Jérôme Mulhbacher

Novel riboswitch ligand analogs as selective inhibitors of guanine-related metabolic pathways.

Riboswitches are regulatory elements modulating gene expression in response to specific metabolite binding. It has been recently reported that riboswitch agonists may exhibit antimicrobial properties by binding to the riboswitch domain. Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 5′-untranslated mRNA structure that causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures. Using this, we have identified a pyrimidine compound (PC1) binding guanine riboswitches that shows bactericidal activity against a subgroup of bacterial species including well-known nosocomial pathogens. This selective bacterial killing is only achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a different metabolic pathway. As a proof of principle, we have used a mouse model to show a direct correlation between the administration of PC1 and the reduction of Staphylococcus aureus infection in mammary glands. This work establishes the possibility of using existing structural knowledge to design novel guanine riboswitch-targeting antibiotics as powerful and selective antimicrobial compounds. Particularly, the finding of this new guanine riboswitch target is crucial as community-acquired bacterial infections have recently started to emerge.

Therapeutic applications of ribozymes and riboswitches.

Therapeutic approaches employing RNA as a tool or as a drug target have recently emerged and have been employed for various applications-ranging from cancer treatment to virus infection. Despite the paucity of its molecular groups compared to proteins, RNA has nevertheless proved to be an excellent choice for researchers who have aspired to develop therapeutic tools. Ribozymes and riboswitches are RNA-based therapeutic tools that are most often employed to knockdown gene expression and to inhibit bacterial infections, respectively. The aim of this review is to summarize recent advances observed in ribozyme- and riboswitch-based therapeutic applications that, in some cases, have reached clinical trials.

Molecular insights into the ligand-controlled organization of the SAM-I riboswitch

S-adenosylmethionine (SAM) riboswitches are widespread in bacteria, and up to five different SAM riboswitch families have been reported, highlighting the relevance of SAM regulation. On the basis of crystallographic and biochemical data, it has been postulated, but never demonstrated, that ligand recognition by SAM riboswitches involves key conformational changes in the RNA architecture. We show here that the aptamer follows a two-step hierarchical folding selectively induced by metal ions and ligand binding, each of them leading to the formation of one of the two helical stacks observed in the crystal structure. Moreover, we find that the anti-antiterminator P1 stem is rotated along its helical axis upon ligand binding, a mechanistic feature that could be common to other riboswitches. We also show that the nonconserved P4 helical domain is used as an auxiliary element to enhance the ligand-binding affinity. This work provides the first comprehensive characterization, to our knowledge, of a ligand-controlled riboswitch folding pathway.

Riboswitches: Ancient and Promising Genetic Regulators

Bait and switch: Metabolite‐sensing riboswitches make use of RNA structural modulation to regulate gene expression, as illustrated in the scheme, in response to subtle changes in metabolite concentrations. This review describes the current knowledge about naturally occurring riboswitches and their growing potential as antibacterial cellular targets and as molecular biosensors.

Ligand recognition determinants of guanine riboswitches

Guanine riboswitches negatively modulate transcription upon guanine binding. The aptamer domain is organized around a three-way junction which forms the ligand binding site. Using currently available 89 guanine aptamer sequences, a consensus secondary structure is deduced and reveals differences from the previously identified aptamer consensus. Three positions are found to display different nucleotide requirements. Using a 2-aminopurine binding assay, we show that variations are allowed depending on the aptamer context. However, changes at position 48 markedly decrease ligand binding in a context-independent fashion. This is consistent with previous observations with the adenine riboswitch in which position 48 was proposed to interact with position 74, which normally base pairs with the ligand. The in vivo transcriptional control of endogenous Bacillus subtilis guanine riboswitches was studied using RT-qPCR assays. The ratio of elongated/terminated transcripts is decreased in presence of a high concentration of guanine but is dependent on the riboswitch analyzed. In general, the aptamer-2AP complex affinity correlates well with the in vivo regulation efficiency of the corresponding riboswitch. These studies suggest that core variations of guanine aptamers are used to produce a spectrum of ligand binding affinities which is used in vivo by host riboswitches to perform gene regulation.

Experimental treatment of Staphylococcus aureus bovine intramammary infection using a guanine riboswitch ligand analog

Staphylococcus aureus is a leading cause of intramammary infections (IMI). We recently demonstrated that Staph. aureus strains express the gene guaA during bovine IMI. This gene codes for a guanosine monophosphate synthetase and its expression is regulated by a guanine riboswitch. The guanine analog 2,5,6-triaminopyrimidine-4-one (PC1) is a ligand of the guanine riboswitch. Interactions between PC1 and its target result in inhibition of guanosine monophosphate synthesis and subsequent death of the bacterium. The present study describes the investigational use of PC1 for therapy of Staph. aureus IMI in lactating cows. The in vitro minimal inhibitory concentration of PC1 ranged from 0.5 to 4 μg/mL for a variety of Staph. aureus and Staphylococcus epidermidis strains and required a reducing agent for stability and full potency. A safety assessment study was performed, whereby the healthy quarters of 4 cows were infused with increasing doses of PC1 (0, 150, 250, and 500 mg). Over the 44 h following infusions, no obvious adverse effect was observed. Ten Holstein multiparous cows in mid lactation were then experimentally infused into 3 of the quarters with approximately 50 cfu of Staph. aureus strain SHY97-3906 and infection was allowed to progress for 2 wk before starting PC1 treatment. Bacterial counts reached then about 10(3) to 10(4) cfu/mL of milk. Infected quarters were treated with 1 of 3 doses of PC1 (0, 250, or 500 mg) after each morning and evening milking for 7d (i.e., 14 intramammary infusions of PC1). During the treatment period, milk from PC1-treated quarters showed a significant reduction in bacterial concentrations. However, this reduction of Staph. aureus count in milk was not maintained during the 4 wk following the end of the treatment and only 15% of the PC1-treated quarters underwent bacteriological cure. The somatic cell count and the quarter milk production were not affected by treatments. Although bacterial clearance was not achieved following treatment with PC1, these results demonstrate that the Staph. aureus guanine riboswitch represents a relevant and promising drug target for a novel class of antibiotics for the animal food industry.

Constitutive Regulatory Activity of an Evolutionarily Excluded Riboswitch Variant

The exquisite specificity of the adenine-responsive riboswitch toward its cognate metabolite has been shown to arise from the formation of a Watson-Crick interaction between the adenine ligand and residue U65. A recent crystal structure of a U65C adenine aptamer variant has provided a rationale for the phylogenetic conservation observed at position 39 for purine aptamers. The G39-C65 variant adopts a compact ligand-free structure in which G39 is accommodated by the ligand binding site and is base-paired to the cytosine at position 65. Here, we demonstrate using a combination of biochemical and biophysical techniques that the G39-C65 base pair not only severely impairs ligand binding but also disrupts the functioning of the riboswitch in vivo by constitutively activating gene expression. Folding studies using single-molecule FRET revealed that the G39-C65 variant displays a low level of dynamic heterogeneity, a feature reminiscent of ligand-bound wild-type complexes. A restricted conformational freedom together with an ability to significantly fold in monovalent ions are exclusive to the G39-C65 variant. This work provides a mechanistic framework to rationalize the evolutionary exclusion of certain nucleotide combinations in favor of sequences that preserve ligand binding and gene regulation functionalities.

Molecular Basis of RNA-Mediated Gene Regulation on the Adenine Riboswitch by Single-Molecule Approaches

The adenine-specific pbuE riboswitch undergoes metal ion-dependent folding that involves a long-range tertiary loop-loop interaction between two stem loops. Fluorescence resonance energy transfer (FRET) and single-molecule FRET studies demonstrate the ability of the loops to interact in the absence of the ligand. Although the riboswitch can fold in the absence of adenine, ligand binding stabilizes this folded conformation by increasing the folding and decreasing the unfolding rates of the riboswitch. The presence of the ligand also decreases the magnesium ion concentration required to promote the loop-loop interaction. Single-molecule FRET studies demonstrate that individual aptamer molecules exhibit great heterogeneity in the rates of folding and unfolding, which is reduced in the presence of adenine. Moreover, single-molecule FRET proposes that riboswitch folding proceeds through a complex landscape that involves a discrete intermediate.

Application of Fluorescent Measurements for Characterization of Riboswitch–Ligand Interactions

Riboswitches are recently discovered messenger RNA motifs involved in gene regulation. They modulate gene expression at various levels, such as transcription, translation, splicing, and mRNA degradation. Because riboswitches exhibit relatively complex structures, they are able to form highly complex ligand-binding sites, which enable the specific recognition of target metabolites in a complex cellular environment. Practically in all studied cases, riboswitches use ligand-induced conformational changes to control gene expression. To monitor the structural reorganization of riboswitches, we use the local fluorescent reporter 2-aminopurine (2AP), which is a structural analog of adenine. The 2AP fluorescence is strongly quenched when the fluorophore is involved in stacking interactions with surrounding bases, and can, therefore, be used to monitor local structural rearrangements. Here, we show specific examples in which 2AP fluorescence can be used to monitor structural changes in the aptamer domain of the S-adenosyl methionine (SAM) riboswitch and where it can be used as a ligand for the guanine riboswitch.

Natural Functional Nucleic Acids: Ribozymes and Riboswitches

Natural functional nucleic acids are of primary importance in most cellular processes. Although artificial RNA motifs with functional properties can routinely be generated in research laboratories, the efficiency of their naturally occurring counterparts is hardly matched. Natural ribozymes and riboswitches are examples of Natures prowess at creating exceedingly good catalysts and ligand-sensing aptamers. This review focuses on natural ribozymes and riboswitches and attempts to highlight how RNA can rival proteins when it comes to show off its capabilities.