: B. pseudomallei; BipD; Melioidosis; SELEX; ssDNA aptamers
• ISSN: 0003-2697; 1096-0309; 1096-0309
• DOI: 10.1016/j.ab.2024.115655

Melioidosis is difficult to diagnose due to its wide range of clinical symptoms. The culture method is time-consuming and less sensitive, emphasizing the importance of rapid and accurate diagnostic tests for melioidosis. Burkholderia invasion protein D (BipD) of Burkholderia pseudomallei is a potential diagnostic biomarker. This study aimed to isolate and characterize single-stranded DNA aptamers that specifically target BipD. The recombinant BipD protein was produced, followed by isolation of BipD-specific aptamers using Systematic Evolution of Ligands by EXponential enrichment. The binding affinity and specificity of the selected aptamers were evaluated using Enzyme-Linked Oligonucleotide Assay. The fifth SELEX cycle showed a notable enrichment of recombinant BipD protein-specific aptamers. Sequencing analysis identified two clusters with a total of seventeen distinct aptamers. AptBipD1, AptBipD13, and AptBipD50 were chosen based on their frequency. Among them, AptBipD1 exhibited the highest binding affinity with a Kd value of 1.0 μM for the recombinant BipD protein. Furthermore, AptBipD1 showed significant specificity for B. pseudomallei compared to other tested bacteria. AptBipD1 is a promising candidate for further development of reliable, affordable, and efficient point-of-care diagnostic tests for melioidosis. [Display omitted] •First study to successfully isolate and characterize DNA aptamers targeting BipD antigen of Burkholderia pseudomallei.•Sanger sequencing data analysis identified 17 candidate aptamers.•The selected aptamers exhibited high binding affinity with dissociation constants (Kd) in the low micromolar range.•All three aptamers displayed good specificity by distinguishing B. pseudomallei from other tested bacteria.•These aptamers could be used as an alternative to antibodies in antigen detection method for melioidosis diagnosis.