In situ click chemistry on RNA

In templated or in situ reactions, bonds form between two fragments that are brought together in the context of a larger molecule such as a protein. We have written previously about dynamic combinatorial chemistry (DCC), which depends on reversible bond formation where the larger molecule shifts the equilibrium toward the linked fragments. For irreversible bond formation, the larger molecule effectively catalyzes the formation of an inhibitor (or at least a binder). In a recent Angew. Chem. Int. Ed. paper Jyotirmayee Dash and colleagues at the Indian Association for the Cultivation of Science describe an application of the latter that uses RNA as the template.

The target of interest was TAR RNA, a short region of viral RNA essential for HIV replication. We have previously highlighted a few examples of fragment screening against RNA (here, here, and here), including TAR, but most of the hits were weak.

The researchers used azide-alkyne cycloaddition, the quintessential click chemistry reaction. They built a small library of four alkynes (only one of which was fragment-sized) and 11 azides. All of these were incubated together (4 µM of each alkyne and 1 µM of each azide) in the presence of 5 µM biotin-labeled TAR RNA for 72 hours. (The reaction is typically slow at room temperatures unless catalyzed by metal ions.) Magnetic streptavidin-coated beads were then used to capture the RNA and any bound ligands, which were identified by HPLC-MS. Control experiments were run with TAR DNA or a mutant form of TAR RNA lacking an essential bulge. The result was one fairly potent compound (below) that was specific for TAR RNA, as well as a couple other molecules that were both weaker and less specific.

The affinity of compound 3ba for TAR RNA was measured by isothermal titration calorimetry (ITC) as well as by a fluorescence assay, which were in good agreement. Importantly, the ITC data suggested 1:1 binding, which is particularly important given that the ligand contains two 2-aminothiazoles, a moiety that has been called a PrAT for its promiscuous behavior. Finally, the ligand could displace the Tat peptide at low micromolar concentrations, suggesting that it is binding at the biologically relevant site of TAR.

I do have a few quibbles. It would have been interesting if the researchers had reported the affinity of the azide and alkyne themselves to see how much of a boost they got by linking. And since the most potent molecule is not always selected from target-guided synthesis, it would have been interesting to make and test other possible cycloaddition products to see if they missed anything useful.

Still, it is nice to see a submicromolar RNA binder come out of an in situ screen. Targeting RNA with small molecules has recently become trendy, and it will be fun to see how far approaches like these can go.