Abstract
A multitude of RNA hairpins are directly implicated in human disease. Many of these RNAs are potentially valuable targets for drug discovery and basic research. However, very little is known about the molecular requirements for achieving sequence-selective recognition of a particular RNA sequence and structure. Although a relatively modest number of synthetic small to medium-sized RNA-binding molecules have been reported, rapid identification of sequence-selective RNA-binding molecules remains a daunting challenge. RNA recognition motif (RRM) domains may represent unique privileged scaffolds for the generation of synthetic proteins that selectively recognize structured disease-relevant RNAs, including RNA hairpins. As a demonstration of this potential, we mutated putative RNA-binding regions within the U1A RRM and a variant thereof and screened these synthetic proteins for affinity to HIV-1 trans-activation response (TAR) element hairpin RNA. Some of these U1A-derived proteins bind TAR with single-digit micromolar dissociation constants, and they do so preferentially over the native protein's original target RNA (U1hpII) and a DNA TAR variant. Binding affinity is not appreciably diminished by addition of 10 molar equivalents of cellular tRNAs from Escherichia coli. Taken together, our findings represent the first synthetic RRMs that selectively bind a disease-relevant RNA hairpin and may represent a general approach for achieving sequence-selective recognition of RNA hairpins, which are the focus of therapeutic discovery and basic research.