Combining capillary electrophoresis and next-generation sequencing for aptamer selection

Abstract

Next-generation sequencing (NGS) machines can sequence millions of DNA strands in a single run, such as oligonucleotide (oligo) libraries comprising millions to trillions of discrete oligo sequences. Capillary electrophoresis is an attractive technique to select tight binding oligos or “aptamers” because it requires minimal sample volumes (e.g., 100 nL) and offers a solution-phase selection environment through which enrichment of target-binding oligos can be determined quantitatively. We describe here experiments using capillary transient isotachophoresis (ctITP)-based nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) as a method for selecting aptamers from a randomized library containing a known (29mer) thrombin-binding aptamer. Our capillary electrophoresis (CE)-selected samples were sequenced by the Ion Torrent Personal Genome Machine (PGM) and analyzed for selection efficiency. We show that a single round of CE selection can enrich a randomer synthetic DNA oligo mixture for thrombin-binding activity from 0.4 % aptamer content before selection to >15 % aptamer content.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-014-8427-y) contains supplementary material, which is available to authorized users.

Facilitating aptamer selection and collection by capillary transient isotachophoresis with laser-induced fluorescence detection

“…Ongoing studies in our laboratory are employing this novel CE-based methodology as a preselection tool for next generation sequencing experiments, as proof-of-principle for a coupled drug discovery system [36]. Preselection enrichment factors must be demonstrated to be sufficient for the ultimate goal of winnowing vast DNA-encoded chemical libraries down to practicable inputs for automated sequencing technologies as key elements of drug discovery systems.…”

“…DNA sequences were used with Ion Torrent TM primer handles in this work in order to demonstrate the compatibility of our methods with the Ion PGM TM next generation sequencing system, which was employed in subsequent studies documented elsewhere [36].…”

Selection of a Novel Aptamer Against Vitronectin Using Capillary Electrophoresis and Next Generation Sequencing

“…33,34 We utilized CE-NGS SELEX to identify an aptamer against VN. 35,36 Herein we describe the selection, characterization, and drug delivery capability of a novel aptamer against VN, VBA-01, and its dimer DVBA-01, using CE-NGS SELEX.…”

In Vitro Selection and Characterization of DNA Aptamers to a Small Molecule Target

“…One recent breakthrough technology is high-throughput sequencing, which has led to advancements even in small molecule selection. Recent examples of high-throughput sequencing applied to aptamer selection include selections for tyramine (Valenzano et al, 2016), thrombin (Riley et al, 2015), and ovarian cancer biomarkers CA125 and HE4 (Eaton et al, 2015;Scoville, Uhm, Shallcross, & Whelan, 2017). Recent examples of high-throughput sequencing applied to aptamer selection include selections for tyramine (Valenzano et al, 2016), thrombin (Riley et al, 2015), and ovarian cancer biomarkers CA125 and HE4 (Eaton et al, 2015;Scoville, Uhm, Shallcross, & Whelan, 2017).…”