Fluorescence Tagged Aptamers for Target Quantification and Cytometry Applications

The breakthrough in genomics and proteomic research has led to demand for identification of fluorescence-labelled ligands for a vast number of target proteins. Fluorescent ligands are used for target protein quantification and studying protein function in a cellular context. Mostly mono- and polyclonal antibodies have been developed to fluorescent ligands. Although being the method of choice for detecting antigen concentration in biological fluids, tissue and cells, antibodies have some limitations, which have led to the search for alternative strategies for the identification of high-affinity ligands for research and diagnostics.

As already mentioned, the capability of aptamers to recognize their target is similar to that of antibodies (Xu and Ellington 1996; Tasset et al. 1997). For instance, aptamers were able to recognize a specific isoform of protein kinase C (Conrad et al. 1994), to differentiate between a phosphorylated and unphosphorylated protein (Seiwert et al. 2000) and to discriminate ATP from other nucleotides (Sassanfar and Szostak 1990). Aptamers are developed by an in vitro selection method and therefore can be evolved against every target, including toxins and compounds, that does not elicit any immune response. The use of RNA aptamers for diagnostic and in vivo applications was limited in the past due to their poor nuclease resistance. These initial limitations have been overcome by the development of modified nucleotides that largely enhance half-times of RNA aptamers in biological fluids (reviewed by Kusser 2000). Post-SELEX modifications of selected aptamers, such as attaching fluorescent reporters, are done at the researcher's will (reviewed by Ulrich et al. 2004).

As an alternative approach, fluorescence-labelled RNA aptamers against ATP were selected from an RNA pool in which UTP had already been substituted for fluorescein-UTP. The initial pool had been synthesized in such a way that uridine residues would be poorly represented. These fluorescein-UTP-labelled aptamers detected ATP at a concentration of 25 pM in complex mixtures (Jhaveri et al. 2000).

An example for the development of fluorescent-tagged RNA aptamers for flow cytometry comes via the work of Davis et al. (1998). Aptamers selected against CD4 were linked to a biotin moiety, coupled to streptavidin-fluorescein or streptavidin-phycoerythrin and then used for separation of CD4-expressing T cells from those lacking CD4. Homann and Göringer (1999) have used fluorescent-labelled RNA ligands to identify a 42-kDa protein within the flagellar pocket of the blood-stream form of Trypanosoma brucei. In addition to being used for target imaging in cells or for target separation, fluorescent-tagged aptamers can substitute antibodies in dot blot, enzyme-linked immunosorbent (ELISA), and Western-blot assays and aptamer-chip based biosensors (Drolet et al. 1996; Ulrich et al. 2004; McCauley et al. 2003). In the future, aptamer-coupling to quantum dots that allow the use of various intensity levels and colours may become important for high-throughput screening in chip-based technologies (reviewed by Ulrich et al. 2004).

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