Selection of aptamers for fluorescent detection of alpha-methylacyl-CoA racemase by single-bead SELEX
Journal
Biosensors and Bioelectronics
Journal Volume
62
Pages
106-112
Date Issued
2014
Author(s)
Abstract
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. 45ng AMACR. Before SELEX, silane chemistry was used to prepare epoxide-functionalized glass microbeads (EGBs, 500μm 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 103nM of AMACR), a low detection limit of 0.44nM (19.5ng/mL), and high AMACR specificity and is promising for clinical AMACR diagnostics. ? 2014 Elsevier B.V.
Subjects
Alpha-methylacyl-CoA racemase (AMACR); DNA aptamer; Enzyme-linked aptamer assay (ELAA); Prostate cancer; SELEX
SDGs
Other Subjects
Cloning; Diseases; Enzymes; Polymerase chain reaction; Aptamer assay; Dna aptamer; Prostate cancers; Racemase; SELEX; Fluorescence; 2 methylacyl coenzyme A racemase; aptamer; DNA aptamer; unclassified drug; alpha-methylacyl-CoA racemase; aptamer; isomerase; tumor marker; article; binding affinity; circular dichroism; combinatorial chemistry; controlled study; DNA base composition; DNA binding; enzyme conformation; fluorescence analysis; fluorescent enzyme linked aptamer assay; ligand binding; limit of detection; molecular evolution; molecular recognition; prediction; protein secondary structure; quantitative analysis; real time polymerase chain reaction; sequence alignment; structure analysis; chemistry; combinatorial chemistry; enzymology; evaluation study; genetic procedures; human; male; molecular genetics; nucleotide sequence; procedures; prostate tumor; Aptamers, Nucleotide; Base Sequence; Biosensing Techniques; Humans; Limit of Detection; Male; Molecular Sequence Data; Prostatic Neoplasms; Racemases and Epimerases; SELEX Aptamer Technique; Tumor Markers, Biological
Type
journal article