Aptamers are small stretches of nucleic acids, which show a high affinity to a large group of target molecules. Aptamers are synthetically derived from nucleic acids and play a significant role as a therapeutic agent. Since their discovery these agents are being largely used to cure a large number of disorders including neurological disorders, wherein drugs have to be potent enough to cross the blood brain barrier. As nucleic acids have a remarkable property of adopting a stable, intricately folded, unique three-dimensional structure, they have been used during generation of aptamers. The structure of nucleic acids provides them a scaffold, to interact with a large number of functional side groups of various ligands, like proteins antibiotics, dyes, drugs etc. As a result, aptamers can be applied in various ways under physiological conditions (Dua, 2008). As an inhibitor, an aptamer inhibits the function of a targeted protein by obtaining a specific three- dimensional structure, which in turn dictates its high-affinity binding to the target protein.
How are aptamers generated?
Aptamers are generated by an iterative in vitro process known as SELEX or systematic evolution of ligands by exponential enrichment. This process was independently established in 1990 by Tuerk and Gold, Ellington and Szostak and Robertson and Joyce (Meyer, 2011). A large group of oligonucleotides (1014-1015) are used for the generation of aptamer. Random regions of DNA are taken and cloned between two defined fragments. The constant region at the 3’ end contains an attachment site for reverse transcriptase primers and a promoter region of T7 RNA polymerase. The constant region at the 5’ end contains the attachment site for the primers of the PCR.
Within a DNA SELEX, a double stranded DNA is converted to single stranded DNA and later to RNA with the help of T7 RNA polymerase through the process of in vitro transcription. A group of RNA molecules are allowed to adopt secondary structures and incubated with target molecules. Those RNA molecules which bind to the targets are selected as aptamers. They are separated from non-aptamers and are reverse transcribed followed by amplification through PCR. This results in a generation of enriched target-specific DNA molecules. This library is made ready for the next round of selection. This entire process is repeated several times so that the molecules present in the final library of aptamers are highly specific and show high affinity towards the ligand. Hence, during this entire procedure the steps of nucleic acid binding, partitioning and amplification are repeated several times to generate a saturated pool of desired aptamers (Dua, 2008).
After the selection process through SELEX, a specific antagonist to a target protein is obtained. This in vitro antagonist has to be tested in various angles to determine its pharmacological effects in animals. It should be chemically synthesized in sufficient quantities, so that it can be used for different in vivo experiments. In order to carry out cost effective chemical synthesis, the size of an aptamer should be minimized (40 nucleotides in length). Strategies for minimizing the size of an aptamer are so chosen that, high affinity towards the target is maintained. In comparison to proteins, the size of most of the aptamers can be successfully minimized with minimal loss of affinity (White, 2000).
Once an aptamer is ready for chemical synthesis, it is subjected to modifications to enhance the bioavailability and to make their delivery easy. The bioavailability of an aptamer basically depends on two key properties: stability in various biological fluids and how well it gets cleared from the system. RNA and DNA oligonucleotides have very short half lives (few seconds in RNA and 30-60 min in DNA), within plasma under in vitro conditions. RNA oligonucleotides are modified by substituting with 2′-amino, 2′-fluoro, or 2′-O-alkyl nucleotides, so that they obtain higher plasma stability. This extends their in vitro half-lives to 5-15 hour range. An aptamer is protected from getting degraded by exonucleases by capping its 3′ end. The ribose and deoxyribose nucleotides of aptamers are substituted with nonnucleotide or modified nucleotides linkers so that aptamers become resistant to the action of endonucleases. Due to their minimized size they get easily cleared off from the kidneys of the animals, however, the clearance rates of aptamers can be easily modified if required (White, 2000).
After rigorous tests on animal models and various rounds of clinical testing, aptamers have successfully entered the market in 2004 in the form of a drug known as Macugen. This drug was successful in treating age-related macular degeneration (AMD) disease. Apatamers are presently being synthesized against a large number of agents; however some of them have to be still clinically tested before they can be used as a drug.
The instability of an aptamer makes the conversion of an aptamer to a therapeutic agent a challenging task. Due to their instability, these aptamers have to be given in multiple doses; hence the cost of synthesis on a large scale is also a point of concern. In spite of these challenges these designer drugs prove to be efficient in targeted therapy.
1. Dua, P., Kim, S., & Lee, D,K. (2008). Patents on SELEX and Therapeutic Aptamers. Recent Pat DNA Gene Seq, 2(3), 172-86.
2. Meyer, C., Hahn, U., & Rentmeister, A. (2011). Cell-Specific Aptamers as Emerging Therapeutics. J Nucleic Acids, 904750.
3. White, R, R., Sullenger, B, A., & Rusconi, C, P. (2000). Developing aptamers into therapeutics. J Clin Invest, 106(8), 929–934.