title: 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 systematic evolution of ligands by exponential enrichment (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 nanomolar range. Inhibition kinetic studies reveal mid nanomolar KI and non-competitive nature of their inhibition, suggesting that they have strong potential as antidotes that can reverse the symptom caused by BoNT/A. More importantly, we observed that the 2'-fluorine-pyrimidine-modified RNA aptamers identified here do not change their binding and biological activities. This observation could lead to a cost-effective way for SELEX, by using regular nucleotide during SELEX, and 2'-fluorine-pyrimidine-modified nucleotide for final application to enhance their RNase-resistance.
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Rd, North Dartmouth, MA, 02747, USA.
authors.forename: Tzuu-Wang
authors.initials: TW
authors.lastname: Chang
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Rd, North Dartmouth, MA, 02747, USA.
authors.forename: Pavithra
authors.initials: P
authors.lastname: Janardhanan
authors.affiliation: US Army Natick Soldier Research, Development & Engineering Center, Natick, MA, 01760, USA.
authors.forename: Charlene M
authors.initials: CM
authors.lastname: Mello
authors.affiliation: Botulinum Research Center, Institute of Advanced Sciences, Dartmouth, MA, 02747, USA.
authors.forename: Bal Ram
authors.initials: BR
authors.lastname: Singh
authors.affiliation: Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Rd, North Dartmouth, MA, 02747, USA. scai@umassd.edu.
authors.forename: Shuowei
authors.initials: S
authors.lastname: Cai
mesh_terms: D052157:Aptamers, Nucleotide / Q000378:metabolism*; D001483:Base Sequence; D019274:Botulinum Toxins, Type A / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011743:Pyrimidines / Q000378:metabolism; D011851:Radioactivity; D052156:SELEX Aptamer Technique; D020349:Surface Plasmon Resonance
chemical_list: D052157:Aptamers, Nucleotide; D011743:Pyrimidines; D010450:Endopeptidases; D019274:Botulinum Toxins, Type A; C030986:pyrimidine
keywords: 2′-fluorine-pyrimidine; Antidote; Aptamer; Botulinum neurotoxin; Endopeptidase; RNA; Surface plasmon resonance