Department of Chemistry, University of Massachusetts Amherst, Amherst, MA 01003, USA.
Org Biomol Chem. 2011 Oct 21;9(20):7151-61. doi: 10.1039/c1ob05963g. Epub 2011 Sep 6.
We have developed a minimally-perturbing strategy that enables labeling and subcellular visualization of endogenous dendritic receptors on live, wild-type neurons. Specifically, calcium-permeable non-NMDA glutamate receptors expressed in hippocampal neurons can be targeted with this novel synthetic tri-functional molecule. This ligand-directed probe was targeted towards AMPA receptors and bears an electrophilic group for covalent bond formation with an amino acid side chain on the extracellular side of the ion channel. This molecule was designed in such a way that the use-dependent, polyamine-based ligand accumulates the chemically-reactive group at the extracellular side of these polyamine-sensitive receptors, thereby allowing covalent bond formation between an electrophilic moiety on the nanoprobe and a nucleophilic amino acid sidechain on the receptor. Bioconjugation of this molecule results in a stable covalent bond between the nanoprobe and the target receptor. Subsequent photolysis of a portion of the nanoprobe may then be employed to effect ligand release allowing the receptor to re-enter the non-liganded state, all the while retaining the fluorescent beacon for visualization. This technology allows for rapid fluorescent labeling of native polyamine-sensitive receptors and further advances the field of fluorescent labeling of native biological molecules.
我们开发了一种微扰策略，可以对活的野生型神经元上的内源性树突状受体进行标记和亚细胞可视化。具体来说，在海马神经元中表达的钙通透性非 NMDA 谷氨酸受体可以用这种新型的合成三功能分子靶向。这种配体导向的探针针对 AMPA 受体，并带有一个亲电基团，用于与离子通道细胞外侧的氨基酸侧链形成共价键。该分子的设计方式是，基于聚胺的使用依赖性配体将反应性基团积聚在这些聚胺敏感受体的细胞外侧，从而允许纳米探针上的亲电部分与受体上的亲核氨基酸侧链之间形成共价键。该分子的生物共轭导致纳米探针和靶受体之间形成稳定的共价键。随后，部分纳米探针的光解可用于实现配体释放，使受体重新进入非配体结合状态，同时保留荧光信标进行可视化。这项技术允许对天然聚胺敏感受体进行快速荧光标记，并进一步推动了荧光标记天然生物分子的领域。
