Aptamer-based analysis of angiogenin by fluorescence anisotropy

Abstract

Recognition and monitoring proteins in real time and in homogeneous solution has always been a difficult task. Here, we introduce a signal transduction strategy for quick protein recognition and real-time quantitative analysis in homogeneous solutions based on a high-affinity aptamer for protein angiogenin (Ang). The method takes advantage of the sensitive anisotropy signal change of fluorophore-labelled aptamer upon protein/aptamer binding. When the labelled aptamer is bound with its target protein Ang, the increased molecular weight causes the rotational motion of the fluorophore attached to the complex to become much slower. Therefore, increasing the amount of Ang results in a raised anisotropy value of the Ang/aptamer. By monitoring the anisotropy change, we are able to detect the binding events between the aptamer and Ang, and measure Ang concentration quantitatively in homogeneous solutions. This assay is highly selective, with a detection limit of 1 nM of Ang. The dissociation constant of the Ang/aptamer binding is determined in the nanomolar range and changes with increasing salt concentration. One can also use our assay to compare the binding affinities of different ligands for the target molecule. Ang in serum samples of malignant lung cancer was also detected. Efficient protein detection using aptamer-based fluorescence anisotropy measurements is expected to find wide applications in protein monitoring, cancer diagnosis, drug screening and other fields.

Recognition of Bungarus multicinctus Venom by a DNA Aptamer against β-Bungarotoxin

“…Two nM of fluorescently labeled aptamer (βB-1, βB-20, βB-19 or βB-32) was firstly incubated with 288 nM of β-BuTx, and then titrated by unlabeled aptamer βB-1. If the unlabeled βB-1 competes for the same binding site as labeled aptamers, the displacement of the aptamer by βB-1 will result in a decrease in anisotropy [47]. As shown in Figure 3, with increase amount of βB-1, a decrease of fluorescene anisotropy was observed.…”

Membrane curvature recognition by C-reactive protein using lipoprotein mimics

“…Fluorescence anisotropy measures the rotational diffusion rate of a fluorescent probe, such as an aptamer, and has been used to measure the binding interactions of biomolecules 27, 33, 34 . For example, the rotation rate of a free probe should be faster (lower anisotropy) compared to the rate of a probe that is bound to its target.…”

Probing the Key Binding Sequence and Improvement of the Stability of a β‐Bungarotoxin‐binding Aptamer in Snake Venom

“…Unlabeled aptamers with higher binding affinity would cause more labeled βB-1 to be replaced and thus, a larger change in anisotropy. 26 Unlabeled βB-1 was also introduced into the competitive displacement assay as a control, and it gave an anisotropy change (Δr) of 0.0201. As shown in Figure S1, the removal of the primer region of the 3 0 terminus did not affect the binding affinity, while removal of the 5 0 terminus primer sequence would lead to loss of binding.…”