Revision as of 09:29, 27 July 2015

The Recognition Element – Aptamers

Since the discovery of the nucleic acid structure, nucleic acids were long thought to have a single function – storage of the heredity information as genetic instructions. Our perception on the function of DNA and RNA changed radically, however, as it was discovered that small RNA molecules could fold into a three-dimensional structure, exposing a surface onto which other small molecules could perfectly fit. Soon after the discovery that nucleic acids could have interaction with other molecules, Craig Tuerk and Larry Gold described a procedure, called SELEX, to isolate high-affinity nucleic acid ligands for proteins through a Darwinian-like evolution process carried out in vitro [1]. This procedure enabled researchers to discover oligonucleotides with high affinities and a perfect fit for arbitrary proteins, cells, small molecules and even viruses. After this perfect fit, these synthetic oligonucleotides were called ‘aptamers’ – stemming from the Latin aptus which can be translated into ‘fit’.

The Rise of Aptamers

25 Years after the discovery of Tuerk’s and Gold’s invention of Systemic Evolution of Ligands by Exponential enrichment (SELEX), aptamers have become available for hundreds of ligands, including proteins, viruses, other small molecules and even whole cells. As such, aptamers have become a viable alternative for biology’s traditional recognition elements, antibodies. Aptamers also provide numerous advantages over antibodies. As mentioned, aptamers are oligonucleotides whereas antibodies are proteins. As such, aptamers have a remarkable stability in a wide range of pH and temperatures, have higher shelf lifes, are non-toxic and lack immunogenicity [2, 3]. Other advantages stem from the fact that aptamers are generated in vitro, whereas antibodies are generated through in vivo enrichment followed by purification through monoclonal cell lines [4]. As a result, generation of aptamers is less laborious, production costs are lower and reproducibility is higher. Finally, in contrast to antibodies, aptamers can be generated against virtually any molecule, including toxins and poor immunogenic targets [5]. This places aptamers amongst the most powerful tools in biotechnology [6]. A major limitation of aptamers in comparison with antibodies is their stability in vivo, where nucleic acids are rapidly degraded. Aptamers which have been used as therapeutic agents, for example, suffered from a half-life of 2 minutes [7]. This problem has, however, partially been overcome by using chemical modifications to the aptamers. An example of these modifications are Spiegelmers [6]. These oligonucleotides’ backbones contain L-Ribose instead of R-Ribose – “spiegel” means mirror. These mirrored aptamers are more stable in vivo, because they suffer less from degradation.

Aptamers in Biotechnology

Since aptamers can be easily modified chemically, they can be attached to numerous surfaces, enabling their way into a wide array of biosensors and drug delivery systems. Generally, the biosensors couple binding of the aptamer to a change in structure in the latter, which generates either a fluorescent or electrical signal. Aptamers have also found their way into drug delivery systems. A particular example of such a system is developed by Wu et al., featuring aptamers targeted at cancer cells with lipid tails. These aptamers could be used as building blocks of micelles, enabling efficient delivery of the micelles to cancerous cells [8].

Aptamers in our Biosensor

Aptamers constitute the ideal sensor domain for our universal membrane sensor: they can be targeted at virtually any molecule, can easily be attached to the membrane using SPAAC chemistry and offer affinities similar to antibodies. Taking into account their remarkable stability and small size, they are unmatched by the traditional recognition elements of biotechnology, antibodies. An essential requirement for our system which limits the choice of aptamers is bifunctionality: the system relies on bringing two membrane proteins in close proximity by binding the ligand with two different moieties. Conceptually, this bifunctionality can be reached either through the use of dual aptamers or through the use of split aptamers.

Figure 2: Bifunctionality is required to bring the membrane proteins into close proximity.

Dual Aptamers

The first way in which bifunctionality can be reached is through the use of dual aptamers acting on different binding sites, called apitopes. A well-known example of a ligand for which dual aptamers are available is human thrombin. A drawback of the use of dual aptamers is that these are in general not available for small molecules and viruses, significantly reducing the available targets for the aptamers. The main target for dual aptamers are proteins.

Split Aptamers

Alternatively, aptamers can be split into two parts. These parts can form heterodimers and thereby self-assemble into the three-dimensional structure required for binding the apitope. A crucial choice in engineering split aptamers is the choice of splitting site [9]. This choice is often not straightforward and the aptamer often suffers from a significantly decreased affinity. Obtaining split aptamers is, however, still in its infancy: of the approximately 100 small-molecule-binding aptamers, only six have been successfully engineered into split aptamers [10].

[1] C. Tuerk and L. Gold, “Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.,” Science, vol. 249, no. 4968, pp. 505–10, Aug. 1990.
[2] D. Musumeci and D. Montesarchio, “Polyvalent nucleic acid aptamers and modulation of their activity: A focus on the thrombin binding aptamer,” Pharmacol. Ther., vol. 136, no. 2, pp. 202–215, 2012.
[3] A. Cibiel, C. Pestourie, and F. Ducongé, “In vivo uses of aptamers selected against cell surface biomarkers for therapy and molecular imaging,” Biochimie, vol. 94, no. 7, pp. 1595–1606, 2012.
[4] L. H. Lauridsen and R. N. Veedu, “Nucleic acid aptamers against biotoxins: a new paradigm toward the treatment and diagnostic approach.,” Nucleic Acid Ther., vol. 22, no. 6, pp. 371–9, 2012.
[5] A. Rhouati, C. Yang, A. Hayat, and J. L. Marty, “Aptamers: A promosing tool for ochratoxin a detection in food analysis,” Toxins (Basel)., vol. 5, no. 11, pp. 1988–2008, 2013.
[6] F. Radom, P. M. Jurek, M. P. Mazurek, J. Otlewski, and F. Jeleń, “Aptamers: Molecules of great potential,” Biotechnol. Adv., vol. 31, no. 8, pp. 1260–1274, 2013.
[7] B. J. Hicke, A. W. Stephens, T. Gould, Y.-F. Chang, C. K. Lynott, J. Heil, S. Borkowski, C.-S. Hilger, G. Cook, S. Warren, and P. G. Schmidt, “Tumor Targeting by an Aptamer,” J. Nucl. Med., vol. 47, no. 4, pp. 668–678, Apr. 2006.
[8] Y. Wu, K. Sefah, H. Liu, R. Wang, and W. Tan, “DNA aptamer-micelle as an efficient detection/delivery vehicle toward cancer cells.,” Proc. Natl. Acad. Sci. U. S. A., vol. 107, no. 1, pp. 5–10, 2010.
[9] W. Zhou, P.-J. J. Huang, J. Ding, and J. Liu, “Aptamer-based biosensors for biomedical diagnostics.,” Analyst, vol. 139, no. 11, pp. 2627–40, 2014.
[10] A. D. Kent, N. G. Spiropulos, and J. M. Heemstra, “General approach for engineering small-molecule-binding DNA split aptamers,” Anal. Chem., vol. 85, no. 20, pp. 9916–9923, 2013.

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 The first way in which bifunctionality can be reached is through the use of dual aptamers acting on different binding sites, called apitopes. A well-known example of a ligand for which dual aptamers are available is human thrombin. A drawback of the use of dual aptamers is that these are in general not available for small molecules and viruses, significantly reducing the available targets for the aptamers. The main target for dual aptamers are proteins. <br />
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 Alternatively, aptamers can be split into two parts. These parts can form heterodimers and thereby self-assemble into the three-dimensional structure required for binding the apitope. A crucial choice in engineering split aptamers is the choice of splitting site [9]. This choice is often not straightforward and the aptamer often suffers from a significantly decreased affinity. Obtaining split aptamers is, however, still in its infancy: of the approximately 100 small-molecule-binding aptamers, only six have been successfully engineered into split aptamers [10]. <br />
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 <img class="imagecolumn" src="https://static.igem.org/mediawiki/2015/e/e4/TU_Eindhoven_design_splitAptamerA.png" />
 <img class="imagecolumn" src="https://static.igem.org/mediawiki/2015/7/78/TU_Eindhoven_design_splitAptamerB.png" />