An aptamer is a short, single-stranded molecule of DNA or RNA that folds into a specific three-dimensional shape, allowing it to bind to a target molecule with high precision. Often described as chemical antibodies, they function like a molecular key designed to fit a particular lock. An aptamer library is a vast collection of these potential keys, containing over a quadrillion unique sequences. This immense diversity is the starting point for a selection process aimed at finding a sequence that can bind to virtually any intended target, from a small chemical compound to an entire cell.
Composition of an Aptamer Library
Each potential aptamer in a library is a synthetically created oligonucleotide, a short strand of nucleic acid, with two distinct parts. The core of the molecule is a central randomized region responsible for the aptamer’s binding capabilities, which typically ranges from 20 to 80 nucleotides in length. The sequence of nucleotide bases—adenine (A), guanine (G), cytosine (C), and thymine (T) in DNA or uracil (U) in RNA—in this region is intentionally varied.
Flanking this variable region on both ends are fixed or constant regions. These sequences are the same for every molecule in the library and serve as universal “handles.” While they do not participate in binding the target, they are necessary for the laboratory techniques used to manipulate the library, particularly for amplification.
Generating an Aptamer Library
The creation of an aptamer library is a process of chemical synthesis. Unlike biological processes that replicate existing genetic material, this initial library is built from the ground up, one nucleotide base at a time, allowing for precise control over each strand’s structure. Chemists synthesize the molecules, paying special attention to the central randomized region.
During the synthesis of this segment, a mixture of all four nucleotide building blocks (A, T/U, C, and G) is supplied at each step. This method ensures that at any given position within the variable region, any of the four bases can be incorporated, resulting in a combinatorial explosion of sequence possibilities. The goal is to create a population so varied that it is statistically likely to contain at least one molecule capable of binding the desired target.
The Selection Process (SELEX)
To find the one sequence out of trillions that binds to a specific target, a method named SELEX is used, which stands for Systematic Evolution of Ligands by Exponential Enrichment. The process is analogous to panning for gold, where a large amount of sand is sifted through repetitive cycles to isolate a few valuable flakes. Each cycle of SELEX enriches the pool of oligonucleotides, progressively increasing the concentration of sequences that bind to the target.
The process involves several steps:
- Incubation: The entire aptamer library is mixed with the target molecule, such as a protein or toxin. During this step, any aptamers that have a three-dimensional shape complementary to the target will bind to it.
- Partitioning: This is a wash phase where all unbound sequences, which constitute the vast majority of the library, are washed away. This leaves only the aptamers that have successfully bound to the target molecules.
- Elution: The bound aptamers are recovered. A change in chemical conditions, such as altering pH or temperature, is used to disrupt the binding and release the aptamers from the target.
- Amplification: This small sample of successful binders is amplified using the Polymerase Chain Reaction (PCR). The fixed-region “handles” on each aptamer are used to make millions of copies, creating a new, enriched library for the next round.
This entire cycle is typically repeated between five and fifteen times. With each successive round, the selection pressure can be increased to favor only the tightest-binding aptamers, ensuring the final product is highly specific.
Applications of Selected Aptamers
The specific molecules isolated through SELEX have a wide range of practical uses across medicine, research, and diagnostics. In diagnostics, aptamers are used in biosensors to detect disease biomarkers, pathogens, or environmental toxins. They can be integrated into systems where this binding event produces a measurable output, such as a change in color or an electrical signal, indicating the presence of the target.
In therapeutics, aptamers can function as drugs by binding to and disrupting the activity of proteins involved in disease pathways. A prominent example is Macugen (pegaptanib), an FDA-approved drug used to treat age-related macular degeneration. This drug works by binding to and inhibiting a protein called VEGF, which promotes abnormal blood vessel growth in the eye.
Aptamers are also valuable research tools. Scientists use them for affinity chromatography, where an aptamer is attached to a solid support to “fish” a specific protein out of a complex biological mixture for study. They can also be attached to fluorescent dyes for bioimaging to visualize the location of specific molecules within living cells.