Streptavidin binding bifunctional aptamers and their interaction with low molecular weight ligands

This paper describes the measurement of the binding affinities of two bifunctional RNA aptamers to their respective ligands. The aptamers comprise either a theophylline or malachite green binding sequence fused to a streptavidin binding sequence. These bifunctional aptamers are shown to bind simultaneously to both the small ligand and to streptavidin whether in free solution or on gold surfaces. Binding isotherms for both interactions were measured by different physiochemical techniques: surface plasmon resonance, fluorescence spectroscopy and dynamic light scattering. Both qualitatively and quantitatively there is little difference in binding affinities between the bifunctional aptamers and their monofunctional components. The respective Kd values for streptavidin binding in the monofunctional aptamer and in the theophylline bifunctional aptamer were 12 nM and 65 nM, respectively whilst the Kd values for theophylline binding in the monofunctional aptamer and the streptavidin bifunctional aptamer were 300 nM and 120 nM. These results are consistent with treating each aptamer sequence as a module that can be combined with others without significant loss of function. This allows for the use of streptavidin based immobilization strategies without either the cost of biotinylated dNTPs or the variable yields associated with the chemical biotinylation of RNA.

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► Aptamers can be made that bind two different ligands to a single RNA sequence.
► Ligand binding is characterized by fluorescence, SPR and dynamic light scattering.
► Affinities are comparable in solution and on gold surfaces for single and dual site aptamers.
► Binding isotherms show that binding of one ligand does not perturb the affinity for the second.
► RNA secondary structure predictions are consistent with experimental data.