Two-dimensional combinatorial screening identifies specific 6′-acylated kanamycin A- and 6′-acylated neamine-RNA hairpin interactions

Herein, we report the RNA hairpin loops from a six-nucleotide hairpin library that bind 6′-acylated kanamycin A (1) and 6′-acylated neamine (2) identified by two-dimensional combinatorial screening (2DCS). Hairpins selected to bind 1 have K_d's ranging from 235 to 1035 nM, with an average K_d of 618 nM. For 2, the selected hairpins bind with K_d's ranging from 135 to 2300 nM, with an average K_d of 1010 nM. The selected RNA hairpin-ligand interactions are also specific for the ligand that they were selected to bind compared with the other arrayed ligand. For example, the mixture of hairpins selected for 1 on average bind 33-fold more tightly to 1 than to 2, while the mixtures of hairpins selected for 2 on average bind 11-fold more tightly to 2 than to 1. Secondary structure prediction of the selected sequences was completed to determine the motifs that each ligand binds, and the hairpin loop preferences for 1 and 2 were computed. For 1, the preferred hairpin loops contain an adenine separated by at least two nucleotides from a cytosine, for example, ANNCNN (two-tailed p-value = 0.0010) and ANNNCN (two-tailed p-value <0.0001). For 2, the preferred hairpin loops contain both 5′GC and 5′CG steps (two-tailed p-value <0.0001). These results expand the information available on the RNA hairpin loops that bind small molecules and could prove useful for targeting RNA.