Using Selection to Identify and Chemical Microarray to Study the RNA Internal Loops Recognized by 6′‐N‐Acylated Kanamycin A

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Herein, we describe our initial steps towards identifying the RNA secondary structure motifs that are recognized by small molecules. We selected members of an RNA 3x3 internal loop motif library that bind kanamycin A, an RNA-binding aminoglycoside antibiotic, by using only one round of selection. A small internal-loop library was chosen because members are likely to be present in other larger, biologically relevant RNAs. We have identified several internal loops of various size and base composition that kanamycin A prefers to bind. The highest affinity structures are two 5'-UU/3'-CU 2x2 internal loops closed by AU pairs. Binding is specific for the selected internal loops with the highest affinities, since binding to the RNA cassette used to display the library or to DNA is >150-fold weaker. Enzymatic mapping experiments also confirm binding of kanamycin A to the internal loops. This method lays the foundation for finding RNA secondary structure elements that bind small molecules and for interrogating factors affecting RNA-ligand interactions. Information from these and subsequent studies will: 1) facilitate the rational and modular design of drugs or probes that bind target RNAs with high affinity, provided the secondary structure of the target is known and 2) give insight into the potential bystander RNAs that aminoglycosides bind.