Macromolecular Interactions: Aptamers


Short nucleic acids can fold into unique three‐dimensional configurations, known as aptamers, to elicit binding of a specific target. The structure of an aptamer, such as stem‐loops, g‐quadruplexes and pseudoknots, directs its ability to recognise and discriminate specific targets. There are significant advantages to aptamers over antibodies, including ease and cost of production as well as lack of immunogenicity. Traditionally, aptamers have been generated by systematic evolution of ligands by exponential enrichment. Aptamers have been developed against a range of targets including proteins, small molecules, intracellular targets, cell‐surface receptors and whole cells, and have thus far resulted in one Food and Drug Administration‐approved therapeutic. Although technical challenges as well as a restrictive intellectual property landscape have hindered the progress of aptamers for direct therapeutic use, they have gained applications as conjugate vehicles for targeted delivery of other therapeutic molecules, in molecular imaging, and as biosensors in increasingly sophisticated detection and monitoring devices.

Key Concepts:

  • Nucleic acid aptamers specifically bind a target molecule, including proteins and small molecules, serving as the functional equivalent of chemical antibodies.

  • Aptamers can adopt a multitude of unique three‐dimensional configurations that are required for specific binding of targets.

  • Aptamers are superior to antibodies with regard to ease of production and lack of immunogenicity.

  • Aptamers are traditionally discovered using systematic evolution of ligands by exponential enrichment.

  • Aptamers have been used for a variety of applications including direct therapeutics, vehicles for targeted delivery of compounds, biosensors, diagnostic imaging and nanomachinery.

Keywords: aptamers; SELEX; diagnostics; therapeutics; microarray; biosensor

Figure 1.

Examples of DNA and RNA aptamers. The target of the aptamer is listed below the aptamer structure. For VEGF and B‐cell receptor (BCR), the name of the aptamer is included (parentheses). Solid red lines represent Watson‐Crick base pairs. Dotted red lines represent wobble (G‐T for DNA, G‐U for RNA) or Hoogsteen (G‐quadruplex interactions in haemagglutinin) base pairing. For VEGF, purple nucleotides represent 2′‐fluorine‐modified pyrimidines and blue nucleotides represent 2‐O‐methylated purines.

Figure 2.

Schematic of a SELEX experiment. The SELEX process (top left) starts with a large nucleic acid library of approximately 1015 different sequences. The nucleic acid pool is incubated with the target compound that is attached to a resin (step 1). The unbound nucleic acids are washed away (step 2) whereas the bound nucleic acids are eluted and retained (step 3). The eluted nucleic acids are then counterscreened against a compound that is similar to the target to ensure specificity of the aptamers (step 4). Aptamers that do not bind the counterscreened compound are kept while the bound aptamers are eluted and discarded (step 5). The aptamers that bound the target compound, but not the counterscreened compound, are then amplified into a new nucleic pool (step 6). Steps 1–6 are repeated with increasingly stringent washes until only a few high‐affinity aptamers remain. The SELEX process is completed when this final pool of aptamers is cloned and sequenced (bottom left).

Figure 3.

Schematic of the iterative APT‐SNAP microarray‐based aptamer discovery system. (a) A target molecule, fluorescently labelled here with Cy5, is incubated with a microarray displaying millions of different aptamers (top). Each feature (square) on the microarray contains a unique aptamer that is repeated millions of times within that feature. Both the shape and sequence composition of the aptamer can be varied. The target molecule will preferentially bind certain aptamers (features) resulting in various intensities (red features, middle). The amount of aptamer bound at each feature is directly related to the affinity of the aptamer for the target molecule (bottom). (b) Through the iterative microarray design, the entire structure and sequence space can be effectively searched rationally rather than randomly as by SELEX. An initial microarray, which contains millions of different aptamer shapes, is used to identify binders to the target molecule (top). General shapes recognised by the target molecule are optimised in the second microarray design (middle). In the final design, the sequence of certain regions within the best binding aptamer shapes are permuted (each colour represents a different sequence) (bottom).

Figure 4.

Aptamer‐directed DNA nanorobots. (a) DNA origami creates a hinged nanobox containing a small‐molecule payload (purple circles). The box is held closed with an aptamer‐locking mechanism (green‐yellow and blue‐orange ovals). (b) Addition of ligand (red ovals), which binds to the aptamer (green and blue segments) and displaces short complementary DNA portion (yellow and orange segments) of the lock, allows the box to open and deliver the payload.

Figure 5.

Aptamer‐tethered DNA nanotrains. (a) The aptamer ‘locomotive’ (left) initiates self‐assembly of ‘boxcar’ DNA strands (centre) into a nanotrain (right), intercalating small‐molecule payload with quenched fluorescence (quenched, pink hexagons). (b) The aptamer ‘locomotive’ carrying the quenched payload directs the ‘boxcars’ to its binding site on cell surface. (c) The target cell internalises the nanotrain, resulting in the diffusion and unquenching of the payload (unquenched, red stars), which allows for detection of targeted delivery (Zhu et al., ).



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Further Reading

de Franciscis V, Rienzo A and Cerchia L (2012) Nucleic acid aptamers for in vivo molecular imaging. In: Schaller B (ed.) Molecular Imaging, pp. 95–116. Rijeka, Croatia: InTech. ISBN: 978‐953‐51–0359‐2. Available at:‐imaging/nucleic‐acid‐aptamers‐for‐in‐vivo‐molecular‐imaging

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Ohuchi S (2012) Cell‐SELEX technology. BioResearch Open Access 1: 265–272.

Song S, Wang L, Li J, Zhao J and Fan C (2008) Aptamer‐based biosensors. Trends in Analytical Chemistry 27(2): 108–117.

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Rodesch, Matthew J, Ozers*, Mary Szatkowski, and Warren, Christopher L(Jun 2014) Macromolecular Interactions: Aptamers. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0003146.pub2]