Nonradioactive fluorescence microtiter plate assay monitoring aptamer selections.

Benchmarks Since its discovery in 1990, aptamer technology (1,2) has emerged as a novel and increasingly important approach to evolve specifically binding nucleic acid molecules (3). As aptamers can be developed to a vast range of target molecules, their potential application is very diverse. They can be used as molecular recognition elements in analytical systems for detection, separation, or purification of target molecules (4–8). They also play an important role in medical therapy (9) and environmental analysis (10,11). Aptamers are usually generated using SELEX (systematic evolution of ligands by exponential enrichment) technology (1,2). In this cyclic, repetitive process, a combinatorial library of synthetic nucleic acids containing approximately 10 15 different molecules is used as a starting pool. In subsequent steps of target binding, removal of unbound sequences, elution of bound sequences and their amplification and purification, the nucleic acid pool is enriched with specifically binding sequences. To monitor the progress of selection, nucleic acids are usually radioactively labeled. This is a rather sensitive method, which enables detection of trace amounts of nucleic acids. Disadvantages of monitoring enrichment radioactively are that appropriate safety precautions have to be taken and that it is rather costly. In a modified selection process for DNA aptamers, named FluMag-SELEX, Stoltenburg and coworkers have inserted fluorescent labels into the DNA via PCR and used it for detecting enrichment (12). Fluorescence allows for a fast and nonradioactive monitoring of selection progress. Since direct integration of fluorescent labels is expensive and might also interfere with the binding properties of the nucleic acid (13), we have developed an easy and sensitive analytical microtiter plate assay, the fluorescent dye-linked aptamer assay, that utilizes the advantages of fluores-cence without being invasive. For monitoring enrichment over the course of selection, we used the fluorescent dye OliGreen ® (Invitrogen GmbH, Karlsruhe, Germany). This reagent is specific for single-stranded DNA and enables the quantification of as little as 100 pg/mL nucleic acid with a standard spectrofluorometer using fluorescein excitation and emission wavelengths (14). For the assay setup, the biotinylated target molecule is immobilized on a streptavidin-coated microtiter plate, incubated with the single-stranded DNA aptamer pool, and while nonbinding sequences are removed, binding sequences are detected (Figure 1). The unbound dye is basically nonfluorescent. Binding assays were performed using streptavidin-coated 96-well microtiter plates (high-binding capacity, black, with SuperBlock blocking buffer; Perbio Science Deutschland GmbH, Bonn, Germany). Between incubation steps, which were done at room temperature and …