Characterization of Aptamers

With surface plasmon resonance spectroscopy, kon and kQa rates of the ap tamer-target complex are determined. These in turn, allow calculation of the KD value [42]. For surface plasmon resonance spectroscopy, one of the two binding partners 

The main difficulty with this technology is the possible interaction of target and/or ap tamer with the chip surface. The nature of this difficulty may be high unspecific interaction or high repulsion. For this reason, the most suitable sensor chip surface and optimal binding conditions must be determined for every target/aptamer combination. 

The modified cellulose filter binding assay is based on the tight binding of proteins to this kind of filter material. When a protein-nucleic acid mixture is filtered, proteins are retained on the filter while nucleic acids are washed through. However, nucleic acids are also retained on the filter if they are bound to proteins. Thus, free and protein-bound nucleic acids can be separated [43]. 

By incubating very small amounts of radioactively labeled nucleic acids (at least 100 times less than the amount of protein) with increasing concentrations of a target protein, the K- value can be calculated after quantification of the free nucleic acids (that is, those that are not bound to the filter) and the nucleic acid-target complexes that are retained on the filter. 

The relatively new method of fluorescence correlation spectroscopy (FCS) is based on the fact that molecules with different molecular weights (usually) exhibit different diffusion times in solution. Thus, small molecules diffuse faster than larger ones. To determine K- values, one component must be labeled with a fluorescent dye. Due to the different molecular weights of the uncomplexed, labeled component and the complex, the diffusion times of the free and complexed molecule differ. This fact allows determining the distribution of free and complexed molecules in the solution. After measuring the distribution in different mixtures with varying ligand concentrations, the K- value can be calculated [44]. 

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After 5 or, up to > 15 rounds of selection, single aptamers can be isolated and identified by cloning and sequencing. The characterization of aptamers can be... 

Finding kon, kos, K, AS, and AH in a fast and accurate fashion is important for assessing the progression of selection as well as for final characterization of aptamers. A single NECEEM electropherogram contains sufficient data for... 

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