SELEX (Systematic Evolution of Ligands by EXponential enrichment) was first described and used in 1990 to isolate a functional oligonucleotide from a random pool of differently-shaped DNA or RNA molecules.1-2SELEX-derived oligonucleotides (RNA or single-stranded DNA) known as aptamers, bind with high affinity, selectivity, and sensitivity to a broad range of targets such as proteins, cells, microorganisms, and small chemical molecules because of their unique three-dimensional shapes.
Like antibodies, aptamers can bind to their targets through structural recognition, but aptamers offer distinct advantages over antibodies including smaller size, better tissue penetration, higher thermal stability, lower immunogenicity, reduced cost of synthesis, and easier modification with different chemical groups. These advantages make aptamers an attractive substitute for antibodies in the fields of biomarker discovery, diagnosis, imaging, and targeted therapy.3
In his interview titled “Rethinking the origin of life,” Dipankar Sen, PhD, professor of molecular biology and biochemistry at Simon Fraser University and a pioneer in the SELEX field notes, “With huge synthetic DNA and RNA libraries now available, we have endless opportunities to create novel enzymes that perform specific therapeutic functions. Ultimately, we hope to synthesize nucleic acid enzymes to help counteract cancers and viral infections.”4 Sen’s ideas became a reality when a group of scientists recently identified two therapeutic DNA aptamers that have the potential to bind and block the SARS-COV-2 interacting region of the human ACE2 receptor.5
This article provides an overview of a SELEX assay with a focus on factors to consider when designing the experiment.
Generating an oligonucleotide library
The first step is the synthesis of largely randomized oligonucleotide sequences, typically up to 1014 to 1015 molecules. To allow for later PCR amplification, scientists add defined regions flanking the random sequences of these oligonucleotides. In the case of RNA SELEX, in vitro transcription of the randomized sequence is applied instead.
Just prior to introducing the target, the oligonucleotide library is denatured by heating the sample to nearly 100°C and then allowing it to cool slowly to create thermodynamically stable secondary and tertiary structures with desired binding properties.
The oligonucleotide library is next incubated with the target. This requires planning the method of immobilizing the target, strategies for subsequently separating the unbound oligonucleotides, the time, temperature, and buffer composition for the incubation, and optimizing the concentrations of the target and the oligonucleotides. The most common target immobilization methods are affinity chromatography columns, nitrocellulose binding assay filters, and paramagnetic beads.6
The conditions of the incubation buffer can be altered depending on the intended target and the desired function of the selected aptamer. For example, if the target is a negatively charged small molecule or protein, it is better to use high salt buffers to increase the chance of binding. If the target is an in vivo protein or a whole cell, then the incubation buffer components and temperature should resemble physiological conditions.
The relative concentrations of target and oligonucleotides should also be adjusted. Usually, an excess of the library oligonucleotides is favored because it boosts the competition between unique sequences for the available binding sites on the target. On the other hand, an excess of target can increase the probability that at least some sequences will bind to the target even if the binding affinity is low.
Washing, elution, and amplification
After an optimized incubation with the desired target for sufficient time, the oligonucleotide library is subjected to a washing step with the same incubation buffer. This step aims to remove the unbound oligonucleotides from the immobilized target and retain the bound ones. This is followed by eluting the specifically bound sequences using denaturing solutions containing urea and EDTA or applying heat to promote oligonucleotide unfolding and detachment.
In the case of DNA SELEX, the eluted oligonucleotides are then collected for PCR amplification or first reverse-transcribed, followed by PCR amplification in the case of RNA SELEX. The amplified DNA sequences are then converted to single-stranded oligonucleotides, which are used as the initial input for the next round of selection.
Negative selection and tracking progress
A negative selection (counter SELEX) step can be added before or immediately after target incubation to eliminate sequences that might have an affinity for the immobilization matrix components. In this step, the oligonucleotide library is incubated with the immobilization matrix, and the unbound sequences are retained. Counter SELEX can also be applied to eliminate sequences that bind target-like molecules or cells by incubating the oligonucleotides library with target analogs, undesired cell types, or non-target proteins.
The progress of a SELEX interaction can be tracked after each round by comparing the total input of oligonucleotides to those that get eluted. The number of eluted oligonucleotides can be assessed by measuring the absorbance at 260 nm or employing fluorescence or radiolabeling methods.
To date SELEX has been used to obtain a considerable number of aptamers that have completed Phase II clinical trials.6 The data from these trials are promising and show that SELEX-based aptamers development is advancing rapidly. Soon we may see aptamers being used as therapeutic tools for new biomarker discovery, earlier diagnosis of diseases, and effective drug delivery.
- Ellington AD, Szostak JW. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990;346(6287):818-822. doi:10.1038/346818a0
2. Tuerk C, Gold L. Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase. Science. 1990;249(4968):505-510. doi:10.1126/science.2200121
3. Afrasiabi S, Pourhajibagher M, Raoofian R, Tabarzad M, Bahador A. Therapeutic applications of nucleic acid aptamers in microbial infections. J Biomed Sci. 2020;27(1):6. doi:10.1186/s12929-019-0611-0
4. Rethinking the origin of life. Sen, D. Molecular Biology and Biochemistry, 2014. [Archived content at Simon Fraser University]
5. Villa A, Brunialti E, Dellavedova J, et al. DNA aptamers masking angiotensin converting enzyme 2 as an innovative way to treat SARS-CoV-2 pandemic. Pharmacol Res. 2022;175:105982. doi:10.1016/j.phrs.2021.105982
6. SELEX methods on the road to protein targeting with nucleic acid aptamers | Elsevier Enhanced Reader. doi:10.1016/j.biochi.2018.09.001