Aptamers are short, single stranded sequences of DNA or RNA that fold into unique tertiary structures allowing them to bind with high affinity and specificity to a target molecule. Aptamers are selected in vitro from random pools of sequences by an iterative method referred to as SELEX (Systematic Evolution of Ligands by Exponential enrichment). Because of their binding ability, aptamers continue to be widely used for analytical detection and diagnostic applications. However, before a selected aptamer can be considered for potential application, its dissociation constant (Kd) needs to be measured. The dissociation constant measures the extent that the aptamer-target complex dissociates into separate components (aptamer and target). For an aptamer to be suitable for analytical applications the dissociation constant needs to be in the nM or pM range. In this study we characterized the Kd for a 40-mer aptamer selected to bind to p57 or Major Soluble Antigen (MSA). This protein is the principal antigen found on the surface of Renibacterium salmoninarum responsible for bacterial kidney disease in salmonids. The binding assay consisted of reacting variable quantities of the aptamer with a fixed amount of protein immobilized on a microplate. After incubating the aptamer with its target protein, the excess unbound aptamer was removed and the the bound aptamer, released by heating the reaction, was quantified by quantitative polymerase chain reaction (q-PCR). This approach avoids use of radioactive labelling and produces results with good accuracy and reproducibility.

Q-PCR approach for characterizing aptamer-protein binding affinity

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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