Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against α-Bungarotoxin

Abstract

BackgroundNucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period.Principal FindingsHerein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM.ConclusionWe have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.

Aptamers: Problems, Solutions and Prospects

“…Similar to antibodies, aptamers can be used to purify target proteins [85, 86]. In contrast to antibodies, aptamers can be selected against non-immunogenic and toxic substances [87, 88]. …”

Devices and approaches for generating specific high-affinity nucleic acid aptamers

“…Using this simple method, an inexpensive and rapid one-step (single cycle) selection was demonstrated. 92 Simply monitoring selections in this way is useful to ensure that binding is taking place and to assess the degree of background binding, which may require additional washing or negative selections. In addition, the use of fluorescently labeled target molecules with the fluorescent library can be used to image both simultaneously and find co-localized spots of aptamers binding specifically to target molecules on the surface.…”

Ideal‐filter capillary electrophoresis: A highly efficient partitioning method for selection of protein binders from oligonucleotide libraries

“…An ultimate solution for problems originating from multiround selection would be a partition method that could enrich binders to the required level (e.g., 99% of binders in the binder‐enriched library) in one step of partitioning. There have been several reports of one‐step selection of oligonucleotide aptamers ; however, they have not been independently confirmed raising doubts in method practicality, transferability, and reliability. We think there are two major reasons for slow progress in creating a practical way of single‐step selection of binders from oligonucleotide libraries.…”

“…The first reason is technological: it is extremely difficult to achieve high efficiencies of partitioning, e.g., due to adsorption of nonbinders to surfaces in solid‐phase selection methods , and non‐uniform migration of the nonbinders in homogeneous CE‐based methods . The second reason is methodological: while high efficiencies of partitioning are the goal, the efficiency of partitioning was not used to guide developments or substantiate claims of one‐step selection; moreover, it was rarely measured .…”