New concept for the development of anti-toxin substances based on aptamer technology : the establishment of a model system
Publish date: 2004-01-01
Report number: FOI-R--1514--SE
Pages: 44
Written in: English
Abstract
The aim of this study was to design a system for the selection of specific inhibitors of neurotoxins. The charybdotoxin (CTX) was used as a model toxin. The concept of the work consisted of two parts; the establishment of in vitro model systems for analysis of the toxin and target interactions, and the decelopment of a system for selection of inhibitory substances. The study describes the development of model systems for measuring the interaction between CTX and its targets in the form of either purified channel proteins or channel proteins inserted into cell membranes. Two recombinant channel proteins were constructed in the prokaryotic KcsA channel protein. A test system, an efflux assay using a recombinant cell line expressing the Kv1.3 channel protein which reflects the interaction of CTX to a membrane-integrated eukaryotic K+ channel, was evaluated. The rubidium efflux assay was demonstrated to be useful in analysing the inhibitory effect of CTX. A protocol for selection of potent toxin inhibitory molecules, aptamers, is also described in this study. The aptamers display high specificity and affinity for their target molecules and have properties that make them suitable for a variety of applications. the library designed for the current study consists of a randomised sequence of 60 nucleotides flanked by defined regions. This library was assessed to be a good basis for the successful selection of CTX-binding aptamers. A modified SELEX method for large-scale selection of aptamers was set up and optimised and it was demonstrated to be useful for the desired purpose.