期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 131, 期 48, 页码 17605-17614出版社
AMER CHEMICAL SOC
DOI: 10.1021/ja905767g
关键词
-
资金
- National Institutes of Health [GM 069773]
- National Science Foundation [CHE-9709183, CHE-0741968]
- Division Of Chemistry
- Direct For Mathematical & Physical Scien [0741968] Funding Source: National Science Foundation
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 angstrom, 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 donors emission quenching or the augmentation of the acceptors sensitized emission, faithfully generate EC50 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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据