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Aptamer
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Aptamer
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Left: Unbound aptamer. Right: the aptamer bound to its target protein. The protein is in yellow. Parts of the aptamer that change shape when it binds its target are in blue, while the unchanging parts are in orange. The parts of the aptamer that contact the protein are highlighted in red.
Breast cancer cells incubated with aptamers that bind selectively to biomarkers on the cancer cells, but not to healthy cells. Aptamers are linked to Alexa Fluor 594, a molecule that glows red under UV light. This type of test allows a doctor or researcher to identify cancer cells in a tissue sample from a patient.

Aptamers are oligomers of artificial ssDNA, RNA, XNA, or peptide that bind a specific target molecule, or family of target molecules. They exhibit a range of affinities (KD in the pM to μM range),[1][2] with variable levels of off-target binding[3] and are sometimes classified as chemical antibodies. 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 measure large numbers of different proteins in a sample. They can be used to identify molecular markers of disease, or can function as drugs, 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 test-tube experiments 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 change the chemistry 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 digestion by DNA- and RNA-destroying enzymes. 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 molecular switches to turn a sensor on and off. Some aptamers are engineered to fit into a biosensor or in a test of a biological sample. It can be useful in some cases for the aptamer to accomplish a pre-defined level or speed of binding. As the yield of the synthesis used to produce known aptamers shrinks quickly for longer sequences,[4] researchers often truncate aptamers to the minimal binding sequence to reduce the production cost.

Etymology

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The word "aptamer" is a neologism coined by Andrew D. Ellington and Jack Szostak in their first publication on the topic. They did not provide a precise definition, stating "We have termed these individual RNA sequences 'aptamers', from the Latin aptus, to fit."[5]

The word itself, however, derives from the Greek word ἅπτω, to connect or fit (as used by Homer (c. 8th century BC)[6][7]) and μέρος, a component of something larger.[8]

Classification

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A typical aptamer is a synthetically generated ligand exploiting the combinatorial diversity of DNA, RNA, XNA, or peptide to achieve strong, specific binding for a particular target molecule or family of target molecules. Aptamers are occasionally classified as "chemical antibodies" or "antibody mimics".[9] However, most aptamers are small, with a molecular weight of 6-30 kDa, in contrast to the 150 kDa size of antibodies, and contain one binding site rather than the two matching antigen binding regions of a typical antibody.

History

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Jack Szostak, Nobel laureate and one of the inventors of SELEX and aptamers.

Since its first application in 1967,[10] directed evolution methodologies have been used to develop biomolecules with new properties and functions. Early examples include the modification of the bacteriophage Qbeta replication system and the generation of ribozymes with modified cleavage activity.[11]

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[12] and the lab of Jack Szostak, selecting RNA ligands against various organic dyes.[5][13] 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[14] and human thrombin,[15] 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.[16][17][18]

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.[19]

Properties

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Structure

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The complex and diverse secondary and tertiary structure of aptamers, as shown in this schematic of an aptamer's secondary structure, is what lets them bind their target strongly and specifically. Complementary base pairing is visible in the black lines connecting some G-C and A-T bases. This is a feature of nucleic acids that does not exist in the amino acids of antibodies. It helps aptamers form these unique structures. Hairpin regions (red), which rely on this base pairing, enhance the aptamer's stability at different temperatures. This image also shows examples of chemical modifications to the base aptamer. Two unnatural bases, which enhance durability, are in yellow. The biotin molecule binds with extreme strength to streptavidin, allowing the aptamer to be anchored to other molecules or to a surface in sensors and assays.

Most aptamers are based on a specific oligomer sequence of 20-100 bases and 3-20 kDa. Some have chemical modifications for functional enhancements or compatibility with larger engineered molecular systems. DNA, RNA, XNA, and peptide aptamer chemistries can each offer distinct profiles in terms of shelf stability, durability in serum or in vivo, specificity and sensitivity, cost, ease of generation, amplification, and characterization, and familiarity to users. Typically, DNA- and RNA-based aptamers exhibit low immunogenicity, are amplifiable via Polymerase Chain Reaction (PCR), and have complex secondary structure and tertiary structure.[20][21][22][23] DNA- and XNA-based aptamers exhibit superior shelf stability. XNA-based aptamers can introduce additional chemical diversity to increase binding affinity or greater durability in serum or in vivo.

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.[24] Chemical modifications of nucleic acid bases or backbones increase the chemical diversity of standard nucleic acid bases.[25]

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.[26] 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.[27] The presence of a target molecule supports the joining of DNA fragments. This can be used as the basis for biosensors.[28] 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.[29] Rapid clearance from serum can also be useful in some applications, such as in vivo diagnostic imaging.[30]

In a study on aptamers[31] 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.

Targets

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Aptamer targets can include small molecules and heavy metal ions, larger ligands such as proteins, and even whole cells.[32][33] These targets include lysozyme,[34] thrombin,[35][36] human immunodeficiency virus trans-acting responsive element (HIV TAR),[37] hemin,[38] interferon γ,[39] vascular endothelial growth factor (VEGF),[40][41] prostate specific antigen (PSA),[42][43] dopamine,[44] and the non-classical oncogene, heat shock factor 1 (HSF1).[45]

Aptamers have been generated against cancer cells,[46] prions,[47] bacteria,[48] and viruses. Viral targets of aptamers include influenza A and B viruses,[49] Respiratory syncytial virus (RSV),[49] SARS coronavirus (SARS-CoV)[49] and SARS-CoV-2.[50]

Aptamers may be particularly useful for environmental science proteomics.[51] 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.[52] 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)[53] and abscisic acid (a plant immune hormone).[54] An aptamer against a-amanitin (the toxin that causes lethal Amanita poisoning) has been developed, an example of an aptamer against a mushroom target.[55]

Aptamer applications can be roughly grouped into sensing, therapeutic, reagent production, and engineering categories. Sensing applications are important in environmental, biomedical, epidemiological, biosecurity, and basic research applications, where aptamers act as probes in assays, imaging methods, diagnostic assays, and biosensors.[32][56][57][58][59][60] In therapeutic applications and precision medicine, aptamers can function as drugs,[61] as targeted drug delivery vehicles,[62] as controlled release mechanisms, and as reagents for drug discovery via high-throughput screening for small molecules[63] and proteins.[64][65] Aptamers have application for protein production monitoring, quality control, and purification.[66][67][68] They can function in molecular engineering applications as a way to modify proteins, such as enhancing DNA polymerase to make PCR more reliable.[69][70][71][72]

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.[73] 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.[74] Hence, specific applications determine the useful range for aptamer affinity.

Antibody replacement

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Aptamers can replace antibodies in many biotechnology applications.[75][52] In laboratory research and clinical diagnostics, they can be used in aptamer-based versions of immunoassays including enzyme-linked immunosorbent assay (ELISA),[76] western blot,[77] immunohistochemistry (IHC),[78] and flow cytometry.[79] As therapeutics, they can function as agonists or antagonists of their ligand.[80] While antibodies are a familiar technology with a well-developed market, aptamers are a relatively new technology to most researchers, and aptamers have been generated against only a fraction of important research targets.[81] Unlike antibodies, unmodified aptamers are more susceptible to nuclease digestion in serum and renal clearance in vivo. Aptamers are much smaller in size and mass than antibodies, which could be a relevant factor in choosing which is best suited for a given application. When aptamers are available for a particular application, their advantages over antibodies include potentially lower immunogenicity, greater replicability and lower cost, a greater level of control due to the in vitro selection conditions, and capacity to be efficiently engineered for durability, specificity, and sensitivity.[82]

In addition, aptamers contribute to reduction of research animal use.[83] 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 monoclonal cell line, avoiding the use of animals entirely.[84]

Controlled release of therapeutics

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The ability of aptamers to reversibly bind molecules such as proteins has generated increasing interest in using them to facilitate controlled release of therapeutic biomolecules, such as growth factors. This can be accomplished by tuning the binding strength to passively release the growth factors,[85] along with active release via mechanisms such as hybridization of the aptamer with complementary oligonucleotides[86] or unfolding of the aptamer due to cellular traction forces.[87]

Tissue Engineering Application

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Aptamer, known for their ability to bind specific molecules reversibly, have been used in 3D bioprinting tissues to precisely deliver growth factors to promote vascularization.[88][89] This controlled delivery allows growth factors to be released at the right place and time, encouraging the formation of localized and complex vascular networks. Additionally, the properties of these networks can be fine-tuned by adjusting how growth factors are released over time, making this approach a powerful tool for creating vascularized engineered tissues.

AptaBiD

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AptaBiD (Aptamer-Facilitated Biomarker Discovery) is an aptamer-based method for biomarker discovery.[90]

Peptide Aptamers

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While most aptamers are based on DNA, RNA, or XNA, peptide aptamers[91] are artificial proteins selected or engineered to bind specific target molecules.

Structure

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Peptide aptamers consist of one or more peptide loops of variable sequence displayed by a protein scaffold. Derivatives known as tadpoles, in which peptide aptamer "heads" are covalently linked to unique sequence double-stranded DNA "tails", allow quantification of scarce target molecules in mixtures by PCR (using, for example, the quantitative real-time polymerase chain reaction) of their DNA tails.[92] The peptides that form the aptamer variable regions are synthesized as part of the same polypeptide chain as the scaffold and are constrained at their N and C termini by linkage to it. This double structural constraint decreases the diversity of the 3D structures that the variable regions can adopt,[93] and this reduction in structural diversity lowers the entropic cost of molecular binding when interaction with the target causes the variable regions to adopt a uniform structure.

Selection

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The most common peptide aptamer selection system is the yeast two-hybrid system. Peptide aptamers can also be selected from combinatorial peptide libraries constructed by phage display and other surface display technologies such as mRNA display, ribosome display, bacterial display and yeast display. These experimental procedures are also known as biopanning. All the peptides panned from combinatorial peptide libraries have been stored in the MimoDB database.[94][95]

Applications

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Libraries of peptide aptamers have been used as "mutagens", in studies in which an investigator introduces a library that expresses different peptide aptamers into a cell population, selects for a desired phenotype, and identifies those aptamers that cause the phenotype. The investigator then uses those aptamers as baits, for example in yeast two-hybrid screens to identify the cellular proteins targeted by those aptamers. Such experiments identify particular proteins bound by the aptamers, and protein interactions that the aptamers disrupt, to cause the phenotype.[96][97] In addition, peptide aptamers derivatized with appropriate functional moieties can cause specific post-translational modification of their target proteins, or change the subcellular localization of the targets.[98]

This assay tests the ability of two different types of aptamers (V and I) to detect their respective protein targets (VEGF and IFN-y). The labels Apt1, Apt2, Apt3, and Apt4 are in decreasing order of binding strength (i.e. Apt1 is the strongest aptamer). The DD, AD, DA, and AA letters mean that they have different combinations of unnatural base pairs. This causes their difference in binding strengths. The "-" columns have no protein, and the "+" columns do have protein. Aptamer with protein (+) and without protein (-) is loaded into wells in a gel and moves down the column lanes. If target is present, they bind and travel more slowly, due to the charge on the aptamer and the mass of the protein. Otherwise, the unbound aptamer moves quickly to the end of the lane. The difference in position between the "+" and "-" bands is the "mobility shift." This allows the researcher to detect the presence or absence of the protein. The darker band in the leftmost V and I lanes show that stronger aptamer-target binding makes it easier to detect the target at a given amount of target protein in the sample. The bottom image includes denaturing urea in the gel that disrupts aptamer-target binding in the weaker I aptamers, showing that the aptamer-protein binding is indeed what caused the mobility shift.

Industry and Research Community

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Commercial products and companies based on aptamers include the drug Macugen (pegaptanib)[99] and the clinical diagnostic company SomaLogic.[100] The International Society on Aptamers (INSOAP), a professional society for the aptamer research community, publishes a journal devoted to the topic, Aptamers. Apta-index[101] is a current database cataloging and simplifying the ordering process for over 700 aptamers.

See also

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References

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Further reading

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

Aptamer

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from Grokipedia
An aptamer is a short oligonucleotide (typically single-stranded DNA or RNA, 25 to 100 nucleotides in length) or peptide molecule that folds into a unique three-dimensional structure enabling it to bind with high affinity and specificity to a diverse array of target molecules, including proteins, small molecules, cells, and viruses. While nucleic acid aptamers are the most common and the focus of much research, peptide aptamers offer similar binding capabilities through different selection methods. These "chemical antibodies" mimic the binding properties of traditional antibodies but offer advantages such as chemical stability, low immunogenicity, and ease of synthesis and modification. The discovery of aptamers traces back to 1990, when the term was independently coined by Andrew Ellington and Jack Szostak in one study for ligands, and the systematic evolution of ligands by exponential enrichment (SELEX) process was developed by Craig Tuerk and Larry Gold in parallel work. SELEX involves iterative selection and amplification from a large random library (often 10^14 to 10^15 unique sequences) over multiple rounds to enrich high-affinity binders. This technique has enabled the identification of aptamers against thousands of targets since its inception. Aptamers have emerged as versatile tools in and , with applications spanning therapeutics, diagnostics, and biosensors. In therapeutics, the first FDA-approved aptamer drug, pegaptanib sodium (Macugen), was authorized in 2004 for treating neovascular age-related by selectively inhibiting (VEGF). In 2023, the FDA approved a second aptamer drug, (Izervay), for secondary to age-related by inhibiting complement component C5. Subsequent developments include aptamers for anticoagulation, cancer targeting, and , leveraging their tunable and ability to be conjugated with nanoparticles or drugs for enhanced delivery. In diagnostics, aptamers facilitate sensitive detection in assays like colorimetric sensors and electrochemical biosensors, often outperforming antibodies due to their thermal stability and rapid dissociation kinetics. Ongoing research focuses on improving aptamer stability through chemical modifications, such as 2'-fluoro or locked nucleic acids, to expand their clinical potential.

Fundamentals

Etymology

The term "aptamer" was coined in 1990 by Andrew D. Ellington and during their pioneering work on the in vitro selection of RNA molecules capable of binding specific ligands with high affinity. This neologism emerged as part of the foundational discovery process, where the researchers sought a descriptor for short, structured nucleic acid sequences that function as targeted binding agents, distinct from naturally occurring biomolecules like antibodies. Etymologically, "aptamer" derives from the Latin word aptus, meaning "to fit," combined with the Greek meros, meaning "part" or "region," emphasizing the molecules' ability to form precise, complementary interactions with target structures, much like a custom-fitted component. In their original publication, Ellington and Szostak explicitly defined the term in this manner to highlight the selective "fit" achieved through evolutionary in vitro processes.

Definition and Characteristics

Aptamers are short, single-stranded oligonucleotides or peptides that fold into specific three-dimensional structures to bind target molecules with high affinity and specificity, often comparable to that of antibodies. Nucleic acid aptamers, the most common type, typically consist of 20-80 nucleotides of DNA or RNA, while peptide aptamers are constrained peptides of 5-20 amino acids usually displayed on a stable protein scaffold such as thioredoxin. These molecules recognize a wide range of targets, including proteins, small molecules, and cells, through non-covalent interactions that enable precise molecular recognition. A defining characteristic of aptamers is their binding affinity, with dissociation constants (Kd) typically in the nanomolar to picomolar range, allowing for strong yet tunable interactions. Their small molecular weight—generally 6-30 for nucleic acid aptamers—facilitates rapid tissue penetration and straightforward , which supports scalable production and modifications such as for enhanced stability. Unlike antibodies, aptamers exhibit low due to their synthetic nature and lack of immune-triggering epitopes, making them suitable for applications. Additionally, their binding is reversible and often conformation-dependent, permitting controlled release or antidote-mediated neutralization in therapeutic contexts. Aptamers are frequently referred to as "chemical antibodies" because they serve as versatile molecular recognition elements in diagnostics, therapeutics, and biosensors, offering advantages in stability and ease of engineering over traditional biologics.

History

Discovery

The discovery of aptamers stemmed from two independent breakthroughs in 1990 that demonstrated the potential of selection to evolve molecules capable of specific binding. At the , Craig Tuerk and Larry Gold initiated the process by starting with a known from T4 gene 43 mRNA, which binds to the T4 , and systematically mutated an 8-nucleotide loop to explore binding variants. Through iterative cycles of binding, partitioning, and amplification from a of variants, they enriched for high-affinity sequences that inhibited the polymerase, marking the first use of a combinatorial approach to evolve functional from a diverse pool. This method, termed Systematic Evolution of Ligands by EXponential enrichment (SELEX), represented a pioneering demonstration of selection from random-sequence to generate -binding . Concurrently, at , Andrew D. Ellington and developed a parallel strategy to isolate molecules that bind small organic dyes, such as Cibacron Blue 3GA, from a large pool of approximately 10^14 random-sequence RNAs. They employed cycles of to partition bound RNAs, followed by reverse transcription, PCR amplification, and transcription to generate enriched libraries for subsequent rounds, ultimately yielding subpopulations with specific, high-affinity binding sites for the dyes. This work independently showcased the feasibility of selecting functional structures from vast random libraries, highlighting the versatility of as a scaffold akin to antibodies. These foundational experiments, published in rapid succession—Tuerk and Gold in Science on August 3, 1990, and Ellington and Szostak in on August 30, 1990—established the core principles of aptamer generation through SELEX, without which later refinements would not have been possible. The term "aptamer," derived from the Latin "aptus" meaning "to fit," was coined by Ellington and Szostak to describe these selected ligands.

Key Developments

Following the initial discovery of aptamers in 1990, the 1990s saw significant refinements to the Systematic Evolution of Ligands by EXponential enrichment (SELEX) process, including optimizations for efficiency and specificity that enabled the generation of aptamers against a broader range of targets. These advancements culminated in the approval of the first therapeutic aptamer, , by the on December 17, 2004, for the treatment of neovascular (wet) age-related macular degeneration. targets , demonstrating aptamers' potential as targeted inhibitors in clinical settings. In the 2000s and , efforts focused on enhancing aptamer stability to overcome degradation, leading to the widespread adoption of chemical modifications such as 2'-fluoro, 2'-O-methyl, and 2'-amino substitutions on the ribose sugar; backbone alterations like phosphorothioate linkages; and mirror-image L-nucleotides (Spiegelmers), which extended serum half-lives from minutes to hours or days. Concurrently, cell-SELEX emerged as a pivotal around 2004, allowing the selection of aptamers against intact cells or complex surface markers without prior of specific targets, facilitating applications in targeting and diagnostics. For aptamers, initial selections using surface display were reported in the late 1990s and early , enabling high-throughput of constrained libraries for intracellular protein interactions. The 2020s marked further clinical progress, with the FDA approving a second aptamer therapeutic, (Izervay), on August 4, 2023, for secondary to age-related macular degeneration, highlighting aptamers' expanding role in . This approval coincided with a surge in aptamer-based clinical trials, including olaptesed pegol (NOX-A12), a inhibitor advancing in phase 1/2 studies for indications such as and , often in combination with radiotherapy or to modulate the .

Classification

Nucleic Acid Aptamers

Nucleic acid aptamers represent the foundational class of aptamers, consisting of single-stranded DNA (ssDNA) or RNA (ssRNA) molecules that fold into unique three-dimensional conformations to recognize and bind specific targets with high affinity and specificity, often comparable to antibodies. These oligonucleotides, typically 20 to 80 nucleotides in length, are derived from vast libraries of random sequences during in vitro selection processes. Unlike peptide aptamers, which utilize protein-derived scaffolds, nucleic acid aptamers rely on the inherent flexibility and base-pairing properties of nucleotides for target interaction. DNA aptamers, composed of deoxyribonucleotide units, offer superior chemical and thermal stability due to the absence of the 2'-hydroxyl group found in , and they can readily form double-stranded helical structures under certain conditions. In contrast, aptamers incorporate ribonucleotides, enabling more intricate three-dimensional folds through additional bonding and steric interactions, which often result in greater structural diversity and adaptability for binding complex targets. Xenonucleic acid (XNA) aptamers extend this category by employing synthetic analogs with alternative sugar moieties, such as or , replacing the natural or ; these variants exhibit markedly enhanced resistance to degradation while maintaining functional binding capabilities. To further bolster stability in biological environments, aptamers frequently undergo chemical modifications, including substitutions at the 2'-position of the sugar (e.g., 2'-fluoro or 2'-O-methyl groups) or incorporation of locked nucleic acids (LNAs), which reduce susceptibility to endonucleases and exonucleases without compromising target affinity. These modifications are particularly crucial for therapeutic applications, as unmodified aptamers can degrade rapidly in serum. Libraries for aptamer selection generally comprise 10^14 to 10^15 unique random sequences flanked by primer-binding regions, allowing for the isolation of high-affinity binders through iterative enrichment. A prominent example of a nucleic acid aptamer is NU172, a 26-nucleotide DNA aptamer designed to bind exosite I of human thrombin, potently inhibiting its fibrin-clotting activity with a dissociation constant in the nanomolar range; it progressed to Phase II clinical trials as an anticoagulant for cardiac surgery patients.

Peptide Aptamers

Peptide aptamers are combinatorial protein reagents consisting of a short, variable peptide sequence, typically 10-20 amino acids long, inserted into a stable scaffold protein to constrain its conformation and enable specific binding to target molecules. This design mimics antibody-antigen interactions but allows intracellular expression, making them suitable for modulating protein function within cells. The scaffold provides structural rigidity, ensuring the peptide loop adopts a defined three-dimensional shape for high-affinity, sequence-specific recognition of target proteins. Unlike the more common nucleic acid aptamers, peptide aptamers are protein-based and selected through genetic libraries rather than in vitro chemical evolution. Subtypes of peptide aptamers are primarily distinguished by the choice of , which influences stability, solubility, and binding efficiency. The most widely used scaffold is A (TrxA) from , where the variable peptide is displayed in the active-site loop to maintain a constrained loop structure. Alternative scaffolds include bacterial proteins like those from other prokaryotes or engineered variants, as well as scaffolds from eukaryotic sources such as Plasmodium falciparum (PfTrx), which offers enhanced immunogenicity for vaccine applications. Peptide aptamers can also be categorized by their display systems during selection, such as the yeast two-hybrid system, which facilitates intracellular screening for protein-protein interactions, or bacterial surface display for high-throughput identification. A prominent example is the E7-binding peptide aptamer developed for human papillomavirus (HPV) research, where aptamers targeting the E7 oncoprotein were selected using a scaffold to inhibit its activity and induce in HPV-positive cervical carcinoma cells. This aptamer, isolated through yeast two-hybrid screening, demonstrated high specificity for E7, disrupting its interaction with cellular targets like and providing a model for antiviral therapeutics. Seminal work also includes early peptide aptamers against (CDK2), which were the first to be genetically selected and shown to block progression by mimicking natural inhibitors. These examples highlight peptide aptamers' utility in dissecting protein networks and validating drug targets.

Structure and Properties

Nucleic Acid Aptamer Structure

Nucleic acid aptamers are short, single-stranded DNA (ssDNA) or RNA (ssRNA) oligonucleotides, typically ranging from 25 to 80 nucleotides in length, consisting of a central randomized sequence flanked by fixed primer regions for amplification during selection. This primary structure provides the sequence diversity essential for evolving specific binding conformations, with the linear chain serving as the scaffold for higher-order folding. At the secondary structure level, aptamers fold through intramolecular base pairing to form motifs such as stem-loops (hairpins), pseudoknots, bulges, and internal loops, which contribute to structural rigidity and specificity. G-quadruplexes, formed in guanine-rich sequences by stacking of G-tetrads stabilized by monovalent cations like potassium, represent another key motif; for instance, the thrombin-binding aptamer (TBA), a 15-nucleotide ssDNA sequence (d(GGTTGGTGTGGTTGG)), adopts an intramolecular antiparallel G-quadruplex with two G-tetrads and a TGT loop, enabling high-affinity target recognition. Pseudoknots, involving crossing of base-paired stems, occur in certain aptamers to create compact architectures that enhance binding pockets, as seen in some RNA aptamers targeting viral proteins. These secondary elements transition to tertiary structures via additional base stacking, hydrogen bonding, and hydrophobic interactions, resulting in well-defined three-dimensional conformations that mimic protein active sites for target interaction. To enhance biostability against degradation and improve , aptamers are often chemically modified using diverse strategies that preserve their ability to fold into specific binding conformations while conferring resistance to nucleases. Common approaches include sugar moiety substitutions such as 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), and 2'-amino (2'-NH₂) groups, which protect against enzymatic cleavage and can stabilize secondary structures; backbone modifications such as phosphorothioate or phosphorodithioate linkages; incorporation of unnatural base pairs; mirror-image L-nucleic acids (Spiegelmers using L-DNA or L-RNA); and protective coatings or conjugations (e.g., PEGylation, oligolysine, or block copolymers). These modifications enhance biostability for applications in biosensing, drug delivery, and therapeutics while preserving specific binding capabilities. For instance, incorporation of 2'-O-methyl groups on the replaces the 2'-OH with a methoxy group to confer resistance while preserving folding. These modifications increase the melting temperature (Tm) of secondary structures—for example, 2'-O-methyl substitutions in aptamers can raise Tm by approximately 1°C per modified position in model duplexes, stabilizing stem-loops without disrupting overall tertiary architecture. , involving conjugation of (PEG) chains to the 5' or 3' terminus, further extends serum by reducing renal clearance, though it minimally affects intrinsic folding but can influence accessibility of binding sites. Mirror-image L-nucleic acid aptamers (Spiegelmers) exhibit exceptional nuclease resistance due to their non-natural chirality while retaining high-affinity and specific target recognition. Other modifications like phosphorothioate linkages in the backbone also bolster stability while maintaining the aptamer's adaptive folding capability.

Peptide Aptamer Structure

Peptide aptamers feature a core design consisting of a short variable peptide loop, typically 5 to 20 in length, inserted into a stable protein scaffold that constrains its conformation for target binding. The prototypical scaffold is A (TrxA) from , where the variable sequence replaces the enzyme's active-site loop between two conserved residues (positions 32 and 35), resulting in a constrained loop of 12 to 20 residues that is presented in a fixed three-dimensional orientation. This insertion disrupts the thioredoxin's activity but preserves its overall fold, allowing the scaffold to solubilize and stabilize the peptide without interfering with its binding function. The scaffold imparts conformational stability to the variable loop by enforcing a specific spatial , often resembling a beta-hairpin or turn structure, which mimics the rigid presentation of complementarity-determining regions (CDRs) in variable domains. This constrained geometry reduces loss upon target binding, thereby enhancing affinity and specificity compared to unconstrained linear peptides, with dissociation constants in the nanomolar range for optimized aptamers. The scaffold's beta-sheet-rich core and solvent-exposed loop position further contribute to this stability, preventing aggregation and enabling intracellular expression in yeast or bacterial two-hybrid systems for selection. Variations in peptide aptamer design include alternative scaffolds beyond thioredoxin A, such as (GFP) or glutathione S-transferase (GST), which offer similar loop constraints but may improve expression in eukaryotic systems or add functional tags for detection. Multi-valent designs incorporate multiple variable loops within a single scaffold or tandem fusions of aptamer units to achieve and higher against multimeric targets. Additionally, fusions with other protein domains, such as protein transduction domains (PTDs) for cellular delivery or regulatory modules like FKBP-FRB for ligand-inducible control, expand their utility while maintaining structural integrity.

Binding Properties

Aptamers exhibit high binding affinity to their , typically characterized by dissociation constants (K_d) in the low nanomolar to high picomolar range, often spanning 10-500 nM for many selected sequences. This affinity arises from non-covalent interactions that enable precise molecular recognition, comparable to that of monoclonal antibodies. The specificity of aptamer binding is primarily driven by shape complementarity between the aptamer's three-dimensional structure and the target molecule, supplemented by hydrogen bonding, electrostatic interactions, and van der Waals forces. For aptamers, these interactions often involve non-Watson-Crick base pairing and other tertiary contacts that allow the formation of unique binding pockets beyond traditional double-helix motifs. This combination ensures selective engagement, minimizing off-target effects even among structurally similar molecules. Aptamers can target a diverse array of entities, including small molecules, proteins, whole cells, and viruses, due to their adaptable folding into conformations that accommodate varied target sizes and chemistries. For instance, they bind small molecules like antibiotics or metabolites through pocket-like enclosures, while interacting with proteins via surface epitopes or even enveloping viral particles to block . Unlike covalent inhibitors that form irreversible bonds, aptamer binding is inherently reversible, governed by association and dissociation kinetics that permit dynamic regulation of target engagement. This reversibility facilitates controlled release or antidote-mediated neutralization, enhancing their utility in tunable therapeutic contexts.

Selection and Generation

SELEX for Nucleic Acid Aptamers

The Systematic Evolution of Ligands by EXponential enrichment (SELEX) is an iterative selection process designed to isolate high-affinity nucleic acid aptamers from large random libraries, independently developed in by two research groups. This method mimics Darwinian by repeatedly enriching sequences with desired binding properties, typically yielding aptamers with dissociation constants in the nanomolar to picomolar range after multiple cycles. The SELEX process commences with the synthesis of a diverse combinatorial library of single-stranded DNA or RNA oligonucleotides, comprising 10^{14} to 10^{15} unique sequences to ensure broad coverage of possible structures. Each sequence features a central random region of 20–80 nucleotides flanked by constant primer-binding sites for subsequent amplification. This library is denatured and incubated with the target molecule under controlled conditions to promote specific binding interactions. Partitioning follows incubation, separating bound aptamer-target complexes from unbound sequences to enrich the pool. For immobilized targets—such as proteins attached to magnetic beads, columns, or microtiter plates—unbound aptamers are washed away, while complexes are retained. Alternative methods include for soluble targets, where protein-nucleic acid complexes are retained on the filter while free aptamers pass through. Bound aptamers are then eluted from the target, commonly by heating to 95°C or altering buffer conditions like or to disrupt interactions without damaging sequences. The recovered single-stranded nucleic acids are amplified via (PCR) to produce double-stranded DNA, which is transcribed to if needed or rendered single-stranded through asymmetric PCR or enzymatic methods, regenerating the enriched library for the next iteration. These cycles of incubation, partitioning, , and amplification are repeated for 8–15 rounds, with progressive increases in selection stringency—such as decreasing target concentration, shortening incubation times, or adding competitors—to favor aptamers with higher affinity and specificity. After the final round, sequences are cloned and sequenced to identify individual aptamers for characterization. Variants of SELEX adapt the standard protocol to specific target types or challenges. In Cell-SELEX, whole living cells serve as targets to select aptamers recognizing complex surface epitopes, such as those on cancer cells; the process involves incubating the library with target cells, separating bound aptamers by or after washing, eluting by heating, and using non-target cells for counter-selection to discard nonspecific binders. Capture-SELEX addresses limitations with small-molecule targets by immobilizing the oligonucleotide library on a solid support (e.g., streptavidin-coated beads via biotinylated sequences) and passing the soluble target through it, capturing and eluting only target-bound aptamers for amplification; this preserves the target's native conformation and is effective for molecules lacking suitable immobilization sites. Optimization through counter-selection enhances specificity by pre-incubating the aptamer pool with non-target molecules, supports, or control cells to remove sequences with off-target binding, followed by discarding bound material without elution; this step, often integrated after initial rounds or before target exposure, is crucial for reducing cross-reactivity in complex matrices.

Selection Methods for Peptide Aptamers

Peptide aptamers are primarily selected using the yeast two-hybrid (Y2H) system, an method that enables intracellular screening against target proteins under physiological conditions. This technique, first demonstrated in 1996, involves combinatorial libraries typically comprising 10^6 to 10^9 variants of randomized peptide sequences constrained within a stable scaffold protein, such as thioredoxin A (TrxA), to confer proper folding and solubility. The Y2H system is particularly suited for identifying high-affinity binders to intracellular targets, as it leverages yeast cellular machinery to detect protein-protein interactions. A variant, the yeast three-hybrid system, extends this approach to select aptamers that modulate interactions involving small molecules or , though it is less commonly used for standard peptide aptamer isolation. The selection process begins with library construction, where degenerate encoding random (usually 8-20 ) are inserted into the scaffold via sites, followed by into a fused to a transcriptional activation domain, such as GAL4-AD. These plasmids are then transformed into yeast host cells, achieving high library diversity through efficient mating with bait-expressing strains. The target protein, fused to a (e.g., GAL4-BD or LexA), is co-expressed; specific binding between the bait and aptamer recruits the activation domain to promoter regions, activating reporter genes like HIS3 or lacZ that confer selectable phenotypes, such as growth on histidine-deficient media or colorimetric output. Enriched clones are isolated after multiple rounds of selection, followed by plasmid recovery, sequencing to identify peptide sequences, and validation through independent binding assays, such as pull-downs or , to confirm specificity and affinity. This stepwise process typically yields aptamers with dissociation constants in the nanomolar range for validated targets. For extracellular targets, where intracellular expression may be unsuitable, alternative display methods like or bacterial display are employed. In , random are fused to coat proteins (e.g., pIII on ), and libraries of 10^8-10^10 variants are iteratively panned against immobilized targets, eluting and amplifying bound phages in E. coli to enrich high-affinity binders. Bacterial display, using systems like E. coli outer membrane proteins (e.g., INPNC or Lpp-OmpA fusions), offers similar extracellular selection with real-time quantification via , facilitating affinity maturation of aptamers. These methods parallel the in vitro partitioning of SELEX for aptamers but adapt to peptide scaffolds for surface-exposed targets.

Emerging Techniques

Computational design of aptamers has emerged as a powerful complement to experimental selection methods, leveraging advances in artificial intelligence and structural biology to predict and optimize aptamer-target interactions. AlphaFold 3, released in 2024 with significant updates in 2025, enables direct modeling of DNA and RNA aptamer structures in complex with protein targets, achieving high accuracy in predicting binding conformations that guide de novo sequence design. Machine learning models, such as AptaTrans, further enhance this process by analyzing large datasets of aptamer sequences to forecast binding affinities and optimize motifs for improved specificity and stability. These computational approaches reduce the reliance on iterative experimental rounds, allowing for rapid screening of vast sequence spaces and integration with generative models that produce novel RNA aptamers tailored to specific targets. In vivo SELEX represents a shift toward selections conducted directly within living animal models, enhancing the physiological relevance of identified aptamers by accounting for complex biological environments such as circulation and tissue barriers. This method involves injecting libraries into organisms like , followed by recovery of enriched sequences from target tissues, yielding aptamers with inherent stability and targeting efficiency. For instance, SELEX has successfully isolated aptamers capable of crossing the blood-brain barrier, as demonstrated in studies using models. Recent advancements in delivery include receptor-targeting aptamers selected via SELEX, with conjugates showing high brain uptake in models. Hybrid methods combine elements of traditional SELEX with innovative partitioning or switching strategies to accelerate discovery and boost specificity. Target-switch SELEX alternates between positive selection against a primary target and negative selection against structurally similar off-targets, enabling the isolation of aptamers with exquisite discrimination, such as those specific to consecutive in peptides. This approach has been applied to generate DNA aptamers that bind with nanomolar affinity while avoiding cross-reactivity, shortening selection timelines to fewer rounds. Complementing this, CE-SELEX employs for high-resolution separation of bound and unbound aptamers, facilitating faster iterations—often completing in 2-4 rounds compared to 8-12 for conventional methods—and higher yields for small-molecule targets. Recent adaptations, including peptide-based CE-SELEX, have extended its utility to cell-surface receptors, isolating aptamers with dissociation constants in the picomolar range.

Applications

Therapeutics and Drug Delivery

Aptamers have emerged as promising direct therapeutics due to their high specificity and ability to antagonize key molecular targets involved in disease progression. The first FDA-approved aptamer drug, (Macugen), is a pegylated aptamer that selectively binds (VEGF), inhibiting its interaction with the to treat neovascular age-related (). Approved in 2004, pegaptanib demonstrated reduced vision loss in clinical trials, marking a milestone for nucleic acid-based antagonists. In 2023, the FDA approved a second aptamer therapeutic, avacincaptad pegol (Izervay), a pegylated aptamer that inhibits complement protein C5 to slow the progression of secondary to . This approval highlighted aptamers' potential in complement-mediated diseases, with phase 3 trials showing a 35% reduction in lesion growth over 12 months compared to . Beyond approved drugs, aptamers are advancing in clinical trials as antagonists for various conditions. ApTOLL, a DNA aptamer targeting (TLR4) to modulate , underwent phase Ib/IIa trials for acute ischemic , where it has shown and reduced mortality at 90 days in early studies when combined with . The completed APRIL trial (2025) demonstrated that ApTOLL (0.2 mg/kg) in combination with mechanical significantly reduced final infarct volume, infarct growth, and at 72 hours post-treatment. Similarly, NOX-A12 (olaptesed pegol), an L-RNA aptamer (Spiegelmer) neutralizing to enhance immune cell infiltration, is in phase II trials for cancers including and , demonstrating and preliminary antitumor activity when combined with radiotherapy or checkpoint inhibitors. In the GLORIA trial for , NOX-A12 with radiotherapy met its primary endpoint and showed increased T-cell infiltration in tumors. In , aptamers serve as targeting ligands in conjugates and carriers to improve specificity and reduce off-target effects. Aptamer- conjugates (ApDCs) link therapeutic payloads to aptamers that bind cell-surface markers overexpressed in diseased tissues, such as nucleolin in cancer cells. A representative example is AS1411-DOX, where the AS1411 aptamer targets nucleolin to deliver selectively to tumor cells, enhancing while minimizing in preclinical models of and . This conjugate inhibited tumor growth more effectively than free by promoting and intracellular drug release. Aptamers also enable controlled release in hydrogels, where their binding affinity sequesters drugs until target-triggered dissociation. Aptamer-functionalized hydrogels have been used for localized delivery of angiogenic factors like VEGF, reducing burst release and systemic in models by achieving sustained release over days. These systems leverage aptamers' reversible binding to create responsive matrices that release payloads in response to disease-specific biomarkers. As of 2025, two aptamer therapeutics remain FDA-approved, with over 20 candidates in clinical trials across , , and , reflecting growing investment in their therapeutic potential. This pipeline includes antagonists and delivery platforms, underscoring aptamers' advantages in precision medicine despite challenges like stability, which and chemical modifications continue to address.

Diagnostics and Biosensors

Aptamers have emerged as key recognition elements in biosensors for diagnostics, enabling sensitive and selective detection of s in point-of-care settings due to their high affinity and stability compared to antibodies. These aptasensors typically operate through conformational changes in the aptamer upon target binding, which transduce signals in various formats. Electrochemical aptasensors, for instance, measure changes in current or impedance, while optical aptasensors detect fluorescence, absorbance, or shifts. A prominent example is the detection of , a for disorders, using DNA aptamers such as the 15-mer TBA (TBA1) with a (Kd) of approximately 100 nM, achieving sub-nanomolar sensitivity in complex samples like serum. Common formats in aptamer-based biosensors include sandwich assays and aptamer beacons, which enhance specificity and signal output. In sandwich assays, two aptamers bind distinct epitopes on the target, such as the exosites of or the receptor-binding domain of viral proteins, often integrated into electrochemical or colorimetric platforms for limits of detection in the femtomolar to picomolar range. Aptamer beacons consist of stem-loop structures where target binding disrupts quenching, releasing a fluorescent signal; for , this format has enabled direct protein detection with minimal sample preparation. Signal amplification is frequently achieved by conjugating aptamers with nanoparticles, such as gold nanoparticles, which increase surface area and catalytic activity, boosting sensitivity in assays by orders of magnitude through enhanced or plasmonic effects. During the 2020s, aptamer biosensors gained traction for rapid diagnostics, particularly through sandwich formats targeting the spike protein. One such assay uses paired aptamers (e.g., Apt1T and Apt5B) in an enzyme-linked format to detect spike at 21 ng/mL (270 pM) in conditions, with potential for adaptation to lateral flow devices for point-of-care use, demonstrating negligible with other coronaviruses. These developments highlight aptamers' role in addressing urgent diagnostic needs with portable, cost-effective platforms.

Imaging and Biomarker Discovery

Aptamers have emerged as versatile probes in due to their high specificity, small size, and ability to be conjugated with various imaging agents. Radiolabeled aptamers, such as those targeting prostate-specific (PSMA), enable (PET) imaging for detection. For instance, the A10-3.2 aptamer, when labeled with gallium-68 ([68Ga]Ga-NOTA-PSMA-Cy5), has demonstrated dual-modality PET/near-infrared (NIRF) imaging in preclinical models, allowing for precise tumor localization and intraoperative guidance. Similarly, fluorescent aptamers facilitate (MRI) contrast enhancement by targeting cell surface markers, offering non-invasive visualization of tumor microenvironments with reduced compared to larger antibody-based probes. A key application of aptamers lies in biomarker discovery through the Aptamer-facilitated Biomarker Discovery (AptaBiD) process, introduced in 2008. AptaBiD involves selecting aptamer libraries against intact cells to isolate high-affinity binders, followed by identification of the bound proteins using , thereby uncovering cell surface biomarkers without prior knowledge of targets. This method has been particularly effective for distinguishing subtle phenotypic differences, such as between immature and mature dendritic cells, where aptamers revealed unique surface proteins like CD209 for maturation states. Recent applications extend AptaBiD to complex diseases, including cancer and immune disorders, enhancing the discovery of novel diagnostic markers through its unbiased, proteome-wide screening approach. Recent advances have integrated aptamers with nanoparticles to improve targeted modalities, particularly in point-of-care (POC) devices. Aptamer-functionalized nanoparticles, such as or carbon dots conjugated with tumor-specific aptamers, enable multimodal with enhanced for cancers like . In 2025, proof-of-concept POC systems using these constructs demonstrated real-time in preclinical settings, achieving sub-millimeter resolution for early detection and paving the way for portable diagnostic tools. These developments underscore aptamers' role in bridging and identification for .

Tissue Engineering and Other Uses

Aptamers have emerged as versatile tools in tissue engineering, particularly for enhancing scaffold functionality through selective cell adhesion and recruitment. By functionalizing biomaterials such as hydrogels and matrices with aptamers, researchers enable precise capture of target cells like mesenchymal stem cells (MSCs) and osteoblasts, promoting regenerative processes. For instance, anti-fibronectin aptamers immobilized on scaffolds facilitate cell attachment by mimicking adhesion sites, leading to improved formation in periodontal defects in rat models. Similarly, di-aptamer systems targeting MSCs have been integrated into scaffolds to accelerate regeneration in calvarial defects, with micro-CT analysis showing significant increases in bone within 8 weeks. In bone tissue engineering, aptamer-modified hydrogels provide controlled environments for osteogenic differentiation. Anti-VEGF receptor-2 aptamers combined with RGD peptides in thiolated hyaluronic acid-poly() diacrylate hydrogels synergize to promote vascularization and osteogenesis in critical-sized defects, enhancing endothelial and mineralized matrix deposition. Aptamer-functionalized DNA hydrogels further support stem recruitment and release, demonstrating superior repair outcomes in rat femoral defects compared to non-functionalized controls. These approaches leverage aptamers' high specificity to avoid non-specific binding, outperforming traditional ligands in selectivity. Aptamers also mediate stem cell differentiation in regenerative contexts. A notable example is the anti-nucleolin DNA aptamer iSN04, which regulates lineage commitment in murine pluripotent stem cells by modulating nucleolin localization and Wnt signaling, inducing cardiomyocyte differentiation when applied post-initial differentiation stages. This 2023 study highlights aptamers' potential for directing stem cell fate without genetic modification, applicable to cardiac tissue repair. Beyond , aptamers find utility in environmental sensing for detecting pollutants. Electrochemical aptasensors employing aptamers against like mercury and lead, or organic contaminants such as and , achieve detection limits in the nanomolar range in water samples, enabling portable monitoring of and . These sensors exploit aptamer conformational changes upon binding to generate measurable electrical signals, offering advantages in sensitivity over conventional methods. In , aptamer-based biosensors facilitate rapid detection. DNA aptamers selected via whole-cell SELEX target bacteria like and Salmonella typhimurium, integrating into lateral flow assays for on-site screening in food matrices with limits of detection as low as 10 CFU/mL. For , biotinylated aptamers enable capture and amplification via qPCR, reducing detection time to under 4 hours compared to culture-based techniques.

Advantages and Challenges

Comparison to Antibodies

Aptamers offer distinct advantages over antibodies in their production methods, physical properties, and overall performance as molecular recognition elements. Unlike monoclonal antibodies, which require complex biological production in cell cultures or animals, aptamers are generated through once their sequence is determined, enabling scalable, reproducible manufacturing without batch-to-batch variability. This synthetic approach also reduces production costs significantly compared to the labor-intensive and expensive processes for antibodies. In terms of physical properties, aptamers are substantially smaller, typically ranging from 6–30 kDa, which facilitates superior tissue penetration and access to cryptic epitopes inaccessible to larger antibodies (approximately 150 kDa). Additionally, aptamers exhibit high thermal and chemical stability; for instance, DNA aptamers can withstand temperatures up to 95°C and repeated denaturation/renaturation cycles without loss of function, in contrast to antibodies that denature at lower temperatures around 60–70°C. Aptamers also demonstrate low or no immunogenicity, minimizing the risk of immune responses that can occur with antibodies, particularly in therapeutic applications. Regarding performance, aptamers achieve binding affinities and specificities comparable to those of antibodies, with dissociation constants (K_d) often in the nanomolar to picomolar range. They are more readily modifiable through chemical conjugation—for example, can extend their plasma half-life from minutes to days (e.g., up to 10 days in the case of ), allowing tailored without the structural constraints faced by antibodies.
PropertyAptamersMonoclonal Antibodies
Size6–30 kDa150–170 kDa
Production CostLower ()Higher (biological production)
ImmunogenicityLow or noneCan induce immune responses

Limitations and Solutions

One major limitation of aptamers, particularly RNA-based ones, is their susceptibility to degradation by nucleases present in biological fluids, which significantly reduces their stability and therapeutic efficacy. This enzymatic breakdown can occur rapidly in serum, limiting the duration of aptamer activity . Another challenge stems from their small molecular size (typically 6–30 kDa), leading to rapid renal clearance and short plasma half-lives, often on the order of minutes, which hinders sustained target engagement. Additionally, while aptamers generally exhibit low compared to protein-based therapeutics, certain sequences can potentially trigger immune activation through Toll-like receptor recognition or induction upon systemic administration. To address nuclease degradation, chemical modifications such as incorporation of locked nucleic acids (LNA) or 2'-fluoro substitutions into the sugar backbone enhance biostability by sterically hindering access, extending serum half-lives from minutes to hours or days without compromising binding affinity. For instance, 2'-fluoro-modified aptamers demonstrate up to 10-fold greater resistance to ribonucleases while maintaining high specificity. A broader range of strategies includes sugar moiety substitutions such as 2'-O-methyl or 2'-amino groups, backbone alterations including phosphorothioate or 2'-5' linkages, incorporation of unnatural base pairs, and the use of mirror-image L-DNA or L-RNA (Spiegelmers), which evade recognition by natural nucleases due to their chirality. Protective coatings and conjugations, such as those involving oligolysine, block copolymers, or PEGylation, can further shield aptamers from enzymatic degradation while preserving specific binding capabilities. To mitigate rapid renal clearance, multimerization strategies—such as linking multiple aptamer units via scaffolds or forming dimers/trimers—increase the overall molecular weight, prolonging circulation times and improving pharmacokinetic profiles in animal models. Capping techniques, including with 20–40 kDa chains at the 5' or 3' ends, further control clearance by reducing glomerular filtration, as evidenced by (Macugen), which achieves a plasma of approximately 9–10 days in humans. Despite these advances, ongoing challenges persist in scaling aptamer applications for use, including difficulties in achieving reproducible high-affinity selections under physiological conditions and ensuring consistent biodistribution across diverse tissues. Off-target effects also remain a concern in complex biological matrices, where nonspecific interactions with abundant proteins or can reduce specificity and lead to unintended binding, necessitating refined selection protocols to minimize such interference.

Research and Commercialization

Industry Landscape

The aptamer industry encompasses a range of firms specializing in the development, manufacturing, and commercialization of aptamer-based reagents for research, diagnostics, and therapeutics. Key players include SomaLogic (acquired by Illumina in 2025), which pioneered SOMAmer (Slow Off-rate Modified Aptamer) reagents—chemically modified DNA aptamers designed for high-specificity protein binding in proteomic applications. SOMAmer technology powers SomaLogic's SomaScan platform, enabling multiplexed analysis of thousands of proteins from small sample volumes, and has been adopted for discovery in clinical studies. Another prominent company is Aptamer Group, which utilizes its proprietary Optimer platform to generate synthetic oligonucleotide-based affinity binders optimized for stability and performance in complex biological matrices, such as ELISA assays and therapeutic conjugates. Base Pair Biotechnologies focuses on custom aptamer discovery services, offering DNA- and RNA-based ligands for , including therapeutic aptamers that target small molecules and penetrate tissues more effectively than antibodies. Commercial products highlight aptamers' transition from research tools to viable alternatives in diagnostics and medicine. The first FDA-approved aptamer therapeutic, (Macugen), a PEGylated aptamer developed by Eyetech Pharmaceuticals and approved in 2004 for intravitreal treatment of neovascular (wet) age-related by selectively inhibiting (VEGF). A second aptamer therapeutic, avacincaptad pegol (Izervay), was approved in 2023 for geographic atrophy secondary to age-related . In diagnostics, aptamer-based kits have entered the market, including lateral flow assay kits from Cytodiagnostics for detecting targets like , and demo/discovery kits from Aptagen demonstrating aptamer capabilities in fluorescence-based detection without capture steps. These products leverage aptamers' advantages in rapid, sensitive binding for . Strategic partnerships with pharmaceutical giants underscore the industry's growth trajectory, particularly in aptamer-drug conjugates and targeted delivery systems. For instance, Aptamer Group has secured multiple contracts in 2025 with top-tier pharma companies, including a £617,000 repeat deal with a top-5 global firm for developing Optimer binders against three drug targets to support development, and a collaboration with to enhance siRNA therapies via cell-specific aptamer targeting. Such alliances, often retaining rights for the aptamer developer, facilitate integration into conjugate platforms for and . The global aptamers market, valued at approximately USD 3.63 billion in 2025, is expanding rapidly due to demand in , driven by aptamers' role in precision diagnostics and targeted therapeutics. This growth, projected at a CAGR exceeding 24% through the early 2030s, reflects increasing adoption by and pharmaceutical sectors, which hold over 38% , amid advancements in manufacturing scalability.

Recent Advances and Future Directions

In 2025, 3 has emerged as a transformative tool for modeling the structures of DNA and aptamers, enabling direct prediction of their three-dimensional conformations and interactions with targets, which accelerates the design of high-affinity sequences without extensive experimental screening. Concurrently, aptamer-functionalized nanoparticles have advanced by enabling targeted delivery of immune-modulating agents to tumor microenvironments, enhancing efficacy against solid tumors while minimizing off-target effects, as demonstrated in recent studies on bispecific aptamer constructs. A notable clinical milestone includes the DNA aptamer ApTOLL, which completed Phase 1b/2a trials for acute ischemic —a disorder—showing positive safety, pharmacokinetic profiles, and reductions in infarct volume and when combined with standard , leading to its licensing by Merck for further development. Looking ahead, AI-accelerated aptamer design is poised to revolutionize discovery through models like DeepAptamer and AptaDiff, which generate and optimize sequences de novo, achieving up to 87.9% improvements in binding affinity and reducing dissociation constants by over twofold compared to traditional SELEX methods. Mirror-image L-aptamers, or spiegelmers, offer enhanced biostability against nucleases, potentially enabling oral delivery routes that bypass injection limitations and improve patient compliance in chronic therapies. Integration of aptamers with systems for gene regulation has shown promise, with aptamer-mediated platforms modulating activity to enable precise base editing, , and transcriptional control, as in recent opto-CRISPR and aptamer-Cas12a hybrids for targeted gene modulation. Despite these strides, aptamer research faces ongoing challenges, including regulatory hurdles from limited long-term clinical data that delay approvals by agencies like the FDA, and high production costs associated with and optimization, which hinder scalability for widespread therapeutic adoption. Addressing these through standardized validation protocols and cost-effective manufacturing innovations will be crucial for translating aptamer technologies into routine clinical use.

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