Processing
Aptamers (Figure 1) are short, 20–80 nt single-stranded DNA or RNA sequences or proteins that bind to target molecules with high affinity and specificity through their 3-dimensional structures. RNA sequences make up the majority of nucleic acid aptamers, perhaps because they can be synthesized by in vitro transcription in the laboratory and, with a 2′-OH, would potentially provide more diverse secondary structure than single-stranded DNA molecules. Nucleic acid aptamers are often identified using an iterative enrichment technique, such as SELEX (Figure 2), where oligos or proteins with increased affinity and specificity to a target molecule are isolated from a sequence pool after several rounds of selection.
Because they have similar target binding affinities for their targets as antibodies, yet offer several advantages over antibody-based affinity molecules, aptamers are often used as substitutes for antibodies. Aptamers are typically easier to produce, especially on a large scale. They are physically more stable, and modifications that increase intracellular stability are easily incorporated, all at a lower cost. Aptamers are also easily purified and typically have low immunogenicity. They penetrate tissues to reach their target sites faster and more effectively than antibodies, due to their smaller size (8–25 kDa nucleic acids vs. ~150 kDa antibodies), and are also able to target molecules with low antigenicity; for example, when protein targets might otherwise not provide enough epitopes for antibody binding because they are fragmented or denatured [1,2].
Like antibodies, aptamers have a broad range of applications, serving as drugs, diagnostic tools, analytic reagents, bio-imaging molecules, and biosensors (aptasensors) [1–5]. Aptamers can also be used in a targeted therapeutic role by delivering nanoparticles, antibodies, and other drugs to cancer cells through conjugation to these molecules [2].
What is SELEX?
SELEX (Systematic evolution of ligands by exponential enrichment) is a recursive nucleic acid aptamer selection technology. It works by isolating sequences with increased affinity and specificity to a target molecule from an oligo sequence pool through several rounds of selection. The process proceeds as follows (also, see Figure 2):
- A library of single-stranded DNA or RNA is generated. These sequences consist of a variable sequence region (usually 30–40 nt) flanked by static binding sites on either side.
- The library is incubated with the selected target molecule. DNA or RNA molecules that bind to the target are prospective aptamers for that target. After unbound sequences are filtered out, the bound sequences are separated from the target and purified. Nitrocellulose membrane filtration, affinity chromatography/magnetic bead, capillary electrophoresis, microfluidic chips, atomic force microscopy (AFM), electrophoretic mobility shift assays (EMSA), and surface plasmon resonance (SPR) are some of the technologies used to separate bound from unbound sequences.
- The bound sequences are then PCR amplified, creating a more specific sequence library. This library is used in a new round of SELEX to further optimize the quality of the aptamers.
The SELEX procedure can fail to produce promising aptamers due to: inadequate library design, non-specific sequence retention, accumulation of amplification artifacts, and use of screening criteria that are not associated with actual application readouts [6]. With the wide range of applications for aptamers, and the limitations of SELEX, improvements and alternatives to SELEX continue to arise (e.g., High-Fidelity SELEX [6], and MAWS—Making Aptamers Without SELEX [7]).
