title: Facilitating aptamer selection and collection by capillary transient isotachophoresis with laser-induced fluorescence detection.
abstract: An efficient separation method that utilizes capillary transient isotachophoresis (ctITP) was developed for the preselection of binding ligands. With the ultimate goal of providing enriched fractions from vast libraries for drug discovery, the preselection process described herein entails three distinct elements, which have been validated using a model thrombin protein (target) and thrombin aptamer (ligand) system. First, a high fidelity, on-column labeling scheme employing the noncovalent, fluorescent reagent SYBR Gold was demonstrated for single-stranded DNA with an 11-fold greater sensitivity than pre-column labeling procedures. Second, this on-column labeling was incorporated into a new ctITP method with laser-induced fluorescence (LIF) detection, which provided greatly enhanced resolution of protein-aptamer complex and free aptamer (in comparison to traditional capillary zone electrophoresis (CZE) methods). Third, this enhanced resolution permitted the subsequent accumulation of bound aptamer fractions via an automated collection method, with the establishment of quantitative measures of DNA accumulation. Preselected aptamer or ligand samples such as these can serve as inputs for subsequent lab-on-bead or next-generation-sequencing technologies, enabling accelerated drug discovery.
authors.affiliation: Department of Chemistry, Wake Forest University, Winston-Salem, NC, 27109, USA.
authors.forename: Kathryn R
authors.initials: KR
authors.lastname: Riley
authors.affiliation: Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Saito
authors.affiliation: Department of Physics, Wake Forest University, Winston-Salem, NC, 27109, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Gagliano
authors.affiliation: Department of Chemistry, Wake Forest University, Winston-Salem, NC, 27109, USA. Electronic address: colyercl@wfu.edu.
authors.forename: Christa L
authors.initials: CL
authors.lastname: Colyer
mesh_terms: D052157:Aptamers, Nucleotide / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D008024:Ligands; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013917:Thrombin / Q000032:analysis
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; C081676:thrombin aptamer; D013917:Thrombin
keywords: Capillary electrophoresis; Capillary transient isotachophoresis; Fluorescence detection; Fraction collection; SYBR Gold; Thrombin aptamer selection