Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0358, USA.
J Am Chem Soc. 2009 Dec 9;131(48):17605-14. doi: 10.1021/ja905767g.
A robust analysis and discovery platform for antibiotics targeting the bacterial rRNA A-site has been developed by incorporating a new emissive U surrogate into the RNA and labeling the aminoglycosides with an appropriate fluorescence acceptor. Specifically, a 5-methoxyquinazoline-2,4(1H,3H)-dione-based emissive uracil analogue was identified to be an ideal donor for 7-diethylaminocoumarin-3-carboxylic acid. This donor/acceptor pair displays a critical Forster radius (R(0)) of 27 A, a value suitable for an A-site-aminoglycoside assembly. Titrating the coumarin labeled aminoglycosides into the emissive A-site construct, labeled at position U1406, shows a decrease in donor emission (at 395 nm) and concurrent increase of the acceptor emission (at 473 nm). Titration curves, obtained by fitting the donor's emission quenching or the augmentation of the acceptor's sensitized emission, faithfully generate EC(50) values. Titration of unlabeled ligands into the preformed FRET complex showed a continuous increase of the donor emission, with a concurrent decrease of the acceptor emission, yielding valuable data regarding competitive displacement of aminoglycosides by A-site binders. Detection of antibiotic binding is therefore not dependent on changes in the environment of a single fluorophore, but rather on the responsive interaction between two chromophores acting as a FRET pair, facilitating the determination of direct binding and competitive displacement events with FRET accuracy.
已经开发出一种针对细菌 rRNA A 位的抗生素的强大分析和发现平台，该平台通过将新的发光 U 取代物引入 RNA 并使用适当的荧光受体标记氨基糖苷来实现。具体而言，确定了基于 5-甲氧基喹唑啉-2,4(1H,3H)-二酮的发光尿嘧啶类似物是 7-二乙氨基香豆素-3-羧酸的理想供体。该供体/受体对显示出关键的Förster 半径（R(0)）为 27 A，这是适合 A 位-氨基糖苷组装的值。将香豆素标记的氨基糖苷滴定到标记位置 U1406 的发光 A 位构建体中，显示出供体发射的降低（在 395nm 处）和受体发射的同时增加（在 473nm 处）。通过拟合供体发射的猝灭或受体敏化发射的增加获得的滴定曲线忠实地产生 EC(50)值。将未标记的配体滴定到预形成的 FRET 复合物中显示出供体发射的连续增加，同时受体发射的减少，从而提供有关 A 位结合物竞争性取代氨基糖苷的有价值数据。因此，抗生素结合的检测不依赖于单个荧光团环境的变化，而是依赖于作为 FRET 对的两个生色团之间的响应相互作用，从而促进了 FRET 准确性下的直接结合和竞争性置换事件的确定。
