FRET Enabled Real Time Detection of RNA-Small Molecule Binding

Abstract

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 Förster radius (R0) of 27 Å, 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 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.

A fluorescence-based screen for ribosome binding antibiotics

“…Previous probes have been incorporated into the bases of a model A-site, allowing changes in the fluorescence to be measured as changes to the RNA occur on drug binding [13,41,42]. In other techniques, fluorescence resonance energy transfer (FRET) analysis is used by labeling the RNA with a donor molecule and labeling an aminoglycoside with an acceptor dye [43,44]. Although these techniques give greater insight into the conformational changes that occur in the A-site on drug binding, these techniques require the specific labeling of the RNA bases for the detection of binding.…”

Studies of RNA Sequence and Structure Using Nanopores

“…These measurements are consistent with previous observations made using an independent FRET-based assay, showing that at 0.5 M NaCl, neomycin has a much higher affinity to the A-site construct than paromomycin, which has a much higher affinity than kanamycin. 37 …”

Stereoselective N-Glycosylation of 2-Deoxythioribosides for Fluorescent Nucleoside Synthesis

“…Following literature procedures, 5-methoxyquinazolin-2,4-(1H,3H)-dione 52 p-Tolyl-3,5-di-O-acetyl-1-thio-2-deoxy-α,β-D-ribofuranoside (2). To a stirring solution of 1,3,5-tri-O-acetyl-2-deoxy-α,β-D-ribose 1 (30.34 g, 116.58 mmol) at −78°C in CH 2 Cl 2 (420 mL) was added pTolSH (14.77 g, 118.91 mmol).…”