title: DNA aptamer beacon assay for C-telopeptide and handheld fluorometer to monitor bone resorption.
abstract: A novel DNA aptamer beacon is described for quantification of a 26-amino acid C-telopeptide (CTx) of human type I bone collagen. One aptamer sequence and its reverse complement dominated the aptamer pool (31.6% of sequenced clones). Secondary structures of these aptamers were examined for potential binding pockets. Three-dimensional computer models which analyzed docking topologies and binding energies were in agreement with empirical fluorescence experiments used to select one candidate loop for beacon assay development. All loop structures from the aptamer finalists were end-labeled with TYE 665 and Iowa Black quencher for comparison of beacon fluorescence levels as a function of CTx concentration. The optimal beacon, designated CTx 2R-2h yielded a low ng/ml limit of detection using a commercially available handheld fluorometer. The CTx aptamer beacon bound full-length 26-amino acid CTx peptide, but not a shorter 8-amino acid segment of CTx peptide which is a common target for commercial CTx ELISA kits. The prototype assay was shown to detect CTx peptide from human urine after creatinine and urea were removed by size-exclusion chromatography to prevent nonspecific denaturing of the aptamer beacon. This work demonstrates the potential of aptamer beacons to be utilized for rapid and sensitive bone health monitoring in a handheld or point-of-care format.
authors.affiliation: Operational Technologies Corporation, 4100 NW Loop 410, Suite 230, San Antonio, TX 78229, USA. john.bruno@otcorp.com
authors.forename: John Gordon
authors.initials: JG
authors.lastname: Bruno
authors.forename: Maria P
authors.initials: MP
authors.lastname: Carrillo
authors.forename: Taylor
authors.initials: T
authors.lastname: Phillips
authors.forename: Douglas
authors.initials: D
authors.lastname: Hanson
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Bohmann
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001483:Base Sequence; D001862:Bone Resorption; D024042:Collagen Type I / Q000032:analysis / Q000737:chemistry*; D005470:Fluorometry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis*; D012984:Software
chemical_list: D052157:Aptamers, Nucleotide; D024042:Collagen Type I; D010455:Peptides; C052929:collagen type I trimeric cross-linked peptide