A reliable, quick and universally applicable method for monitoring aptamer SELEX progress

Abstract

Nucleic acid aptamers for biosensing are developed from a complex ssDNA library through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. Monitoring of SELEX process is crucial for generating high-affinity aptamers. Extant methods for monitoring aptamer selection are either arduous or give false-positive signals, which adversely impact the outcome of selection. We describe a colorimetric, simple and cost-effective, novel method to monitor the progress of in vitro selections. The power of rolling circle amplification (RCA) and inherent Horse Radish Peroxidase (HRP)-mimicking activity of G-quadruplex/hemin DNAzyme were employed to produce a colorimetric signal. A unique extension of DNA population at 3ʹ-OH end by PCR generated concatenated repeats by rolling circle amplification (RCA) reaction. Oxidation of substrate ABTS (2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) in presence of H2O2 and hemin cofactor produced colorimetric signal. Analysis of the signal generated by the DNA pool bound to their target provided a quantitative measurement of SELEX. We demonstrate the reproducibility and accuracy of the method by evaluating the progress of two discrete selections. © 2021 Elsevier B.V.