Potent and selective recognition and modulation of disease-relevant RNAs remains a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition, and showed that as few as one or two mutations can result in moderate affinity (low μM dissociation constant) for the human immunodeficiency virus (HIV) Trans-Activation Response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here we use yeast display and saturation mutagenesis of established RNA binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA binding selectivity; it binds TAR with sub-nanomolar affinity (apparent dissociation constant ~0.5 nM), but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5′-untranslated region, inhibits the interaction between TAR RNA and an HIV Trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents – small molecule, nucleic acid, or protein - reported to date, and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition—and modulation—of a disease-relevant RNA.

Split-GFP reassembly is an operationally simple in vivo technique used to identify and study interactions involving proteins and/or peptides. However, the instability of split-GFP fragments and their susceptibility to aggregation place limitations on the broader use of split-GFP reassembly. Supercharged proteins, including supercharged GFP, are variants with high theoretical negative or positive charge that are resistant to aggregation. We show that a split-superpositive GFP (split-spGFP) variant reassembles faster and more efficiently than previously reported split-sg100 GFP and split-folding-reporter GFP (split-frGFP) systems. In addition, interaction-dependent split-spGFP reassembly is efficient at physiological temperature. The increased efficiency and robustness of split-spGFP reassembly make this reporter system ideal for identifying and studying interactions involving proteins and/or peptides in vivo, and may be particularly useful for identifying or studying interactions involving proteins or peptides that are themselves susceptible to aggregation.

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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 efforts. However, very little is known about the molecular requirements for achieving sequence-selective recognition of a particular RNA sequence and structure. While a relatively modest number of synthetic small to medium-sized RNA-binding molecules have been reported, rapid identification of sequence-selective disease-relevant 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 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. Impressively, some of these U1A-derived proteins bind TAR with single-digit micromolar dissociation constants, and do so preferentially over the native protein's original target RNA (U1hpII) and a DNA TAR variant. For two TAR-binding proteins, 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

U1A binds U1hpII, a hairpin RNA with a 10-nucleotide loop. A U1A mutant (ΔK50ΔM51) binds U1hpII-derived hairpins with shorter loops, making it an interesting scaffold for engineering or evolving proteins that bind similarly sized disease-related hairpin RNAs. However, a more detailed understanding of complexes involving ΔK50ΔM51 is likely a prerequisite to generating such proteins. Toward this end, we measured mutational effects for complexes involving U1A ΔK50ΔM51 and U1hpII-derived hairpin RNAs with seven- or eight-nucleotide loops and identified contacts that are critical to the stabilization of these complexes. Our data provide valuable insight into sequence-selective recognition of seven- or eight-nucleotide loop hairpins by an engineered RNA binding protein.