Selection of aptamers for fluorescent detection of alpha-methylacyl-CoA racemase by single-bead SELEX.

This paper first reports DNA aptamers and a fluorescent enzyme-linked aptamer assay (ELAA) targeting alpha-methylacyl-CoA racemase (AMACR), an emerging prostate cancer biomarker. The aptamers were in vitro selected using a new single-bead SELEX approach, which was rapid and consumed only ca. 45 ng AMACR. Before SELEX, silane chemistry was used to prepare epoxide-functionalized glass microbeads (EGBs, 500 μm in size and manipulated by tweezers) for AMACR coating. Recombinant AMACR was also prepared. During SELEX, the ligand evolution was assured by a differential real-time quantitative PCR assay. After SELEX, the aptamers were identified by the alignment analysis and 2nd structure prediction from the selected, cloned sequences. The circular dichroism (CD) analysis revealed that the aptamers formed stable B-form, stem-loop conformations. The fluorescent ELAA method confirmed the nM-level affinity and high specificity of the aptamers against AMACR. Finally, an aptamer-based fluorescent AMACR assay was demonstrated. The assay featured a wide dynamic range (from 10(-1) to 10(3) nM of AMACR), a low detection limit of 0.44 nM (19.5 ng/mL), and high AMACR specificity and is promising for clinical AMACR diagnostics.