Systematically Studying the Effect of Small Molecules Interacting with RNA in Cellular and Preclinical Models.

The interrogation and manipulation of biological systems by small molecules is a powerful approach in chemical biology. Ideal compounds selectively engage a target and mediate a downstream phenotypic response. Although historically small molecule drug discovery has focused on proteins and enzymes, targeting RNA is an attractive therapeutic alternative, as many disease-causing or -associated RNAs have been identified through genome-wide association studies. As the field of RNA chemical biology emerges, the systematic evaluation of target validation and modulation of target-associated pathways is of paramount importance. In this Review, through an examination of case studies, we outline the experimental characterization, including methods and tools, to evaluate comprehensively the impact of small molecules that target RNA on cellular phenotype.

Characterization of synthetic riboswitch in cell-free protein expression systems.

Riboswitches are RNA-based regulatory elements that utilize ligand-induced structural changes in the 5'-untranslated region of mRNA to regulate the expression of associated genes. The majority of synthetic riboswitches have been selected and tested in cell-based systems. Cell-free protein expression systems (CFPS) have several advantages for the development and testing of synthetic riboswitches, including eliminating interactions with complex cellular networks, and the decoupling of transcription and translation processes. To gain a better understanding of the riboswitch regulatory mechanism, to allow for more efficient riboswitch optimization and use for biosensing applications, we studied the performance of a theophylline-responsive synthetic riboswitch coupled with the superfolder green fluorescent protein (sfGFP) reporter gene in E. coli cellular extract and PURE cell-free systems. To monitor the mRNA dynamics, a malachite green aptamer sequence was added to the 3'-untranslated region of sfGFP mRNA. Performance of the theophylline riboswitch was compared with a constitutively expressed sfGFP (control). Transcription dynamics of the riboswitch mRNA was very similar to the transcription of the control mRNA for all theophylline concentrations tested in both E. coli extract and PURE CFPS. However, sfGFP expression in the riboswitch construct was one order of magnitude lower, even at the highest concentration of theophylline. A mathematical model of riboswitch activation governed by the kinetic trapping mechanism was developed. Two factors - a reduced fraction of mRNA in the 'ON' state and a considerably lower translation initiation rate in the riboswitch - contribute to the much lower level of protein expression in the theophylline riboswitch compared to the control construct.

RSwitch: A Novel Bioinformatics Database on Riboswitches as Antibacterial Drug Targets.

The RSwitch is a MySQL database implemented on a PHP-based server, which also provides various useful tools for analyses of DNA, RNA, and protein sequences applying a user-friendly interface. The RSwitch database currently contains information and annotations of 215 bacterial riboswitches from 16 different types found in 50 human pathogenic bacteria. The riboswitch classes include those sensing FMN, glmS, Cobalamin, Lysine, SAH, SAM, Purine, TPP, c-di-GMPI, c-di-GMPII, Moco, PreQ1, Fluoride, Glycine, Mg2+, and Mn2+ type of bacterial riboswitches. The database provides information about the riboswitch aptamer sequences, the thermodynamic ensemble of the RNA structures on partition function and on free minimum energy function. Additionally, the database presents the centroid structure and the positional entropy for each position of the aptamer sequences. The database also provides the biochemical pathways in which the riboswitches are involved in, as well as multiple sequence alignments, multi-drug resistance bacterial strains and consensus motifs for each type of the switches. The RSwitch database is permanently available online without any restrictions. This bioinformatics database provides for the first time all information needed for assessing the suitability of the presented riboswitches as antibacterial drug targets.

RNA drug discovery: Conformational restriction enhances specific modulation of the T-box riboswitch function.

Antibacterial drug resistance is a global health concern that requires multiple solution approaches including development of new antibacterial compounds acting at novel targets. Targeting regulatory RNA is an emerging area of drug discovery. The T-box riboswitch is a regulatory RNA mechanism that controls gene expression in Gram-positive bacteria and is an exceptional, novel target for antibacterial drug design. We report the design, synthesis and activity of a series of conformationally restricted oxazolidinone-triazole compounds targeting the highly conserved antiterminator RNA element of the T-box riboswitch. Computational binding energies correlated with experimentally-derived Kd values indicating the predictive capabilities for docking studies within this series of compounds. The conformationally restricted compounds specifically inhibited T-box riboswitch function and not overall transcription. Complex disruption, computational docking and RNA binding specificity data indicate that inhibition may result from ligand binding to an allosteric site. These results highlight the importance of both ligand affinity and RNA conformational outcome for targeted RNA drug design.

A modular chromosomally integrated toolkit for ectopic gene expression in Vibrio cholerae.

The ability to express genes ectopically in bacteria is essential for diverse academic and industrial applications. Two major considerations when utilizing regulated promoter systems for ectopic gene expression are (1) the ability to titrate gene expression by addition of an exogenous inducer and (2) the leakiness of the promoter element in the absence of the inducer. Here, we describe a modular chromosomally integrated platform for ectopic gene expression in Vibrio cholerae. We compare the broadly used promoter elements Ptac and PBAD to versions that have an additional theophylline-responsive riboswitch (Ptac-riboswitch and PBAD-riboswitch). These constructs all exhibited unimodal titratable induction of gene expression, however, max induction varied with Ptac > PBAD > PBAD-riboswitch > Ptac-riboswitch. We also developed a sensitive reporter system to quantify promoter leakiness and show that leakiness for Ptac > Ptac-riboswitch > PBAD; while the newly developed PBAD-riboswitch exhibited no detectable leakiness. We demonstrate the utility of the tightly inducible PBAD-riboswitch construct using the dynamic activity of type IV competence pili in V. cholerae as a model system. The modular chromosomally integrated toolkit for ectopic gene expression described here should be valuable for the genetic study of V. cholerae and could be adapted for use in other species.

Riboswitch-Associated Guanidinium-Selective Efflux Pumps Frequently Transmitted on Proteobacterial Plasmids Increase Escherichia coli Biofilm Tolerance to Disinfectants.

Members of the small multidrug resistance (SMR) efflux pump family known as SugE (recently renamed Gdx) are known for their narrow substrate selectivity to small guanidinium (Gdm+) compounds and disinfectant quaternary ammonium compounds (QACs). Gdx members have been identified on multidrug resistance plasmids in Gram-negative bacilli, but their functional role remains unclear, as few have been characterized. Here, we conducted a survey of sequenced proteobacterial plasmids that encoded one or more SugE/Gdx sequences in an effort to (i) identify the most frequently represented Gdx member(s) on these plasmids and their sequence diversity, (ii) verify if Gdx sequences possess a Gdm+ riboswitch that regulates their translation similarly to chromosomally encoded Gdx members, and (iii) determine the antimicrobial susceptibility profile of the most predominate Gdx member to various QACs and antibiotics in Escherichia coli strains BW25113 and KAM32. The results of this study determined 14 unique SugE sequences, but only one Gdx sequence, annotated as "SugE(p)," predominated among the >140 plasmids we surveyed. Enterobacterales plasmids carrying sugE(p) possessed a guanidine II riboswitch similar to the upstream region of E. coligdx Cloning and expression of sugE(p), gdx, and emrE sequences into a low-copy-number expression vector (pMS119EH) revealed significant increases in QAC resistance to a limited range of detergent-like QACs only when gdx and sugE(p) transformants were grown as biofilms. These findings suggest that sugE(p) presence on proteobacterial plasmids may be driven by species that frequently encounter Gdm+ and QAC exposure.IMPORTANCE This study characterized the function of antimicrobial-resistant phenotypes attributed to plasmid-encoded guanidinium-selective small multidrug resistance (Gdm/SugE) efflux pumps. These sequences are frequently monitored as biocide resistance markers in antimicrobial resistance surveillance studies. Our findings reveal that enterobacterial gdm sequences transmitted on plasmids possess a guanidine II riboswitch, which restricts transcript translation in the presence of guanidinium. Cloning and overexpression of this gdm sequence revealed that it confers higher resistance to quaternary ammonium compound (QAC) disinfectants (which possess guanidium moieties) when grown as biofilms. Since biofilms are commonly eradicated with QAC-containing compounds, the presence of this gene on plasmids and its biofilm-specific resistance are a growing concern for clinical and food safety prevention measures.

RNA Drugs and RNA Targets for Small Molecules: Principles, Progress, and Challenges.

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.

Do the P1 and P2 hairpins of the Guanidine-II riboswitch interact?

Riboswitches regulate genes by adopting different structures in responds to metabolite binding. The guanidine-II riboswitch is the smallest representative of the ykkC class with the mechanism of its function being centred on the idea that its two stem loops P1 and P2 form a kissing hairpin interaction upon binding of guanidinium (Gdm+). This mechanism is based on in-line probing experiments with the full-length riboswitch and crystal structures of the truncated stem loops P1 and P2. However, the crystal structures reveal only the formation of the homodimers P1 | P1 and P2 | P2 but not of the proposed heterodimer P1 | P2. Here, site-directed spin labeling (SDSL) in combination with Pulsed Electron-Electron Double Resonance (PELDOR or DEER) is used to study their structures in solution and how they change upon binding of Gdm+. It is found that both hairpins adopt different structures in solution and that binding of Gdm+ does indeed lead to the formation of the heterodimer but alongside the homodimers in a statistical 1:2:1 fashion. These results do thus support the proposed switching mechanism.

Parallel Discovery Strategies Provide a Basis for Riboswitch Ligand Design.

Riboswitches are mRNA domains that make gene-regulatory decisions upon binding their cognate ligands. Bacterial riboswitches that specifically recognize 5-aminoimidazole-4-carboxamide riboside 5'-monophosphate (ZMP) and 5'-triphosphate (ZTP) regulate genes involved in folate and purine metabolism. Now, we have developed synthetic ligands targeting ZTP riboswitches by replacing the sugar-phosphate moiety of ZMP with various functional groups, including simple heterocycles. Despite losing hydrogen bonds from ZMP, these analogs bind ZTP riboswitches with similar affinities as the natural ligand, and activate transcription more strongly than ZMP in vitro. The most active ligand stimulates gene expression ∼3 times more than ZMP in a live Escherichia coli reporter. Co-crystal structures of the Fusobacterium ulcerans ZTP riboswitch bound to synthetic ligands suggest stacking of their pyridine moieties on a conserved RNA nucleobase primarily determines their higher activity. Altogether, these findings guide future design of improved riboswitch activators and yield insights into how RNA-targeted ligand discovery may proceed.

Gram-Negative Antibiotic Active Through Inhibition of an Essential Riboswitch.

Multidrug-resistant Gram-negative (GN) infections for which there are few available treatment options are increasingly common. The development of new antibiotics for these pathogens is challenging because of the inability of most small molecules to accumulate inside GN bacteria. Using recently developed predictive guidelines for compound accumulation in Escherichia coli, we have converted the antibiotic Ribocil C, which targets the flavin mononucleotide (FMN) riboswitch, from a compound lacking whole-cell activity against wild-type GN pathogens into a compound that accumulates to a high level in E. coli, is effective against Gram-negative clinical isolates, and has efficacy in mouse models of GN infections. This compound allows for the first assessment of the translational potential of FMN riboswitch binders against wild-type Gram-negative bacteria.

Conscious uncoupling of riboswitch functions.

Riboswitches alter gene expression in response to ligand binding, coupling sensing and regulatory functions to help bacteria respond to their environment. The structural determinants of ligand binding in the prequeuosine (7-aminomethyl-7-deazaguanine, preQ1) bacterial riboswitches have been studied, but the functional consequences of structural perturbations are less known. A new article combining biophysical and cell-based readouts of 15 mutants of the preQ1-II riboswitch from Lactobacillus rhamnosus demonstrates that ligand binding does not ensure successful gene regulation, providing new insights into these shapeshifting sequences.

High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells.

Synthetic riboswitches mediating ligand-dependent RNA cleavage or splicing-modulation represent elegant tools to control gene expression in various applications, including next-generation gene therapy. However, due to the limited understanding of context-dependent structure-function relationships, the identification of functional riboswitches requires large-scale-screening of aptamer-effector-domain designs, which is hampered by the lack of suitable cellular high-throughput methods. Here we describe a fast and broadly applicable method to functionally screen complex riboswitch libraries (~1.8 × 104 constructs) by cDNA-amplicon-sequencing in transiently transfected and stimulated human cells. The self-barcoding nature of each construct enables quantification of differential mRNA levels without additional pre-selection or cDNA-manipulation steps. We apply this method to engineer tetracycline- and guanine-responsive ON- and OFF-switches based on hammerhead, hepatitis-delta-virus and Twister ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices. In summary, our method enables fast and efficient high-throughput riboswitch identification, thereby overcoming a major hurdle in the development cascade for therapeutically applicable gene switches.

A Theophylline-Responsive Riboswitch Regulates Expression of Nuclear-Encoded Genes.

Riboswitches are small cis-regulatory RNA elements that regulate gene expression by conformational changes in response to ligand binding. Synthetic riboswitches have been engineered as versatile and innovative tools for gene regulation by external application of their ligand in prokaryotes and eukaryotes. In plants, synthetic riboswitches were used to regulate gene expression in plastids, but the application of synthetic riboswitches for the regulation of nuclear-encoded genes in planta remains to be explored. Here, we characterize the properties of a theophylline-responsive synthetic aptazyme for control of nuclear-encoded transgenes in Arabidopsis (Arabidopsis thaliana). Activation of the aptazyme, inserted in the 3' UTR of the target gene, resulted in rapid self-cleavage and subsequent decay of the mRNA. This riboswitch allowed reversible, theophylline-dependent down-regulation of the GFP reporter gene in a dose- and time-dependent manner. Insertion of the riboswitch into the ONE HELIX PROTEIN1 gene allowed complementation of ohp1 mutants and induction of the mutant phenotype by theophylline. GFP and ONE HELIX PROTEIN1 transcript levels were downregulated by up to 90%, and GFP protein levels by 95%. These results establish artificial riboswitches as tools for externally controlled gene expression in synthetic biology in plants or functional crop design.

Protein unties the pseudoknot: S1-mediated unfolding of RNA higher order structure.

Ribosomal protein S1 plays important roles in the translation initiation step of many Escherichia coli mRNAs, particularly those with weak Shine-Dalgarno sequences or structured 5' UTRs, in addition to a variety of cellular processes beyond the ribosome. In all cases, the RNA-binding activity of S1 is a central feature of its function. While sequence determinants of S1 affinity and many elements of the interactions of S1 with simple secondary structures are known, mechanistic details of the protein's interactions with RNAs of more complex secondary and tertiary structure are less understood. Here, we investigate the interaction of S1 with the well-characterized H-type pseudoknot of a class-I translational preQ1 riboswitch as a highly structured RNA model whose conformation and structural dynamics can be tuned by the addition of ligands of varying binding affinity, particularly preQ1, guanine, and 2,6-diaminopurine. Combining biochemical and single molecule fluorescence approaches, we show that S1 preferentially interacts with the less folded form of the pseudoknot and promotes a dynamic, partially unfolded conformation. The ability of S1 to unfold the RNA is inversely correlated with the structural stability of the pseudoknot. These mechanistic insights delineate the scope and limitations of S1-chaperoned unfolding of structured RNAs.

Riboswitch distribution, structure, and function in bacteria.

Riboswitches are gene control elements that directly bind to specific ligands to regulate gene expression without the need for proteins. They are found in all three domains of life, including Bacteria, Archaea, and Eukaryota. Riboswitches are mostly spread in bacteria and archaea. In this paper, we discuss the general distribution, structure, and function of 28 different riboswitch classes as we focus our attention on riboswitches in bacteria. Bacterial riboswitches regulate gene expression by four distinct mechanisms. They regulate the expression of a limited number of genes. However, most of these genes are responsible for the synthesis of essential metabolites without which the cell cannot function. Therefore, riboswitch distribution is also important for antibacterial drug development.

Theophylline-inducible riboswitch accurately regulates protein expression at low level in Escherichia coli.

OBJECTIVES: Fine-tuning of enzyme expression at low levels is an important challenge for metabolic engineers. Here, theophylline-inducible riboswitch for translational regulation was evaluated. The background expression, translation rate, and time delay for its induction was reported. RESULTS: To evaluate the effect of the amount of mRNA on its translation rate, transcription of the riboswitch RNA with red fluorescent protein (RFP) was controlled by the lac system with addition of isopropyl ß-D-1-thiogalactopyranoside in Escherichia coli. Regardless of the amount of riboswitch mRNA, the translation of RFP was completely suppressed without theophylline during both growth and stationary phases. Furthermore, a strong positive correlation between theophylline concentration (0 to 1 mM) and specific RFP production rate was observed. The specific RFP production rate with the riboswitch was approximately 2.3% of that without the riboswitch. Furthermore, 60 min of time delay for RFP expression was observed after adding theophylline during the stationary phase. CONCLUSION: Theophylline-inducible riboswitch precisely controls protein translation at low expression levels with significantly low background expression. It can emerge as a powerful tool for fine tuning of enzyme expression.

Visualizing RNA Conformational Changes via Pattern Recognition of RNA by Small Molecules.

Conformational changes in RNA play vital roles in the regulation of many biological systems, yet these changes can be challenging to visualize. Previously, we demonstrated that Pattern Recognition of RNA by Small Molecules (PRRSM) can unbiasedly cluster defined RNA secondary structure motifs utilizing an aminoglycoside receptor library. In this work, we demonstrate the power of this method to visualize changes in folding at the secondary structure level within two distinct riboswitch structures. After labeling at three independent positions on each riboswitch, PRRSM accurately classified all apo and ligand-bound riboswitch structures, including changes in the size of a structural motif, and revealed modification sites that prevented folding and/or led to a mixture of states. These data underscore the utility and robustness of the PRRSM assay for rapid assessment of RNA structural changes and for gaining ready insight into nucleotide positions critical to RNA folding.

Magnesium controls aptamer-expression platform switching in the SAM-I riboswitch.

Investigations of most riboswitches remain confined to the ligand-binding aptamer domain. However, during the riboswitch mediated transcription regulation process, the aptamer domain and the expression platform compete for a shared strand. If the expression platform dominates, an anti-terminator helix is formed, and the transcription process is active (ON state). When the aptamer dominates, transcription is terminated (OFF state). Here, we use an expression platform switching experimental assay and structure-based electrostatic simulations to investigate this ON-OFF transition of the full length SAM-I riboswitch and its magnesium concentration dependence. Interestingly, we find the ratio of the OFF population to the ON population to vary non-monotonically as magnesium concentration increases. Upon addition of magnesium, the aptamer domain pre-organizes, populating the OFF state, but only up to an intermediate magnesium concentration level. Higher magnesium concentration preferentially stabilizes the anti-terminator helix, populating the ON state, relatively destabilizing the OFF state. Magnesium mediated aptamer-expression platform domain closure explains this relative destabilization of the OFF state at higher magnesium concentration. Our study reveals the functional potential of magnesium in controlling transcription of its downstream genes and underscores the importance of a narrow concentration regime near the physiological magnesium concentration ranges, striking a balance between the OFF and ON states in bacterial gene regulation.

Molecular mechanisms for dynamic regulation of N1 riboswitch by aminoglycosides.

A synthetic riboswitch N1, inserted into the 5'-untranslated mRNA region of yeast, regulates gene expression upon binding ribostamycin and neomycin. Interestingly, a similar aminoglycoside, paromomycin, differing from neomycin by only one substituent (amino versus hydroxyl), also binds to the N1 riboswitch, but without affecting gene expression, despite NMR evidence that the N1 riboswitch binds all aminoglycosides in a similar way. Here, to explore the details of structural dynamics of the aminoglycoside-N1 riboswitch complexes, we applied all-atom molecular dynamics (MD) and temperature replica-exchange MD simulations in explicit solvent. Indeed, we found that ribostamycin and neomycin affect riboswitch dynamics similarly but paromomycin allows for more flexibility because its complex lacks the contact between the distinctive 6' hydroxyl group and the G9 phosphate. Instead, a transient hydrogen bond of 6'-OH with A17 is formed, which partially diminishes interactions between the bulge and apical loop of the riboswitch, likely contributing to riboswitch inactivity. In many ways, the paromomycin complex mimics the conformations, interactions, and Na+ distribution of the free riboswitch. The MD-derived interaction network helps understand why riboswitch activity depends on aminoglycoside type, whereas for another aminoglycoside-binding site, aminoacyl-tRNA site in 16S rRNA, activity is not discriminatory.

Canonical translation-modulating OFF-riboswitches with a single aptamer binding to a small molecule that function in a higher eukaryotic cell-free expression system.

We have found that OFF-riboswitches that ligand-dependently downregulate the canonical translation in a higher eukaryotic expression system (wheat germ extract) can be easily created by inserting a single aptamer into the 5' untranslated region (UTR) of mRNA, even if its ligand is as small as theophylline. The key is the position of the inserted aptamer: the 5' end (+0 position) is much better than other positions for inhibiting canonical translation with the aptamer-ligand complex. The data showed that ribosome loading is suppressed by a rigid structure in the 5' end, and this suppression is dependent on the structure's stability but not on its size. Although this preference of aptamer insertion point contradicts the results in a lower eukaryote, it accords with the fact that the 5'-end structural hindrance is more effective for blocking the ribosome in higher eukaryotes. Therefore, the present type of OFF-riboswitch would function in various higher eukaryotic expression systems.