Selection of RNA Aptamers Against Botulinum Neurotoxin Type A Light Chain Through a Non-Radioactive Approach

Abstract

Botulinum neurotoxin (BoNT), a category A agent, 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, a non-radioactive based SELEX process is developed by utilizing surface plasmon resonance to monitor the binding enrichment. Two RNA aptamers have been identified as strong binders against light chain of botulinum neurotoxin type A. These two aptamers showed strong inhibition activity on LCA, with IC50 in nM range. Inhibition kinetic studies reveal mid nanomolar KI and non-competitive nature of their inhibition, suggesting they have strong potential as antidotes that can reverse the symptom caused by BoNT/A. More importantly, we observed that 2′-fluorine-pyrimidines modified RNA aptamers identified here do not change their binding and biological activities. This observation could lead to a cost-effective way for Systematic Evolution of Ligands by EXponential enrichment (SELEX), by using regular nucleotide during SELEX, and 2′-fluorine-pyrimidines modified nucleotide for final application to enhance their RNase-resistance.

Selection of DNA Aptamers Against Botulinum Neurotoxin E for Development of Fluorescent Aptasensor

“…Ever since the first aptamer‐based methodology was reported in 1990 by Tuerk and Gold, aptamers have been extensively identified and analyzed in various applications including target validation, diagnostics, and therapeutics . In an effort, various research groups have been specifically targeted for aptamers against BoNT type A (BoNTA) with low nanomolar binding affinities and published elsewhere . In addition, the identified aptamers also been successfully used to develop sensitive biosensors …”