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It has been shown that selected aptamers can be used to make the catalytic activity dependent on the presence of a ligand.<x-ref>Soukup1999</x-ref> Fusions like that are called aptazymes. For this a catalytic domain has to interact via a communication module with an aptamer (Fig. 6). In presence of the ligand the aptamer undergoes conformational changes which results in an activation or inactivation of the catalytic activity. In our project we use different aptamers and combine them with catalytic nucleic acids to use them in various applications. In order to develop the necessary tools we need a fast and efficient way to generate new aptamers and aptazymes. To meet these needs we have developed two parts of software: One that generates aptamers from scratch (MAWS) and another one that joins them with an existing catalytic nucleic acid(JAWS) in an optimized way. This method not only is faster but also cheaper than existing methods.
 
It has been shown that selected aptamers can be used to make the catalytic activity dependent on the presence of a ligand.<x-ref>Soukup1999</x-ref> Fusions like that are called aptazymes. For this a catalytic domain has to interact via a communication module with an aptamer (Fig. 6). In presence of the ligand the aptamer undergoes conformational changes which results in an activation or inactivation of the catalytic activity. In our project we use different aptamers and combine them with catalytic nucleic acids to use them in various applications. In order to develop the necessary tools we need a fast and efficient way to generate new aptamers and aptazymes. To meet these needs we have developed two parts of software: One that generates aptamers from scratch (MAWS) and another one that joins them with an existing catalytic nucleic acid(JAWS) in an optimized way. This method not only is faster but also cheaper than existing methods.
 
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Revision as of 21:14, 18 September 2015

Aptamers

Figure 4. Theophylline Aptamer

Aptamers are RNA or DNA sequences that can selectively bind to a ligand or target. Scientists have selected a variety of aptamers for small molecules ranging from theophylline (Fig. 4A) being one of the first described aptamersjenison1994 over ATPsassanfar1993 to an aptamer for the protein thrombinbock1992.

Both catalytic nucleic acids and aptamers are as for today selected via systematic evolution of ligands by exponential enhancementJoyce1989ellington1990tuerk1990bartel1993 also known as SELEX (Fig. 5). It is a very time consuming and expensive process. Many cycles of selection from a random pool of sequences have to be performed in order select the best candidates in each round. A variable starting pool of random sequences has to be prepared and after every cycle the candidates have to be mutated to further improve the binding to the target. All in all the process is rather inefficient. Probably this is the reason why there are still many small molecules and proteins for which no aptamer has been published yet.

Figure 5. SELEX

Systematic Evolution of Ligands by EXponential Enrichment, zu deutsch: Systematische Evolution von Liganden durch exponentielle Anreicherung (SELEX): Current method to select functional nucleic acids from a random library. Figure adapted from :Source

It has been shown that selected aptamers can be used to make the catalytic activity dependent on the presence of a ligand.Soukup1999 Fusions like that are called aptazymes. For this a catalytic domain has to interact via a communication module with an aptamer (Fig. 6). In presence of the ligand the aptamer undergoes conformational changes which results in an activation or inactivation of the catalytic activity. In our project we use different aptamers and combine them with catalytic nucleic acids to use them in various applications. In order to develop the necessary tools we need a fast and efficient way to generate new aptamers and aptazymes. To meet these needs we have developed two parts of software: One that generates aptamers from scratch (MAWS) and another one that joins them with an existing catalytic nucleic acid(JAWS) in an optimized way. This method not only is faster but also cheaper than existing methods.

Figure 6. Aptazyme

An aptamer connected via a communication module to an catalytic sequence; The communication module translates the interaction of the ligand with the aptamer to the catalytic part of the aptazyme.

Besides the above describes functional nucleic acids SELEX revealed aptamers which not only bind to a ligand but also are able to turn on fluorescence of different non-fluorescent dyes. An interesting example for an aptamer with such a special function is the Malachite Green Aptamer.Grate1999 In presence of malachite green the aptamer turns on the fluorescence of the dye.babendure2003 In 2011 Jaffrey selected the famous Spinach RNA aptamer (Fig. 7). It binds a small molecule that resembles the fluorophore of the green fluorescent protein (GFP).paige2011 Upon binding of the small molecule to the Spinach a green fluorescence comparable to that of the GFP is emitted. In our project we use variants of these fluorescent aptamers and engineer them to be dependent on small molecules of our interest.

Figure 7. Spinach Aptamer

The Spinach Aptamer turns on fluorescence of its ligand upon bind it.

Though we so far mainly discussed functional RNA there are also both DNA aptamers and catalytic DNA which were selected using SELEX as well. As for today the concept of functional nucleic acids in nature has only been found for RNA. Nevertheless DNA aptamers and also catalytic DNA were selected in vitro.