Systematic Evolution of Ligands by EXponential Enrichment (SELEX) is a laboratory technique that isolates specific nucleic acid sequences. It identifies unique DNA or RNA strands that bind precisely to a chosen target molecule, much like finding a key for a lock. This process discovers molecular tools with diverse applications.
The SELEX Process
The SELEX procedure begins with a vast library of synthetic DNA or RNA sequences, estimated at 10^13 to 10^15 random sequences. Each is of a fixed length, flanked by constant primer sites for later amplification. These random sequences are designed to fold into unique three-dimensional shapes, offering a wide array of potential binding structures.
The nucleic acid sequences are then mixed with the target molecule of interest, such as a protein, a small organic compound, or even a whole cell. During incubation, some sequences bind to the target due to their three-dimensional conformations and chemical interactions.
Next, a partitioning step separates the bound sequences from those that did not bind to the target. Techniques like affinity chromatography or paramagnetic beads wash away unbound sequences, leaving only target-bound complexes. This filters out non-binding sequences.
The bound nucleic acid sequences are then collected, a process known as elution. These isolated sequences are copied millions of times using amplification techniques like Polymerase Chain Reaction (PCR) for DNA aptamers or reverse transcription-PCR for RNA aptamers. This ensures sufficient quantities for the next selection round.
This cycle of incubation, partitioning, elution, and amplification repeats multiple times, typically 10 to 20 rounds. Each successive round increases selection stringency, retaining only sequences with stronger, more specific binding affinities. This iterative enrichment process gradually refines the pool, leading to a collection dominated by sequences that bind most effectively to the target.
Understanding Aptamers
Aptamers are specific single-stranded DNA or RNA molecules emerging from the SELEX process. Engineered in the laboratory, they bind with high specificity and affinity to a given target, acquiring unique three-dimensional structures. Their binding capabilities are comparable to antibodies, making them molecular tools.
A distinction between aptamers and antibodies lies in their origin and characteristics. Antibodies are proteins produced biologically by an animal’s immune system. Aptamers, in contrast, are chemically synthesized in a laboratory, allowing precise control over their sequence and structure, and eliminating the need for animal immunization. This chemical synthesis also leads to lower production costs and ensures high batch-to-batch consistency, reducing variability seen with biologically derived reagents.
Aptamers are much smaller than antibodies; a typical aptamer (30-80 nucleotides) weighs 12 to 30 kilodaltons, while an IgG antibody is 150 to 170 kilodaltons. This smaller size allows for improved tissue penetration and access to targets in dense tissues, which is limited for larger antibodies. Aptamers also exhibit high stability, tolerating a wider range of temperatures and pH, and can be reversibly denatured and refolded without losing binding activity.
Applications of Aptamer Technology
Aptamers generated through SELEX have found utility across various scientific and medical fields. Their ability to bind specifically to diverse targets, from small molecules to complex cellular structures, makes them versatile. Applications range from disease detection to therapeutic interventions.
In diagnostics, aptamers are employed in biosensors to detect biomarkers, pathogens, or toxins with high sensitivity. Aptamer-based biosensors can identify cancer cells or viral infections at early stages, offering rapid and precise detection. They can be incorporated into point-of-care devices, streamlining diagnostics and contributing to personalized medicine.
Aptamers also have therapeutic applications, acting as targeted drugs or drug delivery vehicles. They can block disease-causing proteins, such as in age-related macular degeneration, where pegaptanib was approved to inhibit vascular endothelial growth factor (VEGF). Aptamers can also be conjugated to therapeutic agents to deliver drugs directly to specific cells, like cancer cells, minimizing off-target effects and enhancing treatment efficacy.
Beyond diagnostics and therapeutics, aptamers serve as research tools in laboratory settings. They purify specific proteins from complex mixtures, facilitating isolation and characterization. Aptamers can also visualize molecules within cells, providing insights into cellular processes and molecular interactions.
Variations of the SELEX Technique
The SELEX process has undergone adaptations to address complex target challenges and expand its capabilities. These variations allow for aptamer selection under conditions that more closely mimic their intended biological environments. This evolution highlights the versatility of the SELEX principle.
Cell-SELEX
Cell-SELEX uses whole living cells as the target for aptamer selection. Instead of purified molecules, the random nucleic acid library is incubated directly with target cells, such as cancer cells. This approach discovers aptamers that recognize specific cell surface markers in their native conformation, even if the marker’s precise molecular identity is unknown. Cell-SELEX often incorporates a negative selection step, exposing the library to non-target cells to remove non-specific binders, enhancing selectivity for the desired cell type.
In Vivo SELEX
In Vivo SELEX performs aptamer selection within a living organism. The nucleic acid library is introduced into an animal model, allowing sequences to circulate and interact with targets in their natural physiological environment. Aptamers binding to specific tissues or organs are retained, while unbound sequences are cleared. This approach identifies aptamers that function effectively within the complex biological milieu, considering factors like cellular uptake, metabolism, and interactions with endogenous molecules.