Technical Document: Aptamer Screening – Principles, Applications, and Advances Document Version: 1.0 Date: October 26, 2023 Subject: Overview of the methodologies and diverse applications of aptamer screening technologies. 1.0 Executive Summary Aptamer screening, primarily through the Systematic Evolution of Ligands by EXponential enrichment (SELEX) process, is a high-throughput in vitro technology for identifying single-stranded DNA or RNA oligonucleotides (aptamers) with high affinity and specificity for a target molecule. This document outlines the core principles of aptamer screening and its transformative applications across diagnostics, therapeutics, biotechnology, and environmental monitoring. Aptamers, often termed "chemical antibodies," offer advantages such as in vitro synthesis, low immunogenicity, and ease of modification, making them powerful tools in molecular recognition. 2.0 Introduction to Aptamer Screening Aptamers are short, synthetic oligonucleotides that fold into defined three-dimensional structures, enabling them to bind to targets ranging from small ions and organic molecules to proteins, cells, and even whole organisms. The process of discovering these binding sequences is called aptamer screening. The gold standard method is SELEX, a repetitive cycle of: Incubation: A vast, random oligonucleotide library (10^14–10^15 sequences) is exposed to the target. Partitioning: Target-bound sequences are separated from unbound ones. Amplification: The bound sequences are amplified via PCR (for DNA) or RT-PCR (for RNA). Conditioning: The enriched pool is prepared for the next selection…
Aptamer Screening Methods Introduction Aptamers are single-stranded DNA or RNA oligonucleotides that bind to specific target molecules with high affinity and specificity. The Systematic Evolution of Ligands by EXponential enrichment (SELEX) process is the primary method for aptamer development. The choice of screening strategy depends critically on the nature of the target—its size, structure, chemical properties, and available functional groups for immobilization. This document outlines established and emerging SELEX methodologies tailored for different target classes: small molecules, proteins, and whole cells. 1. Screening Methods for Small Molecule Compounds Small molecule targets (MW < 1000 Da, e.g., toxins, antibiotics, hormones) present unique challenges due to their simple structure, limited binding sites, low affinity for nucleic acids, and difficulty in separation from unbound sequences. Screening strategies often require immobilization of the target or the library, with optimized separation techniques. 1.1. Agarose Affinity Chromatography SELEX Principle: The small molecule target is covalently coupled to cross-linked agarose beads packed into a chromatography column. A nucleic acid library is passed through; bound sequences are retained and later eluted for amplification. Process: Typically requires 3–18 selection rounds. Applications: Early and successful selection of aptamers for dyes, ATP, S-adenosylhomocysteine, L-arginine, coenzyme A, kanamycin, and benzylpenicillin. Advantages: Mature, reliable technology. Limitations: Requires large…
In recent years, various screening methods have been developed to generate aptamers more reliably and efficiently. These include Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and its various derivative techniques, such as magnetic bead SELEX, capture SELEX, graphene oxide SELEX, cell-SELEX, capillary electrophoresis SELEX, and atomic force microscopy SELEX. This review summarizes these methods and analyzes the key characteristics, advantages, and limitations of each SELEX approach (see Table 1). The wide application of aptamers has driven the continuous development of SELEX technology. Over recent decades, numerous new methods for screening aptamers have emerged, significantly reducing the screening time from weeks to just hours. Table 1. Advantages and disadvantages of SELEX method currently used. Method Key Aspects Advantages Disadvantages Target small molecule compounds Agarose affinity chromatography SELEX The aptamers that can bind to the target can be isolated by fixing the small molecular target with agarose affinity chromatography column The most traditional aptamer screening method with the longest application time Low separation efficiency, requires relatively large amounts of elution materials MB-SELEX SsDNA or target was fixed with magnetic beads, and the bound sequence was separated from the unbound sequence by magnetic field. Avoid changes in the inherent structure of the target…
