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. 2015 Jul 14;43(21):e144. doi: 10.1093/nar/gkv718

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© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Determination of the binding affinity between various probes and DNB. (A) Dissociation constants (K_D) between DNB and various probes. The K_D values were determined by titrating increasing amounts of aptamer with a fixed concentration of probe (100 nM) and measuring the fluorescence increase upon binding. The K_D values were found to be 8.55 ± 0.53 μM for FL-DN-DNB (red), 4.48 ± 0.60 μM for RG-DN-DNB (green), 0.35 ± 0.05 μM for TMR-DN-DNB (blue), 0.80 ± 0.1 μM for SR-DN-DNB (black) and 18.0 ± 1.8 μM for TR-DN-DNB (pink). (B) The dissociation constant between dinitroaniline-amine (ligand used for SELEX) and DNB was calculated in a competition based assay where the DNB aptamer (100 nM) was saturated with SR-DN (1 μM) and the fluorescence decrease was measured upon addition of dinitroaniline-amine. The K_D was determined from the IC₅₀ obtained by the competition assay. K_D was calculated to be 100 ± 14 nM.