Background

Nucleic acids as specific ligand binders

Aptamers are highly affine nucleic acids that are able to bind other molecules by the key-lock principle through formation of a sequence-dependent three dimensional structure. (See fig. 1) Aptamers were first isolated by Gold and Tuerk in 1989 using the in vitro selection procedure SELEX (Systematic Evolution of Ligands by EXponential Enrichment). SELEX is employed for the identification of RNA or DNA molecules that bind to their target molecule with high affinity. Starting with combinatorial libraries with up to 10¹⁵ different molecules, the specific binders are isolated by an iterative process of ligand binding, washing, recovery (elution), and amplification (see fig. 2). This has yielded aptamers with affinities ranging form sub-picomolar to nanomolar affinities thus comparable to other well established biomolecules like monoclonal antibodies. Several hundreds of aptamers have already been identified to various kinds of targets as small organic molecules, proteins, virus particles up to entire cells and tissues. (see Ellington Lab Aptamer Database: http://aptamer.icmb.utexas.edu/) In comparison to other binding molecules, aptamers have the advantage to not elicit unwanted immune responses and are able to easily penetrate biological tissues in vivo. In addition, as oligonucleotides they can be easily chemically synthesised thus highly reproducible and allow the facile introduction of further modifications. As such the use of steric isomers of the biogenic D-ribose, so called spiegelmers are resistant to the ubiquitous nucleases and therefore to degradation in various environments including the human body. This spiegelmer technology has been developed and patented by Fürste, Bald, and Erdmann at the FU Berlin. Aptamer applications are ranging from therapeutics and diagnostics to biosensors, nanotechnology, and affinity chromatography. Despite this enormous potential only few aptamers are commercialised like Macugen (Eyetech and Pfizer), a treatment for neovascular age-related macular degeneration. Depending on the application, different properties and selection methods may be required for optimal solutions.

fig. 1	fig. 2

Nucleic acid binders in nature

Aptamers are not solely artificial molecules. They have been discovered to exist as so-called riboswitches in nature. These are found mainly in untranslated regions of mRNA that regulates its expression by modulating either transcription or translation upon ligand binding. Additionally, genomic DNA sequences mostly in promoter regions are acting as natural receptors for important regulatory proteins like transcription factors. Genomic SELEX can be employed to identify the sequences involved in the binding of such transcription factors. Ideally, only one round of selection may be necessary to enrich a population of specifically binding DNA sequences.

Caveats

Despite the advantages of aptamers as being easily chemically synthesised, modified for various applications, and cheaply available some drawbacks remain. Most importantly, negatively charged molecules are poor targets because of electrostatic repulsion by the likewise negatively charged nucleic acids. Many binders have also shown to be poorly active upon immobilisation and proper controls are necessary to ensure that true binders have been obtained (see fig. 3). These shortcomings are partially reflected by the fact that most publications demonstrating proof-of principle applications of this technology are conducted with robust ssDNA aptamers binding to thrombin or ATP.

fig. 3

Our task

In order to cope with these problems, a high throughput selection could be employed to quickly yield and evaluate binders to a wide range of targets. Thus it is the scope of our group to establish this technology in high throughput and optimise the selection procedures to obtain the most useful nucleic acid binders for each given application. Special emphasis is put on reproducibility, monitoring, and streamlining of our automated selections.