Biochemical and Thermodynamic Characterization of Compounds That Bind to RNA Hairpin Loops: Toward an Understanding of Selectivity

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Abstract
Elucidation of the molecular forces governing small molecule-RNA binding is paramount to the progress of rational design strategies. The extensive characterization of the aminoglycoside-16S rRNA A-site interaction has deepened our understanding of how aminoglycosides bind to their target and exert their antimicrobial effects. However, to date no other RNA binding compounds have undergone such rigorous evaluation, and in general the origins of small molecule-RNA binding remain a mystery. We recently reported the identification of small molecules, dimers of 2-deoxystreptamine, which are able to bind selectively to RNA tetraloops and octaloops, respectively [Thomas, Liu, and Hergenrother (2005) J. Am. Chem. Soc. 127, 12434-12435]. Described herein is the biochemical and biophysical characterization of the RNA binding properties of the most selective compound, B-12, as well as closely related analogues. These studies further substantiate that B-12 is indeed selective for RNA octaloop sequences and indicate that the origin of this selectivity may lie in B-12's unusual binding mode, in which entropic factors are major contributors to the overall binding energy. In fact, isothermal titration calorimetry (ITC) experiments indicate that the binding of B-12 and most of its analogues is associated with a strong entropic contribution to the total binding energy. This is in stark contrast to the aminoglycosides, for which favorable enthalpy typically provides the driving force for binding. These studies are the first to examine small molecule-RNA hairpin loop binding in detail and are a necessary step toward the design of compounds that are specific binders for a given RNA sequence.