Departments of Ophthalmology and Visual Sciences, Bioengineering, Neurology and Rehabilitation, University of Illinois at Chicago, 1855 W. Taylor St., Chicago, IL 60612, USA.
Bioconjug Chem. 2010 Aug 18;21(8):1455-64. doi: 10.1021/bc100050s.
Highly fluorescent CdSe quantum dots (qdots) can serve as a platform for tethering multiple copies of a receptor-targeted ligand, affording study of how the level of multivalency affects receptor binding. We previously showed that qdots conjugated with long PEG chains terminated by muscimol, a known GABA(C) agonist, exhibit specific binding to the surface membrane of GABA(C) receptor-expressing Xenopus oocytes. The present report addresses the effect of varying the number, i.e., valency, of muscimol- (M-) terminated PEG chains attached to the qdot on binding of the resulting conjugate to GABA(C) receptors. M-PEG-qdots of differing muscimol valency were prepared by conjugating AMP-CdSe/ZnS qdots with muscimol-terminated and methylamine-terminated PEG chains in proportions designed to yield varying percentages of muscimol-terminated chains among the total approximately 150-200 chains bound to the qdot. The investigated valencies represented 0%, approximately 25%, approximately 50%, and 100% loading with muscimol (preparations termed M-PEG-qdot0, M-PEG-qdot25, M-PEG-qdot50, and M-PEG-qdot100, respectively. Binding of a given conjugate to surface membranes of GABA(C) receptor-expressing oocytes was analyzed by quantitative fluorescence microscopy following defined incubation with approximately 30 nM of the conjugate. With 5-20 min incubation, the fluorescence signal resulting from incubation with M-PEG-qdot25 exceeded, by approximately 6-fold, the fluorescence level obtained with M-PEG-qdot preparations that lacked muscimol-terminated chains (M-PEG-qdot0). M-PEG-qdot50 yielded a net signal roughly similar to that of M-PEG-qdot25, and that produced by M-PEG-qdot100 exceeded, by approximately 30-50%, those for M-PEG-qdot25 and M-PEG-qdot50. The time course of changes in oocyte surface membrane fluorescence resulting from the introduction of and removal of M-PEG-qdots in the medium bathing the oocyte indicated only a modest dependence of both binding and wash-out kinetics on muscimol valency. The results demonstrate a dependence of the binding activity of the M-PEG-qdot conjugates on muscimol valency, presumably reflecting higher GABA(C) avidity and/or affinity of the muscimol at high valency, and provide insight on the interactions of membrane receptor proteins with qdot conjugates containing multiple copies of a receptor-targeting ligand.
高度荧光的 CdSe 量子点 (qdots) 可以作为连接多个受体靶向配体的平台，从而研究多价效应对受体结合的影响。我们之前已经表明，连接有长 PEG 链的 qdots，PEG 链的末端是毒蕈碱，一种已知的 GABA(C) 激动剂，能够特异性地与表达 GABA(C) 受体的非洲爪蟾卵母细胞膜结合。本报告探讨了连接到 qdot 上的毒蕈碱 (M-) 终止 PEG 链的数量，即价数，对所得缀合物与 GABA(C) 受体结合的影响。通过将 AMP-CdSe/ZnS qdots 与毒蕈碱终止和甲胺终止的 PEG 链以设计的比例进行缀合，制备了具有不同毒蕈碱价数的 M-PEG-qdots，使得连接到 qdot 上的总约 150-200 个链中，毒蕈碱终止链的百分比各不相同。所研究的价数分别代表 0%、约 25%、约 50%和 100%的毒蕈碱负载（分别称为 M-PEG-qdot0、M-PEG-qdot25、M-PEG-qdot50 和 M-PEG-qdot100）。通过用约 30 nM 的缀合物进行定义的孵育后，通过定量荧光显微镜分析给定缀合物与表达 GABA(C) 受体的卵母细胞膜的结合。在 5-20 分钟的孵育过程中，与缺乏毒蕈碱终止链的 M-PEG-qdot 制剂（M-PEG-qdot0）相比，M-PEG-qdot25 孵育产生的荧光信号增加了约 6 倍。M-PEG-qdot50 产生的净信号大致与 M-PEG-qdot25 相似，而 M-PEG-qdot100 产生的净信号比 M-PEG-qdot25 和 M-PEG-qdot50 高约 30-50%。从中介液中引入和去除 M-PEG-qdots 引起卵母细胞膜荧光变化的时间过程表明，结合和冲洗动力学仅对毒蕈碱价数有适度的依赖性。结果表明，M-PEG-qdot 缀合物的结合活性依赖于毒蕈碱价数，这可能反映了高价毒蕈碱的更高 GABA(C) 亲合力和/或亲和力，并为研究含有多个受体靶向配体的膜受体蛋白与 qdot 缀合物的相互作用提供了深入了解。
