Selection of RNA Aptamers Against Botulinum Neurotoxin Type A Light Chain Through a Non-Radioactive Approach.
Selection of RNA Aptamers Against Botulinum Neurotoxin Type A Light Chain Through a Non-Radioactive Approach.
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DOI：
10.1007/s12010-016-2081-0
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发表时间：
2016-09
影响因子：
3
通讯作者：
Cai, Shuowei
中科院分区：
文献类型：
--
作者：
Chang, Tzuu-Wang;Janardhanan, Pavithra;Mello, Charlene M.;Singh, Bal Ram;Cai, Shuowei
关键词：
肉毒神经毒素（BoNT）是A类毒素，是人类已知的毒性最强的分子. BoNT轻链结构域的内肽酶活性是抑制神经递质释放和导致肉毒中毒致死性的弛缓性麻痹的原因。目前，还没有解毒剂可以逆转BoNT引起的弛缓性麻痹。在本研究中，一个非放射性的基础上的SELEX过程，利用表面等离子体共振监测结合富集。两种RNA适体已被鉴定为针对A型肉毒杆菌神经毒素轻链的强结合剂。这两种适体对LCA表现出较强的抑制活性，IC 50在nM范围内。抑制动力学研究揭示了中纳摩尔KI和其抑制的非竞争性性质，表明它们具有作为解毒剂的强大潜力，可以逆转BoNT/A引起的症状。更重要的是，我们观察到，2′-氟嘧啶修饰的RNA适体在这里鉴定不改变其结合和生物活性。这一发现为利用指数富集进行配体系统进化（SELEX）提供了一种经济有效的方法，即在SELEX过程中使用常规核苷酸，并在最终应用时使用2′-氟嘧啶修饰的核苷酸来增强其RNA酶抗性。
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.
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