Quantitative affinity electrophoresis of RNA-small molecule interactions by cross-linking the ligand to acrylamide.

We show that the affinity electrophoresis analysis of RNA-small molecule interactions can be made quantifiable by cross-linking the ligand to the gel matrix. Using an RNA-aminoglycoside model system to verify our method, we attached an acryloyl chloride molecule to the aminoglycosides paromomycin and neomycin B to synthesize an acrylamide-aminoglycoside monomer. This molecule was then used as a component in gel polymerization for affinity electrophoresis, covalently attaching an aminoglycoside molecule to the gel matrix. To test RNA binding to the cross-linked aminoglycosides, we used the aminoglycoside binding RNA molecule derived from thymidylate synthase messenger RNA (mRNA) that contains a C-C mismatch. Binding is indicated by the difference in RNA mobility between gels with cross-linked ligand, with ligand embedded during polymerization, and with no ligand present. Critically, the predicted straight line relationship between the reciprocal of the relative migration of the RNA and the ligand concentration is obtained when using cross-linked aminoglycosides, whereas a straight line is not obtained using embedded aminoglycosides. Average apparent dissociation constants are determined from the slope of the line from these plots. This method allows an easy quantitative comparison between different nucleic acid molecules for a small molecule ligand.

Engineering a structure switching mechanism into a steroid-binding aptamer and hydrodynamic analysis of the ligand binding mechanism.

The steroid binding mechanism of a DNA aptamer was studied using isothermal titration calorimetry (ITC), NMR spectroscopy, quasi-elastic light scattering (QELS), and small-angle X-ray spectroscopy (SAXS). Binding affinity determination of a series of steroid-binding aptamers derived from a parent cocaine-binding aptamer demonstrates that substituting a GA base pair with a GC base pair governs the switch in binding specificity from cocaine to the steroid deoxycholic acid (DCA). Binding of DCA to all aptamers is an enthalpically driven process with an unfavorable binding entropy. We engineered into the steroid-binding aptamer a ligand-induced folding mechanism by shortening the terminal stem by two base pairs. NMR methods were used to demonstrate that there is a transition from a state where base pairs are formed in one stem of the free aptamer, to where three stems are formed in the DCA-bound aptamer. The ability to generate a ligand-induced folding mechanism into a DNA aptamer architecture based on the three-way junction of the cocaine-binding aptamer opens the door to obtaining a series of aptamers all with ligand-induced folding mechanisms but triggered by different ligands. Hydrodynamic data from diffusion NMR spectroscopy, QELS, and SAXS show that for the aptamer with the full-length terminal stem there is a small amount of structure compaction with DCA binding. For ligand binding by the short terminal stem aptamer, we propose a binding mechanism where secondary structure forms upon DCA binding starting from a free structure where the aptamer exists in a compact form.

Identification of RNA-ligand interactions by affinity electrophoresis.

We have developed an affinity electrophoresis method to screen for RNA-ligand interactions. Native polyacrylamide gels were polymerized in the absence and presence of different RNA binding molecules. Binding is indicated by a difference in mobility between the gel with ligand present and the gel with no ligand present. The utility of this method was demonstrated using the known interaction between the Escherichia coli ribosomal A-site RNA and different aminoglycoside ligands. The RNA-aminoglycoside interaction observed is dose dependent, and the affinity mirrors what is observed in solution. In addition, we used this method to gauge the affinity to different aminoglycoside molecules of an RNA molecule derived from the thymidylate synthase mRNA construct that contains a CC mismatch.

Finding the path in an RNA folding landscape.

In this issue of Structure, Reymond et al. (2010) combine molecular and computational biology approaches to provide structural details for intermediates in the folding pathway of the hepatitis delta virus ribozyme.