In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine receptor.
In vitro selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine receptor.
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DOI：
10.1073/pnas.95.24.14051
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发表时间：
1998-11
影响因子：
11.1
通讯作者：
Henning Ulrich;J. Ippolito;O. Pagán;V. Eterović;R. Hann;Hua Shi;J. Lis;M. Eldefrawi;G. P. Hess
中科院分区：
文献类型：
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作者：
Henning Ulrich;J. Ippolito;O. Pagán;V. Eterović;R. Hann;Hua Shi;J. Lis;M. Eldefrawi;G. P. Hess
烟碱乙酰胆碱受体（AChR）控制神经系统细胞间的信号传递。滥用药物如可卡因会抑制这种受体。瞬态动力学研究表明，抑制剂降低了通道打开平衡常数[Hess， g.p. & Grewer， C. (1998) Methods enzymes . 291, 443-473]。是否可以找到与抑制剂竞争结合位点但不改变通道打开平衡的化合物？利用指数富集方法对RNA配体的系统进化和加利福尼亚鱼雷电鳗膜中的AChR进行了研究，以发现可以取代受体抑制剂的RNA。RNA配体的选择分两个连续的步骤进行：(i)凝胶转移选择与电传膜中AChR结合的高亲和力配体，以及（ii）随后使用硝化纤维素过滤器，膜结合受体和RNA都与之强烈结合，但从中所需的RNA可以被高亲和力的AChR抑制剂苯环利定从受体中置换出来。经过9轮筛选，分离出两类具有纳摩尔亲和力的与AChR结合的RNA分子并对其进行测序。这两类RNA分子都被苯环利定和可卡因从它们在AChR上的结合位点取代。通过使用全细胞电流记录技术确定，I类分子是BC3H1肌肉细胞中AChR活性的有效抑制剂。II类分子虽然与AChR抑制剂竞争，但在本试验中不影响受体活性；这种化合物或衍生物可能有助于减轻数百万吸毒者所经历的毒性。
The nicotinic acetylcholine receptor (AChR) controls signal transmission between cells in the nervous system. Abused drugs such as cocaine inhibit this receptor. Transient kinetic investigations indicate that inhibitors decrease the channel-opening equilibrium constant [Hess, G. P. & Grewer, C. (1998) Methods Enzymol. 291, 443-473]. Can compounds be found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of RNA ligands by exponential enrichment methodology and the AChR in Torpedo californica electroplax membranes were used to find RNAs that can displace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: (i) a gel-shift selection of high-affinity ligands bound to the AChR in the electroplax membrane, and (ii) subsequent use of nitrocellulose filters to which both the membrane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibitor, phencyclidine. After nine selection rounds, two classes of RNA molecules that bind to the AChR with nanomolar affinities were isolated and sequenced. Both classes of RNA molecules are displaced by phencyclidine and cocaine from their binding site on the AChR. Class I molecules are potent inhibitors of AChR activity in BC3H1 muscle cells, as determined by using the whole-cell current-recording technique. Class II molecules, although competing with AChR inhibitors, do not affect receptor activity in this assay; such compounds or derivatives may be useful for alleviating the toxicity experienced by millions of addicts.