In vitro selection of RNA aptamers that inhibit the activity of type A botulinum neurotoxin

Abstract

The category A agent, botulinum neurotoxin (BoNT), is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, we have identified three RNA aptamers through SELEX process, which bind strongly to the light chain of type A BoNT (BoNT/A) and inhibit the endopeptidase activity, with IC 50 in low nM range. Inhibition kinetic studies reveal low nM K I and non-competitive nature of their inhibition. Aptamers are unique group of molecules as therapeutics, and this is first report of their development as an antidote against botulism. These data on K I and IC 50 strongly suggest that the aptamers have strong potential as antidotes that can reverse the symptom caused by BoNT/A. KeywordsRNA; aptamer; botulinum neurotoxin; antidote; endopeptidase Botulinum neurotoxins, produced by Clostridia botulinum, are the most toxic substances known to mankind, the cause of the deadly disease, botulism, and can serve as a bioweapon [1,2]. They are also being used as medical therapeutics, treating an array of neuromuscular diseases, and are used for cosmetic applications [3]. Currently, there are seven known serotypes of BoNT, designated as A to G. Of those, type A, B, E are the most common cause for human botulism, and type A is the most potent among the seven serotypes.BoNTs are 150 kDa proteins comprised of three functional domains: the light chains (LCs) of neurotoxins are zinc-endopeptidase, which collectively cleave several proteins involved in synaptic vesicle docking and fusion, and therefore, block the release of acetylcholine. The * Correspondence should be addressed to: Shuowei Cai Department of Chemistry and Biochemistry University of Massachusetts Dartmouth 285 Old Westport Rd North Dartmouth, MA 02747 Phone: 508-999-8807 Fax: 508-999-8451 scai@umassd.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. heavy chain (HC) plays an accessory role of binding to the target nerve cells (through its Cterminus) and the translocation of the LC into the cell cytoplasm (through its N-terminus) [1,3]. NIH Public AccessCurrent therapy for botulism involves respiratory supportive care and the administration of antitoxin. However, to date there is no treatment that can reverse the flaccid muscle paralysis caused by BoNT after the symptom has developed [2][3][4]. Because the toxin has widespread medical therapeutic use...

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