Development of Aptamer-Based TID Assays Using Thermophoresis and Microarrays

Abstract

Aptamers are single-stranded oligonucleotides which can be used as alternative recognition elements for protein detection, because aptamers bind their targets with a high affinity similar to antibodies. Due to the targetinduced conformational changes of aptamers, these oligonucleotides can be applied in various biosensing platforms. In this work, aptamers directed against the vascular endothelial growth factor (VEGF) were used as a model system. VEGF plays a key role in physiological angiogenesis and vasculogenesis. Furthermore, VEGF is involved in the development and growth of cancer and other diseases like agerelated macular degeneration, rheumatoid arthritis, diabetes mellitus, and neurodegenerative disorders. Detecting the protein biomarker VEGF is therefore of great importance for medical research and diagnostics. In this research, VEGFbinding aptamers were investigated for the systematic development of a targetinduced dissociation (TID) assay utilizing thermophoresis and microarrays. The established aptamer-microarray allowed for the detection of 0.1 nM of VEGF. Furthermore, the systematic development of the TID method using the VEGF model protein could help to develop further TID assays for the detection of various protein biomarkers.

Characterization of an Aptamer Directed against 25-Hydroxyvitamin D for the Development of a Competitive Aptamer-Based Assay

“…As soon as the most suitable oligonucleotide candidate was determined, competitive assays using immobilized aptamers were carried out. We have recently used this strategy (the development of a TID assay via MST and its transfer to an aptamer-microarray format) successfully for a protein target [20]. Here, we demonstrate the suitability of the approach for aptamers directed against small molecules.…”

“…The CD spectroscopy indicated that the target might bind to a preformed structural motif of the aptamer. In other published competitive assays, we have already been able to successfully use the 10-nt length [15,20]. Moreover, it could be shown that longer oligonucleotides lead to decreased complementary strand displacement and consequently decreased sensing performance [25].…”

Anti‐VEGF DNA‐based aptamers in cancer therapeutics and diagnostics

“…Schematic representation of the (A) aptamer‐based four‐color nanoprobe for simultaneous sensing and imaging of multiple tumor‐associated proteins in living cells and (B) aptamer‐based VEGF detection by TID of the fluorescently labeled complementary oligonucleotide. Figures were reproduced from Xu et al 245 (A) and Kurth et al 246 (B) with permission. AFP, ɑ‐fetoprotein; CEA, carcinoembryonic antigen; HER2, human epidermal growth factor receptor 2; TID, target‐induced dissociation; VEGF, vascular endothelial growth factor [Color figure can be viewed at wileyonlinelibrary.com]…”

“…In the presence of VEGF 165 , the formation of the aptamer‐protein complex favored the release of the complementary strand, resulting in a detectable decrease of the fluorescence intensity (Figure 13B). The proposed aptamer‐microarray allowed a LOD of 0.1 nM for VEGF 165 246 …”

“…The LODs were 0.45 nM for AFP, 0.30 nM for VEGF 165 , 0.62 nM for CEA, and 0.96 nM for HER2 245. Kurth et al246 developed a V7t1-based target-induced dissociation (TID) assay for the detection of VEGF 165 , anchoring the amino-modified V7t1 aptamer on the 3D-aldehyde-surface of a microarray and hybridizing it with a Cy5-labeled oligonucleotide, complementary to V7t1. In the presence of VEGF 165 , the formation of the aptamerprotein complex favored the release of the complementary strand, resulting in a detectable decrease of the fluorescence intensity (…”