Mark A. Boerneke

Functional conservation despite structural divergence in ligand-responsive RNA switches.

Significance RNA viruses, including the human pathogenic hepatitis C virus (HCV), use a structured untranslated region of their genome to hijack host cell ribosomes for the synthesis of viral proteins. These genome regions are termed internal ribosome entry site (IRES) elements and are encoded by distinct sequences in different viruses but share common functional RNA motifs. This study shows that viral IRES elements contain conformationally flexible RNA switches, whose state can be captured by the binding of a common ligand. Conformational switching plays a role in the function of the IRES elements. These new RNA switches are smaller than previously discovered “riboswitches” and may be the simplest form of ligand-responsive mechanical modules in nucleic acids. An internal ribosome entry site (IRES) initiates protein synthesis in RNA viruses, including the hepatitis C virus (HCV). We have discovered ligand-responsive conformational switches in viral IRES elements. Modular RNA motifs of greatly distinct sequence and local secondary structure have been found to serve as functionally conserved switches involved in viral IRES-driven translation and may be captured by identical cognate ligands. The RNA motifs described here constitute a new paradigm for ligand-captured switches that differ from metabolite-sensing riboswitches with regard to their small size, as well as the intrinsic stability and structural definition of the constitutive conformational states. These viral RNA modules represent the simplest form of ligand-responsive mechanical switches in nucleic acids.

Crystal-Structure-Guided Design of Self-Assembling RNA Nanotriangles.

RNA nanotechnology uses RNA structural motifs to build nanosized architectures that assemble through selective base-pair interactions. Herein, we report the crystal-structure-guided design of highly stable RNA nanotriangles that self-assemble cooperatively from short oligonucleotides. The crystal structure of an 81 nucleotide nanotriangle determined at 2.6 Å resolution reveals the so-far smallest circularly closed nanoobject made entirely of double-stranded RNA. The assembly of the nanotriangle architecture involved RNA corner motifs that were derived from ligand-responsive RNA switches, which offer the opportunity to control self-assembly and dissociation.

2-Aminobenzoxazole ligands of the hepatitis C virus internal ribosome entry site

2-Aminobenzoxazoles have been synthesized as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The compounds were designed to explore the less basic benzoxazole system as a replacement for the core scaffold in previously discovered benzimidazole viral translation inhibitors. Structure-activity relationships in the target binding of substituted benzoxazole ligands were investigated.

Aryl-substituted aminobenzimidazoles targeting the hepatitis C virus internal ribosome entry site.

We describe the exploration of N1-aryl-substituted benzimidazoles as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The design of the compounds was guided by the co-crystal structure of a benzimidazole viral translation inhibitor in complex with the RNA target. Structure-binding activity relationships of aryl-substituted benzimidazole ligands were established that were consistent with the crystal structure of the translation inhibitor complex.

Ligand-responsive RNA mechanical switches.

Ligand-responsive RNA mechanical switches represent a new class of simple switching modules that adopt well-defined ligand-free and bound conformational states, distinguishing them from metabolite-sensing riboswitches. Initially discovered in the internal ribosome entry site (IRES) of hepatitis C virus (HCV), these RNA switch motifs were found in the genome of diverse other viruses. Although large variations are seen in sequence and local secondary structure of the switches, their function in viral translation initiation that requires selective ligand recognition is conserved. We recently determined the crystal structure of an RNA switch from Seneca Valley virus (SVV) which is able to functionally replace the switch of HCV. The switches from both viruses recognize identical cognate ligands despite their sequence dissimilarity. Here, we describe the discovery of 7 new switches in addition to the previously established 5 examples. We highlight structural and functional features unique to this class of ligand-responsive RNA mechanical switches and discuss implications for therapeutic development and the construction of RNA nanostructures.

Structure of the Ribosomal RNA Decoding Site Containing a Selenium-Modified Responsive Fluorescent Ribonucleoside Probe

Abstract Comprehensive understanding of the structure–function relationship of RNA both in real time and at atomic level will have a profound impact in advancing our understanding of RNA functions in biology. Here, we describe the first example of a multifunctional nucleoside probe, containing a conformation‐sensitive fluorophore and an anomalous X‐ray diffraction label (5‐selenophene uracil), which enables the correlation of RNA conformation and recognition under equilibrium and in 3D. The probe incorporated into the bacterial ribosomal RNA decoding site, fluorescently reports antibiotic binding and provides diffraction information in determining the structure without distorting native RNA fold. Further, by comparing solution binding data and crystal structure, we gained insight on how the probe senses ligand‐induced conformational change in RNA. Taken together, our nucleoside probe represents a new class of biophysical tool that would complement available tools for functional RNA investigations.

Conformational flexibility of viral RNA switches studied by FRET.

The function of RNA switches involved in the regulation of transcription and translation relies on their ability to adopt different, structurally well-defined states. A new class of ligand-responsive RNA switches, which we recently discovered in positive strand RNA viruses, are distinct from conventional riboswitches. The viral switches undergo large conformational changes in response to ligand binding while retaining the same secondary structure in their free and ligand-bound forms. Here, we describe FRET experiments to study folding and ligand binding of the viral RNA switches. In addition to reviewing previous approaches involving RNA model constructs which were directly conjugated with fluorescent dyes, we outline the design and application of new modular constructs for FRET experiments, in which dye labeling is achieved by hybridization of a core RNA switch module with universal DNA fluorescent probes. As an example, folding and ligand binding of the RNA switch from the internal ribosome entry site of hepatitis C virus is studied comparatively with conventional and modular FRET constructs.

Design and Crystallography of Self-Assembling RNA Nanostructures

Biological RNA architectures are composed of autonomously folding modules which can be tailored as building blocks for the construction of RNA nanostructures. Designed base pair interactions allow complex nano-objects to self-assemble from simple RNA motifs. X-ray crystallography plays an important role in both the design and analysis of such RNA nanostructures. Here, we describe methods for the design and X-ray crystallographic structure analysis of an RNA square and two different triangles, which self-assemble from short oligonucleotides and serve as a platform for building functional nano-sized nucleic acid architectures.