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Aptamers are oligomers of artificial ssDNA, RNA, XNA, or peptide that ligand, or family of target molecules. They exhibit a range of affinities (KD in the pM to μM range), with variable levels of Antitarget binding and are sometimes classified as antibody mimetic. Aptamers and antibodies can be used in many of the same applications, but the nucleic acid-based structure of aptamers, which are mostly oligonucleotides, is very different from the amino acid-based structure of antibodies, which are proteins. This difference can make aptamers a better choice than antibodies for some purposes (see antibody replacement).
Aptamers are used in biological lab research and medical tests. If multiple aptamers are combined into a single assay, they can proteomics. They can be used to identify molecular markers of disease, or can function as RNA therapeutics, drug delivery systems and controlled drug release systems. They also find use in other molecular engineering tasks.
Most aptamers originate from SELEX, a family of in vitro for finding useful aptamers in a massive pool of different DNA sequences. This process is much like natural selection, directed evolution or artificial selection. In SELEX, the researcher repeatedly selects for the best aptamers from a starting DNA library made of about a quadrillion different randomly generated pieces of DNA or RNA. After SELEX, the researcher might mutate or bioconjugation of the aptamers and do another selection, or might use rational design processes to engineer improvements. Non-SELEX methods for discovering aptamers also exist.
Researchers optimize aptamers to achieve a variety of beneficial features. The most important feature is specific and sensitive binding to the chosen target. When aptamers are exposed to bodily fluids, as in serum tests or aptamer therapeutics, it is often important for them to resist hydrolysis by nucleases. Therapeutic aptamers often must be modified to clear slowly from the body. Aptamers that change their shape dramatically when they bind their target are useful as to turn a sensor on and off. Some aptamers are engineered to fit into a biosensor or in a assay. It can be useful in some cases for the aptamer to accomplish a pre-defined level or rate constant of binding. As the yield of the synthesis used to produce known aptamers shrinks quickly for longer sequences, researchers often truncate aptamers to the minimal binding sequence to reduce the production cost.
The word itself, however, derives from the Greek language word ἅπτω, to connect or fit (as used by Homer (c. 8th century BC)) and μέρος, a component of something larger.
In 1990, two teams independently developed and published SELEX ( Systematic Evolution of Ligands by EXponential enrichment) methods and generated RNA aptamers: the lab of Larry Gold, using the term SELEX for their process of selecting RNA ligands against T4 DNA polymerase and the lab of Jack Szostak, selecting RNA ligands against various . Two years later, the Szostak lab and Gilead Sciences, acting independently of one another, used in vitro selection schemes to generate DNA aptamers for organic dyes and human thrombin, respectively. In 2001, SELEX was automated by J. Colin Cox in the Ellington lab, reducing the duration of a weeks-long selection experiment to just three days.
In 2002, two groups led by Ronald Breaker and Evgeny Nudler published the first definitive evidence for a riboswitch, a nucleic acid-based genetic regulatory element, the existence of which had previously been suspected. Riboswitches possess similar molecular recognition properties to aptamers. This discovery added support to the RNA World hypothesis, a postulated stage in time in the origin of life on Earth.
As 22 genetically encoded and over 500 naturally occurring amino acids exist, peptide aptamers, as well as antibodies, have much greater potential combinatorial diversity per unit length relative to the 4 nucleic acids in DNA or RNA. Chemical modifications of nucleic acid bases or backbones increase the chemical diversity of standard nucleic acid bases.
Split aptamers are composed of two or more DNA strands that are pieces of a larger parent aptamer that has been broken in two by a molecular nick. The ability of each component strand to bind targets will depend on the location of the nick, as well as the secondary structures of the daughter strands. The presence of a target molecule supports the joining of DNA fragments. This can be used as the basis for biosensors. Once assembled, the two separate DNA strands can be ligated into a single strand.
Unmodified aptamers are cleared rapidly from the bloodstream, with a half-life of seconds to hours. This is mainly due to nuclease degradation, which physically destroys the aptamers, as well as clearance by the kidneys, a result of the aptamer's low molecular weight and size. Several modifications, such as 2'-fluorine-substituted pyrimidines and polyethylene glycol (PEG) linkage, permit a serum half-life of days to weeks. PEGylation can add sufficient mass and size to prevent clearance by the kidneys in vivo. Unmodified aptamers can treat coagulation disorders. The problem of clearance and nuclease digestion is diminished when they are applied to the eye, where there is a lower concentration of nuclease and the rate of clearance is lower. Rapid clearance from serum can also be useful in some applications, such as in vivo diagnostic imaging.
/ref> designed to bind with proteins associated with Ebola infection, a comparison was made among three aptamers isolated for their ability to bind the target protein EBOV sGP. Although these aptamers vary in both sequence and structure, they exhibit remarkably similar relative affinities for sGP from EBOV and SUDV, as well as EBOV GP1.2. Notably, these aptamers demonstrated a high degree of specificity for the GP gene products. One aptamer, in particular, proved effective as a recognition element in an electrochemical sensor, enabling the detection of sGP and GP1.2 in solution, as well as GP1.2 within a membrane context.The results of this research point to the intriguing possibility that certain regions on protein surfaces may possess aptatropic qualities. Identifying the key features of such sites, in conjunction with improved 3-D structural predictions for aptamers, holds the potential to enhance the accuracy of predicting aptamer interaction sites on proteins. This, in turn, may help identify aptamers with a heightened likelihood of binding proteins with high affinity, as well as shed light on protein mutations that could significantly impact aptamer binding.This comprehensive understanding of the structure-based interactions between aptamers and proteins is vital for refining the computational predictability of aptamer-protein binding. Moreover, it has the potential to eventually eliminate the need for the experimental SELEX protocol.
Aptamers have been generated against cancer cells, prions, bacteria, and viruses. Viral targets of aptamers include influenza A and B viruses, Respiratory syncytial virus (RSV), SARS coronavirus (SARS-CoV) and SARS-CoV-2.
Aptamers may be particularly useful for environmental science proteomics. Antibodies, like other proteins, are more difficult to sequence than nucleic acids. They are also costly to maintain and produce, and are at constant risk of contamination, as they are produced via cell culture or are harvested from animal serum. For this reason, researchers interested in little-studied proteins and species may find that companies will not produce, maintain, or adequately validate the quality of antibodies against their target of interest. By contrast, aptamers are simple to sequence and cost nothing to maintain, as their exact structure can be stored digitally and synthesized on demand. This may make them more economically feasible as research tools for underfunded biological research subjects. Aptamers exist for plant compounds, such as theophylline (found in tea) and abscisic acid (a plant immune hormone). (2026). 9781509050789 ISBN 9781509050789 An aptamer against a-amanitin (the toxin that causes lethal Amanita poisoning) has been developed, an example of an aptamer against a mushroom target.
Aptamer applications can be roughly grouped into sensing, therapeutic, reagent production, and engineering categories. Sensing applications are important in environmental, biomedical, Epidemiology, biosecurity, and basic research applications, where aptamers act as probes in assays, imaging methods, diagnostic assays, and biosensors.Agnivo Gosai, Brendan Shin Hau Yeah, Marit Nilsen-Hamilton, Pranav Shrotriya, "Label free thrombin detection in presence of high concentration of albumin using an aptamer-functionalized nanoporous membrane", Biosensors and Bioelectronics, Volume 126, 2019, pp. 88–95, , . In therapeutic applications and precision medicine, aptamers can function as drugs, as targeted drug delivery vehicles, as controlled release mechanisms, and as reagents for drug discovery via high-throughput screening for small molecules and proteins. Aptamers have application for protein production monitoring, quality control, and purification. They can function in molecular engineering applications as a way to modify proteins, such as enhancing DNA polymerase to make PCR more reliable.
Because the affinity of the aptamer also affects its dynamic range and limit of detection, aptamers with a lower affinity may be desirable when assaying high concentrations of a target molecule. Affinity chromatography also depends on the ability of the affinity reagent, such as an aptamer, to bind and release its target, and lower affinities may aid in the release of the target molecule. Hence, specific applications determine the useful range for aptamer affinity.
In addition, aptamers contribute to reduction of research animal use. While antibodies often rely on animals for initial discovery, as well as for production in the case of polyclonal antibodies, both the selection and production of aptamers is typically animal-free. However, phage display methods allow for selection of antibodies in vitro, followed by production from a Monoclonality cell line, avoiding the use of animals entirely.
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