Please use this identifier to cite or link to this item: https://scholarbank.nus.edu.sg/handle/10635/19125
Title: Generation of aptamers against the ganglioside-binding proteins via single bead selex
Authors: WU YUZHOU
Keywords: Botulinum neurotoxins, heavy chain, aptamer, SELEX,inhibitor, single bead
Issue Date: 18-Jun-2010
Citation: WU YUZHOU (2010-06-18). Generation of aptamers against the ganglioside-binding proteins via single bead selex. ScholarBank@NUS Repository.
Abstract: Botulinum neurotoxins (BoNTs) are widely regarded as the ¿poison of all poisons¿. BoNTs are built upon two primary domains, the light chain (LC) and the heavy chain (HC). The LC constitutes a zinc metalloprotease which cleave the SNARE proteins, whereas the HC domain transports the LC into the neuronal cytosol via the acidic endosome. Numerous researchers have explored the design of potent BoNTs inhibitors targeting the LC domain. However, in nearly all cases, low cell uptake or high cytotoxicity was limiting factors preventing their potential for in vivo applications. Due to their mechanism of action, inhibitors targeting the HC domain would not need to cross cell membranes in order to reach their targets since they would interfere with BoNTs in the blood stream and thus prevent BoNTs uptake. Up to now, no rational approach for the identification of HC inhibitors has been reported. Such an approach is also considered challenging due to the necessity to address both neuroselective binding sites with so far unknown small molecule inhibitors. Therefore, the present project is focused on the development of macromolecular aptamer inhibitors ideally targeting both binding sites of the HC domain. Aptamers are particularly attractive as potential antibody replacements due to their high binding specificities and affinities and improved accessibilities and stabilities. We have chosen the SELEX process to generate novel aptamers against the ganglioside binding peptide or protein reflecting the native membrane binding sites from the HC domain of BoNT/A. A single bead SELEX technique has been established successfully in the course of this project by using the 19mer ganglisoside binding peptide from the amino acid sequence at one of the essential membrane binding site (gangalioside binding site) from BoNT/A HC domain as the first target. Aptamers were found with about 0.8uM binding affinity to this peptide after 3 cycles of SELEX. After the peptide SELEX process has been successfully established, the ganglioside binding protein (gsbp), which is part of the HC domain containing both essential binding sites for BoNT/A membrane recognition was used as the final target. This protein contained the native, active conformation and was a particular challenge to identify the optimal conditions to maintain this conformation during the whole SELEX process. Therefore, the SELEX procedure was further modified and adjusted to this very sensitive protein which facilitated the identification of novel aptamers that were able to interact with the protein surface. Aptamer with 5uM binding affinity was successfully identified after 5 cycles of SELEX. These aptamers will be very promising candidates to interact with BoNT/A and they might serve as valuable tools to allow a first proof-of-concept study and assess the therapeutic potential of blocking BoNT/A cell uptake, which has not been explored yet. Therefore, such aptamers pave the way to therapeutic aptamers displaying higher binding affinities which might serve as an efficient treatment of botulism at the early stage. The ability of these aptamers to prevent BoNT/A uptake will be in the focus of future investigations.
URI: http://scholarbank.nus.edu.sg/handle/10635/19125
Appears in Collections:Master's Theses (Open)

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