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 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.

Figure 1

(a) Binding of aminoglycosides to the bacterial A-site impacts the placement and dynamics of the unpaired A1492 and 1493 residues. (b) By replacing one of the nucleosides in the A-site with an isosteric emissive nucleoside analogue as a donor (D) and tagging the antibiotics with an appropriate acceptor (A), binding and displacement events can be accurately monitored using FRET.

Figure 2

Structure of the donor 1 and acceptor 2, as well as 3 (the parent donor heterocycle) and 4 (an isomeric methoxy substituted heterocycle).

Figure 3

Normalized absorption (---) and emission (—) spectra of 1 (black) and 2 (red) in water.

Figure 4

Normalized absorption (---) and emission (—) spectra of 4 (black), 3 (red), and 1 (blue) in water.

Figure 5

Unmodified 9 and modified 10 A-site constructs.

Figure 6

(a) Example of binding study. 11 is titrated into 10. (b) Example of displacement study. Tobramycin is titrated into 10 saturated with 12. Conditions: 10 (1 × 10⁻⁶ M), cacodylate buffer pH 7.0 (2.0 × 10⁻² M), NaCl (1.0 × 10⁻¹ M).

Figure 7

Fractional fluorescence saturation of the donor (■) in the labeled A-site and the emissive acceptor (○) in the labeled aminoglycosides in the following experiments: (a) titration of 10 with 11; (b) titration of 10 with 12; (c) displacement of the A-site bound 11 with neomycin; (d) displacement of the A-site bound 12 with neomycin; (e) displacement of the A-site bound 11 with tobramycin; (f) displacement of the A-site bound 12 with tobramycin. Conditions: 10 (1.0 × 10⁻⁶ M), cacodylate buffer pH 7.0 (2.0 × 10⁻² M), NaCl (1.0 × 10⁻¹ M).

Figure 8

Aminoglycosides used for displacement studies.

Figure 9

A model of neomycin bound to the A-site (PDB 2ET4). The distance from the center of U1406 (green) to the primary 5′ hydroxymethyl group on the ribose (orange) is less than 10 Å.

Scheme 1

Synthesis of the Modified Nucleoside 5 and Its Phosphoramidite 8^{a a} Reagents: (a) NaOCN, NaOH, conc. HCl, water, 90%. (b) (i) N,O-bis(trimethylsilyl)acetamide, CF₃SO₃Si(CH₃)₃, β-D-ribofuranose 1-acetate 2,3,5-tribenzoate, CH₃CN; (ii) conc. NH₄OH, 81%. (c) DMTrCl, Et₃N, pyridine, 85%. (d) iPr₂NEt, nBu₂SnCl₂, (iPr₃SiO)CH₂Cl, ClCH₂CH₂Cl, 30%. (e) iPr₂NEt, (iPr₂N)P(Cl)O–CH₂CH₂CN, ClCH₂CH₂Cl, 60%.

Scheme 2

Structure of the Aminoglycosides Used for Binding and Displacement Studies (11 and 12) and the Synthesis of Coumarin-Labeled Neomycin 11^{a a} Reagents: (a) 7-(Et₂N)coumarin-3-carboxylic acid, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, 4-dimethylaminopyridine, iPr₂EtN, Cl₂CH₂, 84%. (b) Trifluoroacetic acid, triisopropylsilane, CH₂Cl₂ 82%.