Aptabody–aptatope interactions in aptablotting assays. Issue 22 (22nd May 2017)

• Record Type: Journal Article
• Authors: Sekhon, Simranjeet Singh; Um, Hyun-Ju; Shin, Woo-Ri; Lee, Sang-Hee; Min, Jiho; Ahn, Ji-Young; Kim, Yang-Hoon
• Is Part Of: Nanoscale. Volume 9:Issue 22(2017)
• Page Start: 7464
• Page End: 7475
• Publication Date: 2017-05-22
• Subjects: Nanoscience -- Periodicals; Nanotechnology -- Periodicals; 620.505
• DOI: 10.1039/c7nr01827d
• Languages: English
• ISSNs: 2040-3364

Abstract

Abstract : An aptabody based aptablotting technique involving a direct and an indirect aptabody–aptabody complex is reported. The aptabody is successfully screened by in vitro SELEX and its specific molecular recognition is achieved by dot-blot and a SDS-PAGE/electroblotting analysis. Abstract : We demonstrate an aptablotting assay method that involves direct and indirect aptabody recognition. Nanoscale single-stranded DNA aptamers against GST and DIG-tags are utilized as aptabodies (GST-2 and DIG-1, respectively), and the GST-2 aptabody binding site, or aptatope, as predicted by a MOE-docking simulation of the protein–aptamer complex, shows the interaction of the GST-2 aptabody at the catalytically active region. The aptabody–aptatope interaction was evaluated by an in vitro enzyme inhibitory analysis. The binding capacity of the GST-2 aptabody was assessed by dot-blot, EMSA and SDS-PAGE/electroblot analyses, and the results showed that the aptabodies interact with both the native mono-/dimeric form and the denatured GST form on a membrane. The use of aptabodies can overcome the obstacles of current immunoblot assays, and these molecules are easily assessable via ELISA systems. Moreover, the hybridization of aptabodies and antibodies (hybrid-aptablotting) may have considerable impacts on the design of bioassay platforms.