Using pyrene-labeled HIV-1 TAR to measure RNA-small molecule binding.
To quantitatively understand the binding affinity and target selectivity of small-molecule RNA interactions, it is useful to have a rapid, highly reproducible binding assay that can be readily generalized to different RNA targets. To that end, an assay has been developed and validated for measuring the binding of low-molecular weight ligands to RNA by monitoring the fluorescence of a covalently incorporated fluorophore. As a test system, the fluorescence of a pyrene-derivatized HIV-1 TAR (transactivating response element) RNA was measured upon titration with aminoglycoside antibiotics. The binding isotherms thus obtained fit well with a model for a 1:1 interaction and yield an accurate measure of the equilibrium dissociation constant. Among a series of natural aminoglycosides, the binding affinity correlates with the number of amines, supporting an electrostatic compensation model for binding. Furthermore, the ionic strength dependence confirms that much of the binding energy is electrostatic. Finally, by measuring the binding affinity in the presence of nucleic acid competitors, we confirm that although aminoglycosides show high RNA to DNA selectivity, their selectivity among different RNA targets is sub- optimal. We conclude that this newly developed assay can be generalized to measure the binding affinities and selectivities of a variety of small molecules to a specific RNA target.
D000617:Aminoglycosides / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004722:Endoribonucleases / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D005601:Framycetin / Q000378:metabolism; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics; D008958:Models, Molecular; D009355:Neomycin / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D011721:Pyrenes / Q000737:chemistry*; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D055672:Static Electricity
D000617:Aminoglycosides; D005456:Fluorescent Dyes; D011721:Pyrenes; D005601:Framycetin; D012313:RNA; D004722:Endoribonucleases; C032825:ribonuclease V(1); D009355:Neomycin