Aptamers, first reported in 1990, are chemical alternatives to antibodies. They offer several advantages for scientific research, as well as for clinical diagnostics and therapeutic applications, including their small size, synthetic production method, and functional stability when stored at room temperature.
What are aptamers?
Aptamers are short single-stranded DNA or RNA molecules that can bind with high affinity and specificity to their cognate targets. They achieve this by folding into complex three-dimensional (3D) structures like loops and stems to attain similar binding properties to antibodies.
These molecules can serve as a substitute for antibodies used to identify a specific target. The sequences are often modified to enhance stability during in vitro and in vivo use.
How are amptamers synthesized?
Aptamers are usually synthesized as highly complex single stranded DNA libraries, consisting of > 1015 different members. These libraries are then used in a sequential screening process to isolate specific aptamers which bind to a target of choice.
Typically, aptamer lengths in libraries vary between 20 and 220 nucleotides, with constant flanking sequences (used for e.g. amplification and sequencing of individual aptamers) and a randomized core sequence (with affinity to one or multiple target molecules). In theory, aptamer libraries can be extremely complex. For example, an aptamer of 100 nucleotides would allow for a library of potentially 1060 different members. Due to experimental constraints, aptamer libraries in use are significantly smaller. Still, they represent some of the most complex libraries available and there is a high potential for identifying an aptamer with strong affinity to virtually any biological molecule.
When using RNA aptamers, stability is a major concern since RNAs are quickly degraded in vivo. The use of specific modifcations, such as 2'-Fluor and 2'-Amino-mononulceotide triphosphates enables the generation of nuclease-resistant aptamer libraries, which can also be used in biological fluids, such as blood or urine.
Key considerations for synthesis
When designing aptamer libraries there are a few key points to take into consideration:
- Fidelity: especially when synthesizing longer aptamer sequences you want as few errors in the constant regions as possibly, to ensure high efficiency amplification of the library and efficient sequencing of selected aptamers
- Proper randomization: truly randomized central sequences with as little bias as possible are key for generating aptamer libraries of high complexity
- Stability: when synthesizing RNA libraries, the use of modified nucleotides ensures that the final aptamers are stable under a range of different conditions and in different environments, including biological fluids
Integrated DNA Technologies (IDT) is one of the world's biggest manufacturers of nucleic acid products and has proven expertise in generating high quality DNA and RNA aptamer libraries. Explore their offering:
How are aptamers identified?
Aptamers are often identified using the in vitro technique called SELEX (Systematic Evolution of Ligands by EXponential enrichment), where oligos with increased affinity and specificity to the target molecules are isolated from the sequence pool after several rounds of selection. This involves iteratively incubating a library of nucleic acid sequences with the target of interest before eluting any bound sequences for PCR amplification. By increasing the stringency of the elution conditions with each cycle, it is possible to isolate the tightest binders, which can then be characterized with techniques such as ELISA and surface plasmon resonance (SPR). In recent years, variations of SELEX have been developed, including methods that eliminate the need for immobilization.
With the identification of critical structural patterns, it is now possible using computer modeling to bypass SELEX altogether and design aptamers from scratch, although a limitation of this approach is that it prohibits the use of whole cells as targets. Critical factors to consider during the aptamer design process include which chemical modifications are necessary to prevent degradation by nucleases and whether specific functional groups must be added to enable a particular application.
Once aptamers with favorable properties have been identified, they must usually be shortened to improve their stability. This involves using computer modeling to predict how truncation will affect the 2D and 3D structure. In silico methods are also used for optimization purposes, such as to predict docking and molecular dynamics or identify any structural patterns that are critical for the aptamer-target interaction.
Aptamers vs. antibodies
They have similar affinities as antibodies for their targets and provide several advantages, including greater stability, easier large-scale production, low immunogenicity, and the ability to target molecules with low antigenicity. Like antibodies, aptamers have a broad range of research applications.
Aptamers have several advantages over antibodies, not least the fact that they can be produced quickly and easily without the need for animal use. Aptamers also benefit from low production costs, high batch-to-batch consistency, and functional stability when stored at room temperature, which gives them a long shelf-life and simplifies both transportation and storage. In addition, the low immunogenicity of aptamers makes them valuable tools for in vivo applications, while their small size compared to antibodies allows them to better penetrate cells and tissues. This can be especially useful when studying difficult-to-access targets such as those found within the tumor microenvironment. On the flipside, aptamers are poorly suited to applications in which it is desirable to stimulate an immune response and may undergo rapid clearance in vivo unless they have been modified to prevent this.
Applications of aptamers
Aptamers can be used for many of the same research applications as antibodies, including flow cytometry, western blotting, and lateral flow immunoassay. They are particularly of interest as antibody replacements in enzyme-linked immunosorbent assay (ELISA), where they now form the basis of novel methods known as enzyme linked apta-sorbent assay (ELASA) and enzyme-linked oligonucleotide assay (ELONA). Like ELISA, these newer assay formats can adopt a direct, indirect, or sandwich configuration, using either aptamers on their own or in combination with antibodies. For most immunoassay-like uses, the aptamer is labeled with a reporter molecule (e.g., an enzyme or fluorescent dye) or a protein tag that can be recognized by existing secondary antibody reagents. It is worth nothing that, unlike antibodies, aptamers can be conjugated to labels with a 1:1 stoichiometric ratio.
Beyond scientific research, aptamers also have promising roles in diagnostics and as therapeutic agents. Diagnostic applications of aptamers that are currently being explored include their use as antibody alternatives for pathogen detection, cancer recognition, and monitoring of environmental contaminants. Often, for these types of uses, the aptamers are immobilized onto biosensors to reduce hands-on time and streamline workflows. Potential therapeutic applications of aptamers include their use as drug delivery systems (e.g., aptamer-drug conjugates) and bispecifics, such as those capable of binding both a cancer cell and an immune cell to facilitate targeted tumor destruction.
LubioScience represents some of the most trusted brands in research and works closely with our partners to bring aptamer-based products to the scientific community. Contact us today to discuss how we can support your project.
Suppliers
IDT - Integrated DNA Technologies
With over 30 years experience as a manufacturer, IDT offers innovative tools for NGS, CRISPR, qPCR and PCR. IDT offers superior quality DNA and RNA oligos, genes, gene fragments, Cas nucleases and more, with fast turnaround times!
Antibodies.com
Antibodies.com supplies high-quality biological reagents to life science researchers at cost-effective prices. They are a trusted supplier of antibodies, proteins, immunoassays, and other companion reagents. Headquartered in Cambridge (UK), they support life scientists worldwide by supplying the same high-quality products as the industry leading suppliers, that are sourced from the same primary manufacturers, but at more affordable prices.
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References
Figures
- https://blog.biosearchtech.com/where-are-all-the-therapeutic-aptamers
- Catuogno, Silvia & Esposito, Carla. (2017). Aptamer Cell-Based Selection: Overview and Advances. Biomedicines. 5. 49. 10.3390/biomedicines5030049.
- Kim, DH., Seo, JM., Shin, KJ. et al. Design and clinical developments of aptamer-drug conjugates for targeted cancer therapy. Biomater Res 25, 42 (2021). https://doi.org/10.1186/s40824-021-00244-4