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Aptamers with primer binding sites are necessary for the SELEX (Systematic Evolution of Ligands by EXponential enrichment) process. Such primer sequences cause non-specific binding by their nature and raise the incidence of false positives. Thus, the use of aptamers without primer or with minimum primer, to shorten the length of selected aptamers, has become an interesting research topic. Recently, a primer-free aptamer selection protocol based on the magnetic-assisted rapid aptamer selection (MARAS) technology has been reported to generate primer-free aptamers with high affinity and specificity. Adversely, the yield of a suitable aptamer was low and the advantage of MARAS was deteriorated. In this study, multiple negative selection runs were used in the MARAS procedure to remove aptamers with non-specific binding to unwanted biomolecules. Smart aptamers with predetermined affinity for C-reactive protein were isolated by window-MARAS. The specificity of aptamers was validated by using blind serum samples and compared with those using monoclonal antibody-based nephelometry analysis. The relative specificity of aptamers with or without primers was evaluated, revealing that the specificity of the aptamer with primer is similar to that of the aptamer without primer. By using a randomized library with primers, the simplicity and rapidity of MARAS in generating aptamers are preserved, and by implementing multiple negative selection, MARAS could efficiently select aptamers with high binding affinity and specificity for clinical applications.

Evaluation of aptamer specificity with or without primers using clinical samples for C-reactive protein by magnetic-assisted rapid aptamer selection