CELL-SELEX METHODOLOGY APPLIED TO MICROORGANISMS

Aptamers are single-stranded DNA or RNA molecules that fold into three-dimensional structures capable of binding diverse molecular targets with high affinity and specificity. They are generated through the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technique, first described in 1990. Aptamers function similarly to antigen-antibody interactions and have applications in clinical diagnostics, disease research, drug development, and biomarker detection, as well as in environmental, agricultural, and food sectors. Notably, Macugen® (pegaptanib) is the first FDA-approved aptamer for treating age-related macular degeneration. Aptamers offer advantages over traditional antibodies, including smaller size, low toxicity, and cost-effective, reproducible in vitro synthesis, enabling binding to non-immunogenic targets. However, standardizing laboratory protocols for purification, visualization, and manipulation remains challenging due to their small size, comparable to primers. This work aimed to standardize and develop a SELEX protocol for selecting aptamers against bacterial surface proteins using Escherichia coli as an initial model. The protocol followed the classical SELEX method with adaptations, including cycles of incubating the oligonucleotide library with the target, partitioning, and amplification by symmetric and asymmetric PCR, repeated for 8 to 12 rounds until enrichment plateau, followed by Sanger sequencing. Verifi polymerase and Applied Biosystems Veriti thermocycler were used, along with protein purification by FPLC and immobilization on ELISA plates. In cell-SELEX, 20 pmol of the library and 107 bacteria/mL were used. Asymmetric PCR proved effective in generating ssDNA, preceded by conventional pre-amplification. Immobilization assays of microbial proteins were validated for evaluating aptamer binding. Sodium acetate precipitation was optimized, including the use of linear polyacrylamide, and restriction enzymes will be tested to improve the selection process. Subsequent rounds will focus on increasing specificity, followed by sequencing. The standardized protocol proved efficient for generating aptamers against bacterial targets, with methodological refinements planned to enhance specificity and applicability in diagnostics and therapy.

This work was supported by Coordination for the Improvement of Higher Education Personnel (CAPES
- Finance Code 001).