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4.1.1

Introduction to Aptamers

Aptamers are DNA or RNA single strands that have been selected from random pools based on their ability to bind ligands. Like antibodies, aptamers are highly specific to their targets, and thus have many potential uses in medicine and other areas of technology. The list of targets includes metal ions [73], small molecules like cocaine [74], proteins like streptavidin or thrombin [75], dyes like malachite green [76], and organic superstructures like the HI-virus [77].

G G G G G G G G

Figure 4.1: Structure of thrombin-aptamer complex, derived from X-ray analysis. The representation of the aptamer on the right side displays the two G-quadruplexes of the armchair-like binding motif.

It is important to mark the difference between classical DNA binding structures and aptamers. DNA binding motifs of proteins or intercalating molecules are shaped to interact with the native B-DNA double strand, while an aptamer uses the affinity of a specially formed tertiary structure of a DNA single strand to an object that does not bind normally to a double helix. This tertiary structures can consist of regular Watson-Crick base pairs or more exotic structures like G-quadruplexes in one of the thrombin aptamers. Even though the secondary structure of a DNA or RNA strand can be derived from its sequence with acceptable accuracy, the development of aptamers is still a matter of trial and error. Interactions of aptamer and target are very hard to predict and in most cases only X-ray or NMR analysis can unveil the structure of the aptamer-target complex. Specific aptamer structures are evolved in a so called SELEX (Systematic evolution of ligands by exponen- tial enrichment) process [78]. A library of nucleic acid sequences is tested for their binding specificity or catalytic function. The most suitable sequences are selectively amplified and the test is repeated with stricter criteria. After several cycles the remaining sequences that were best in the competition are analyzed.

4.1 Aptamers 29

4.1.2

Reference [3]: Design Variations of an Aptamer-based DNA

Nanodevice

The main applications for aptamers are immobilization of proteins on a surface and de- tection or deactivation of proteins in bulk liquid. Immobilization of proteins for detection or synthesis is a key technique for portable analytical devices like proteomics chips, it is a challenging task. Any influence on the fragile structure of the protein can corrupt its func- tion or lead to denaturation. A common method of tethering proteins to a chip surface is to attach a biotinylated protein to a polyethylene glycol (PEG) linker via streptavidin. PEG itself is modified with a silane or thiol group to bind to gold or silicon dioxide substrates. This method requires many elaborate steps, but sometimes it still causes the protein to denature. Together with many other methods, it also has the disadvantage that the link is permanent, the protein cannot be removed easily once it has bound. A better result can be achieved with less effort with the help of aptamers. They can be bound to a gold or silane layer as well and since the binding site and spacer to the surface are parts of the same molecule, no assembly steps are necessary. The only effort is the SELEX process to find the appropriate sequence for every new protein.

Additional to immobilization on a surface, deactivation of proteins in solution can be a task mastered by aptamers. When bound to the active site of the protein or by causing a conformational change it can modulate the function of the protein reversibly. This was demonstrated in vivo by Rusconi et al.: The blood coagulation process was influenced by the interaction of an aptamer with a blood clotting factor protein [79]. This way an aptamer can be employed as a switchable drug: It prevents thrombosis when active, but can be turned off to stop uncontrolled bleeding from a wound.

The work presented here aims at the extension of the functionality of the aptamer device previously designed in our lab [13]. The device is based on a short thrombin binding nucleic acid sequence [75]. The binding motif assumes a three dimensional structure with two central G-quadruplexes, stabilized by potassium ions. A “toehold” of 12 bases is attached to the 5’ end of the aptamer to enable the switching to an off state. Without this expansion the operating strand has no leverage to bind and to remove the protein by hybridizing to the aptamer sequence.

To investigate the integration of this aptamer in other devices the influence of modifica- tions of the aptamer on its binding abilities was analyzed in [3]. Dye molecules at the ends of the aptamer are used for detecting the binding of a protein in FRET experiments, but it was not clear, if they change the binding behavior. The other variations were extensions of the strand by additional nucleic acid bases (Fig. 4.2). So far it was known to be uncritical to extend the 5’ end, but in order to obtain an additional handle to be used within an aptamer device it would be necessary to extend the 3’ end as well.

The binding capabilities were tested in gel shift experiments. The binding of thrombin is indicated by the development of an additional band containing the aptamer-protein com- plex, heavier that the one of the aptamer alone (Fig. 2 in [3]).

Figure 4.2: Design variations of a thrombin-binding aptamer device. The basic aptamer (red) was extended by additional bases (blue) and fluorophores (green).

It showed that the presence or position of the dye had almost no influence on the function of the aptamer, therefore FRET experiments can be performed without further concern. But while the extension of the 5’ end did not influence the binding constant, the addition of 20 bases to the 3’ end prevented binding completely. A new design needs to be found if a second arm at the aptamer is needed.