Guanine Riboswitch Research Articles

Exploring the Lower Limit of Target Concentration in Capture-SELEX Using Guanine as a Model Target.

During an aptamer selection, using a lower target concentration may result in aptamers with a higher binding affinity. Consequently, this begs the question of whether there is a lower limit for target concentration. In this work, we conducted three aptamer selections using 5 µM, 500 nM and 50 nM guanine as the targets, respectively. Successful enrichment of the same guanine aptamers was achieved at both 5 µM and 500 nM guanine, but not with 50 nM. Using 5 µM guanine, the aptamer was enriched in eight rounds of selection, compared to that for 500 nM, which was accomplished in 17 rounds. We discuss the relation of optimal target concentration to the observed Kd value of the resulting aptamers, of which the highest affinity aptamer had a measured Kd of 200 nM. Additionally, we investigated the binding of the aptamers through mutation studies, revealing a critical cytosine. Mutating this cytosine to a thymine switched the selectivity from guanine to adenine, which is reminiscent of the guanine riboswitch. This study revealed a limit in using low target concentration, and the insights described in this article will be useful for guiding the choice of target concentration during capture-SELEX.

Rationally Designed Pyrimidine Compounds: Promising Novel Antibiotics for the Treatment of Staphylococcus aureus-Associated Bovine Mastitis.

Staphylococcus aureus is one of the major pathogens causing bovine mastitis, and antibiotic treatment is most often inefficient due to its virulence and antibiotic-resistance attributes. The development of new antibiotics for veterinary use should account for the One Health concept, in which humans, animals, and environmental wellbeing are all interconnected. S. aureus can infect cattle and humans alike and antibiotic resistance can impact both if the same classes of antibiotics are used. New effective antibiotic classes against S. aureus are thus needed in dairy farms. We previously described PC1 as a novel antibiotic, which binds the S. aureus guanine riboswitch and interrupts transcription of essential GMP synthesis genes. However, chemical instability of PC1 hindered its development, evaluation, and commercialization. Novel PC1 analogs with improved stability have now been rationally designed and synthesized, and their in vitro and in vivo activities have been evaluated. One of these novel compounds, PC206, remains stable in solution and demonstrates specific narrow-spectrum activity against S. aureus. It is active against biofilm-embedded S. aureus, its cytotoxicity profile is adequate, and in vivo tests in mice and cows show that it is effective and well tolerated. PC206 and structural analogs represent a promising new antibiotic class to treat S. aureus-induced bovine mastitis.

Exploiting natural riboswitches for aptamer engineering and validation.

Over the past three decades, researchers have found that some engineered aptamers can be made to work well in test tubes but that these same aptamers might fail to function in cells. To help address this problem, we developed the 'Graftamer' approach, an experimental platform that exploits the architecture of a natural riboswitch to enhance in vitro aptamer selection and accelerate in vivo testing. Starting with combinatorial RNA pools that contain structural features of a guanine riboswitch aptamer interspersed with regions of random sequence, we performed multiplexed in vitro selection with a collection of small molecules. This effort yielded aptamers for quinine, guanine, and caffeine that appear to maintain structural features of the natural guanine riboswitch aptamer. Quinine and caffeine aptamers were each grafted onto a natural guanine riboswitch expression platform and reporter gene expression was monitored to determine that these aptamers function in cells. Additionally, we determined the secondary structure features and survival mechanism of a class of RNA sequences that evade the intended selection strategy, providing insight into improving this approach for future efforts. These results demonstrate that the Graftamer strategy described herein represents a convenient and straightforward approach to develop aptamers and validate their in vivo function.

Variants of the guanine riboswitch class exhibit altered ligand specificities for xanthine, guanine, or 2′-deoxyguanosine

The aptamer portions of previously reported riboswitch classes that sense guanine, adenine, or 2′-deoxyguanosine are formed by a highly similar three-stem junction with distinct nucleotide sequences in the regions joining the stems. The nucleotides in these joining regions form the major features of the selective ligand-binding pocket for each aptamer. Previously, we reported the existence of additional, rare variants of the predominant guanine-sensing riboswitch class that carry nucleotide differences in the ligand-binding pocket, suggesting that these RNAs have further diversified their structures and functions. Herein, we report the discovery and analysis of three naturally occurring variants of guanine riboswitches that are narrowly distributed across Firmicutes. These RNAs were identified using comparative sequence analysis methods, which also revealed that some of the gene associations for these variants are atypical for guanine riboswitches or their previously known natural variants. Binding assays demonstrate that the newfound variant riboswitch representatives recognize xanthine, guanine, or 2′-deoxyguanosine, with the guanine class exhibiting greater discrimination against related purines than the more common guanine riboswitch class reported previously. These three additional variant classes, together with the four previously discovered riboswitch classes that employ the same three-stem junction architecture, reveal how a simple structural framework can be diversified to expand the range of purine-based ligands sensed by RNA.

In Vitro Selection of RNAs That Can Detect Hypoxanthine: A Surprising Technical Issue

The goal of this project is to find RNA molecules that detect hypoxanthine with great sensitivity and specificity. Hypoxanthine is the product of a family of RNA editing enzymes called ADARs. A sensor that can detect hypoxanthine will facilitate the study of the regulation of ADAR activity. There are naturally occurring guanine sensors called guanine riboswitches. We are using in vitroselection to find variants of a guanine riboswitch that can detect hypoxanthine rather than guanine. While performing the selection experiment, we encountered a technical problem. Instead of isolating hypoxanthine sensors, a group of related aberrant RNAs dominated the selected RNA pool. These RNAs had many sequence changes and deletions compared to the sequence of the guanine riboswitch, and they could not detect hypoxanthine. The aberrant RNAs were produced not by the selection process, but as a consequence of multiple rounds of amplification. Here we report our attempts to understand why this happened and to prevent the formation of the aberrant RNAs.

Structure and mechanism of the methyltransferase ribozyme MTR1.

RNA-catalysed RNA methylation was recently shown to be part of the catalytic repertoire of ribozymes. The methyltransferase ribozyme MTR1 catalyses the site-specific synthesis of 1-methyladenosine (m1A) in RNA, using O6-methylguanine (m6G) as methyl group donor. Here we report the crystal structure of MTR1 at a resolution of 2.8 Å, which reveals a guanine binding site reminiscent of natural guanine riboswitches. The structure represents the postcatalytic state of a split ribozyme in complex with the m1A-containing RNA product and the demethylated cofactor guanine. The structural data suggest the mechanistic involvement of a protonated cytidine in the methyl transfer reaction. A synergistic effect of two 2'-O-methylated ribose residues in the active site results in accelerated methyl group transfer. Supported by these results, it seems plausible that modified nucleotides may have enhanced early RNA catalysis and that metabolite-binding riboswitches may resemble inactivated ribozymes that have lost their catalytic activity during evolution.

An in silico study of the strand invasion mechanism of the yxjA guanine riboswitch

The yxjA guanine riboswitch is a structural element located in the 5'-untranslated region of messenger RNA that controls gene expression by regulating transcription. In the absence of guanine, the downstream expression platform undergoes strand invasion and forms an antiterminator, allowing transcription to continue. In the presence of guanine, the downstream expression platform forms a terminator, ending transcription. The molecular basis for the binding of guanine to the aptamer domain is well established.

Inactivation of the riboswitch-controlled GMP synthase GuaA in Clostridioides difficile is associated with severe growth defects and poor infectivity in a mouse model of infection

ABSTRACT Clostridioides difficile is the main cause of nosocomial antibiotic-associated diarrhoea. There is a need for new antimicrobials to tackle this pathogen. Guanine riboswitches have been proposed as promising new antimicrobial targets, but experimental evidence of their importance in C. difficile is missing. The genome of C. difficile encodes four distinct guanine riboswitches, each controlling a single gene involved in purine metabolism and transport. One of them controls the expression of guaA, encoding a guanosine monophosphate (GMP) synthase. Here, using in-line probing and GusA reporter assays, we show that these riboswitches are functional in C. difficile and cause premature transcription termination upon binding of guanine. All riboswitches exhibit a high affinity for guanine characterized by Kd values in the low nanomolar range. Xanthine and guanosine also bind the guanine riboswitches, although with less affinity. Inactivating the GMP synthase (guaA) in C. difficile strain 630 led to cell death in minimal growth conditions, but not in rich medium. Importantly, the capacity of a guaA mutant to colonize the mouse gut was significantly reduced. Together, these results demonstrate the importance of de novo GMP biosynthesis in C. difficile during infection, suggesting that targeting guanine riboswitches with analogues could be a viable therapeutic strategy.

Riboswitches as Drug Targets for Antibiotics.

Riboswitches reside in the untranslated region of RNA and regulate genes involved in the biosynthesis of essential metabolites through binding of small molecules. Since their discovery at the beginning of this century, riboswitches have been regarded as potential antibacterial targets. Using fragment screening, high-throughput screening and rational ligand design guided by X-ray crystallography, lead compounds against various riboswitches have been identified. Here, we review the current status and suitability of the thiamine pyrophosphate (TPP), flavin mononucleotide (FMN), glmS, guanine, and other riboswitches as antibacterial targets and discuss them in a biological context. Further, we highlight challenges in riboswitch drug discovery and emphasis the need to develop riboswitch specific high-throughput screening methods.

High Affinity Binding of N2-Modified Guanine Derivatives Significantly Disrupts the Ligand Binding Pocket of the Guanine Riboswitch

Riboswitches are important model systems for the development of approaches to search for RNA-targeting therapeutics. A principal challenge in finding compounds that target riboswitches is that the effector ligand is typically almost completely encapsulated by the RNA, which severely limits the chemical space that can be explored. Efforts to find compounds that bind the guanine/adenine class of riboswitches with a high affinity have in part focused on purines modified at the C6 and C2 positions. These studies have revealed compounds that have low to sub-micromolar affinity and, in a few cases, have antimicrobial activity. To further understand how these compounds interact with the guanine riboswitch, we have performed an integrated structural and functional analysis of representative guanine derivatives with modifications at the C8, C6 and C2 positions. Our data indicate that while modifications of guanine at the C6 position are generally unfavorable, modifications at the C8 and C2 positions yield compounds that rival guanine with respect to binding affinity. Surprisingly, C2-modified guanines such as N2-acetylguanine completely disrupt a key Watson–Crick pairing interaction between the ligand and RNA. These compounds, which also modulate transcriptional termination as efficiently as guanine, open up a significant new chemical space of guanine modifications in the search for antimicrobial agents that target purine riboswitches.

Ligand Binding Mechanism and Its Relationship with Conformational Changes in Adenine Riboswitch.

Riboswitches are naturally occurring RNA aptamers that control the expression of essential bacterial genes by binding to specific small molecules. The binding with both high affinity and specificity induces conformational changes. Thus, riboswitches were proposed as a possible molecular target for developing antibiotics and chemical tools. The adenine riboswitch can bind not only to purine analogues but also to pyrimidine analogues. Here, long molecular dynamics (MD) simulations and molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) computational methodologies were carried out to show the differences in the binding model and the conformational changes upon five ligands binding. The binding free energies of the guanine riboswitch aptamer with C74U mutation complexes were compared to the binding free energies of the adenine riboswitch (AR) aptamer complexes. The calculated results are in agreement with the experimental data. The differences for the same ligand binding to two different aptamers are related to the electrostatic contribution. Binding dynamical analysis suggests a flexible binding pocket for the pyrimidine ligand in comparison with the purine ligand. The 18 μs of MD simulations in total indicate that both ligand-unbound and ligand-bound aptamers transfer their conformation between open and closed states. The ligand binding obviously affects the conformational change. The conformational states of the aptamer are associated with the distance between the mass center of two key nucleotides (U51 and A52) and the mass center of the other two key nucleotides (C74 and C75). The results suggest that the dynamical character of the binding pocket would affect its biofunction. To design new ligands of the adenine riboswitch, it is recommended to consider the binding affinities of the ligand and the conformational change of the ligand binding pocket.

Graphene biosensor as affinity biosensors for biorecognition between Guanine riboswitch and ligand

Guanine riboswitch (GR) is located in the untranslated region of mRNA that performs important biological functions by interactions with specific ligand molecules. The present standard methods for detecting mRNA has been widely used, but suffer from extra labeling steps, high cost, low throughput, low sensitivities and limited dynamic ranges. The field effect transistor (FET) biosensors showed high performance in detecting a large number of biological analytes. However, the detection of analytes with low charge and small molecule still remains a challenge. In this paper, a label-free graphene field effect transistor (G-FET) biosensor has been developed to detect the binding interactions between GR and four purine analog ligand molecules (GUA, 6GU, 2BP, XAN). We demonstrated that the G-FET biosensor were highly sensitive in detecting the electric-neutral and low molecular weight ligands by using GR as probe. The sensing mechanisms for electric-neutral molecule is revealed by molecular dynamics simulation. The equilibrium dissociation constants KD and binding free energy ΔG of GR-GUA, GR-6GU, GR-2BP and GR-XAN were obtained by the G-FET biosensor, and the results are comparable with the calculated by molecular dynamics simulation and previous reports. We demonstrated that the G-FET biosensor can be used as affinity biosensors to quantify biomolecular interactions.

Effect of mutations on binding of ligands to guanine riboswitch probed by free energy perturbation and molecular dynamics simulations

Riboswitches can regulate gene expression by direct and specific interactions with ligands and have recently attracted interest as potential drug targets for antibacterial. In this work, molecular dynamics (MD) simulations, free energy perturbation (FEP) and molecular mechanics generalized Born surface area (MM-GBSA) methods were integrated to probe the effect of mutations on the binding of ligands to guanine riboswitch (GR). The results not only show that binding free energies predicted by FEP and MM-GBSA obtain an excellent correlation, but also indicate that mutations involved in the current study can strengthen the binding affinity of ligands GR. Residue-based free energy decomposition was applied to compute ligand-nucleotide interactions and the results suggest that mutations highly affect interactions of ligands with key nucleotides U22, U51 and C74. Dynamics analyses based on MD trajectories indicate that mutations not only regulate the structural flexibility but also change the internal motion modes of GR, especially for the structures J12, J23 and J31, which implies that the aptamer domain activity of GR is extremely plastic and thus readily tunable by nucleotide mutations. This study is expected to provide useful molecular basis and dynamics information for the understanding of the function of GR and possibility as potential drug targets for antibacterial.

Changing the Ligand Specificity of a Guanine Riboswitch by In Vitro Selection

Riboswitches are RNA elements in messenger RNAs that regulate gene expression by undergoing a ligand‐triggered conformational change. Riboswitches bind tightly and specifically to their ligands, so they have the potential to serve as highly effective sensors in vitro. We previously modified several purine riboswitches for use in vitro and, indeed, they could detect their ligands with high sensitivity and specificity (1). To extend the utility of our sensor design, we developed an in vitro selection strategy to isolate variants of riboswitches that have altered ligand specificity. We are attempting to find variants of the guanine riboswitch that can detect hypoxanthine rather than guanine. This is a challenging task because hypoxanthine is identical to guanine except for the absence of an exocyclic amino group. We partially randomized the sequence of our guanine sensor, and used hypoxanthine as the ligand during selection. Our first experiment produced only the original, wild‐type guanine riboswitch. In hindsight, this was not surprising since the guanine riboswitch can detect hypoxanthine (albeit poorly), and it was the most abundant RNA in our initial partially‐randomized RNA pool. To solve this problem we designed a new experiment based on the C74U mutant of the guanine riboswitch. C74 forms a Watson‐Crick base pair with the bound guanine ligand. Changing C74 to U results in a riboswitch that cannot bind to guanine or hypoxanthine but can bind weakly to adenine (2). We performed a second selection in which we partially randomized the sequence of the C74U mutant, except every variant retained a U at position 74. Since the C74U mutant cannot bind to hypoxanthine, we reasoned that we could select variants that could do so by allowing hypoxanthine to form a “wobble” pair with U74. We also anticipated that we could isolate hypoxanthine sensors that discriminate against guanine due to steric clashes between the sensor and the exocyclic amino group of guanine. After 11 rounds of selection, the selected RNA pool could no longer detect adenine but could detect hypoxanthine and guanine. The sensitivity for hypoxanthine and guanine was much lower than that of the wild‐type guanine riboswitch. However, the selectivity for hypoxanthine versus guanine was slightly improved, suggesting that the pool may contain individual variants that can discriminate against guanine. We are currently determining the sequence of a few variants in the selected pool to identify RNAs that we would like to test individually for their ability to specifically detect hypoxanthine.Support or Funding InformationDefense Threat Reduction Agency [MIPR HDTRA1620511 to D.M.] Office of Naval Research Chemistry Department of the U.S. Naval AcademyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Using Rosetta for RNA homology modeling.

The three-dimensional structures of RNA molecules provide rich and often critical information for understanding their functions, including how they recognize small molecule and protein partners. Computational modeling of RNA 3D structure is becoming increasingly accurate, particularly with the availability of growing numbers of template structures already solved experimentally and the development of sequence alignment and 3D modeling tools to take advantage of this database. For several recent "RNA puzzle" blind modeling challenges, we have successfully identified useful template structures and achieved accurate structure predictions through homology modeling tools developed in the Rosetta software suite. We describe our semi-automated methodology here and walk through two illustrative examples: an adenine riboswitch aptamer, modeled from a template guanine riboswitch structure, and a SAM I/IV riboswitch aptamer, modeled from a template SAM I riboswitch structure.

Variant Bacterial Riboswitches Associated with Nucleotide Hydrolase Genes Sense Nucleoside Diphosphates.

The ykkC RNA motif was a long-standing orphan riboswitch candidate that has recently been proposed to encompass at least five distinct bacterial riboswitch classes. Most ykkC RNAs belong to the subtype 1 group, which are guanidine-I riboswitches that regulate the expression of guanidine-specific carboxylase and transporter proteins. The remaining ykkC RNAs have been organized into at least four major categories called subtypes 2a-2d. Subtype 2a RNAs are riboswitches that sense the bacterial alarmone ppGpp and typically regulate amino acid biosynthesis genes. Subtype 2b riboswitches sense the purine biosynthetic intermediate PRPP and frequently partner with guanine riboswitches to regulate purine biosynthesis genes. In this study, we examined ykkC subtype 2c RNAs, which are found upstream of genes encoding hydrolase enzymes that cleave the phosphoanhydride linkages of nucleotide substrates. Subtype 2c representatives mostly recognize adenosine and cytidine 5'-diphosphate molecules in either their ribose or deoxyribose forms (ADP, dADP, CDP, and dCDP). Other nucleotide-containing compounds, especially nucleoside 5'-triphosphates, are strongly rejected by some members of this putative riboswitch class. High ligand concentrations in vivo are predicted to turn on expression of hydrolase enzymes, which presumably function to balance cellular nucleotide pools. These results further showcase the striking functional diversity derived from the structural scaffold shared among all ykkC motif RNAs, which has been adapted to sense at least five different types of natural ligands. Moreover, riboswitches for nucleoside diphosphates provide additional examples of the numerous partnerships observed between natural RNA aptamers and nucleotide-derived ligands, including metabolites, coenzymes, and signaling molecules.

Changing the Ligand Specificity of a Riboswitch from Guanine to Hypoxanthine

Riboswitches are RNA elements in messenger RNAs that regulate gene expression by undergoing a ligand‐triggered conformational change. Previously, we modified several riboswitches for use as sensitive and specific sensors for their ligands. To extend the utility of our sensor design, we developed an in vitro selection strategy to isolate variants of riboswitches that have altered ligand specificity. We initially tested our strategy by attempting to change the specificity of a guanine riboswitch from guanine to hypoxanthine. Our first selection experiment produced only the original, wild‐type guanine riboswitch. In hindsight, this was not surprising since the guanine riboswitch can detect hypoxanthine (albeit poorly), and it was the most abundant RNA in our initial partially‐randomized RNA pool. For our second experiment, we reduced the abundance of the wild‐type riboswitch by increasing the degree of sequence randomization. This experiment resulted in isolating RNAs with many sequence changes and deletions that failed to detect hypoxanthine. We reasoned that these aberrant RNAs arose during PCR with Taq DNA polymerase. Here, we describe the results of a new selection experiment in which we used a proofreading DNA polymerase and a redesigned partially‐randomized RNA pool.Support or Funding InformationDefense Threat Reduction Agency (DTRA)Office of Naval Research (ONR)Chemistry Department, United States Naval AcademyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sensitive and specific detection of ligands using engineered riboswitches

Riboswitches are RNA elements found in non-coding regions of messenger RNAs that regulate gene expression through a ligand-triggered conformational change. Riboswitches typically bind tightly and specifically to their ligands, so they have the potential to serve as highly effective sensors in vitro. In B. subtilis and other gram-positive bacteria, purine nucleotide synthesis is regulated by riboswitches that bind to guanine. We modified the xpt-pbuX guanine riboswitch for use in a fluorescence quenching assay that allowed us to specifically detect and quantify guanine in vitro. Using this assay, we reproducibly detected as little as 5 nM guanine. We then produced sensors for 2′-deoxyguanosine and cyclic diguanylate (c-diGMP) by appending the P1 stem of the guanine riboswitch to the ligand-binding domains of a 2′-deoxyguanosine riboswitch and a c-diGMP riboswitch. These hybrid sensors could detect 15 nM 2′-deoxyguanosine and 3 nM c-diGMP, respectively. Each sensor retained the ligand specificity of its corresponding natural riboswitch. In order to extend the utility of our approach, we developed a strategy for the in vitro selection of sensors with novel ligand specificity. Here we report a proof-of-principle experiment that demonstrated the feasibility of our selection strategy.

Folding behaviors of purine riboswitch aptamers

Most riboswitches are characterized by two components, an aptamer domain that folds into a unique ligand binding pocket to interact with the ligand, and an expression platform that converts folding changes in the aptamer into changes in gene expression. Using the recently developed systematic helix-based computational method, we theoretically studied the refolding and co-transcriptional folding behaviors of the purine riboswitch aptamers from Bacillus subtilis xpt-pbuX guanine riboswitch and Vibrio vulnificus add adenine riboswitch. Despite several intermediate structures persisting a short time during the transcription, helices P2, P3 and P1 fold in turn for both aptamers. Although some misfolded structures are observed during the refolding process, the RNAs can fold into the ligand binding pocket structure containing helices P2, P3 and P1 within a few seconds, suggesting the aptamer domains are highly evolved. The purine riboswitch aptamers can quickly fold into the ligand binding pocket structure even at a high transcription speed, possibly because formation of this structure is the necessary prerequisite for the riboswitch to bind its ligand and then regulate relevant gene expression.

Purine analogs targeting the guanine riboswitch as potential antibiotics against Clostridioides difficile

Riboswitches recently emerged as possible targets for the development of alternative antimicrobial approaches. Guanine-sensing riboswitches in the bacterial pathogen Clostridioides difficile (formerly known as Clostridium difficile) constitute potential targets based on their involvement in the regulation of basal metabolic control of purine compounds. In this study, we deciphered the structure-activity relationship of several guanine derivatives on the guanine riboswitch and determined their antimicrobial activity. We describe the synthesis of purine analogs modified in ring B as well as positions 2 and 6. Their biological activity was determined by measuring their affinity for the C. difficile guanine riboswitch and their inhibitory effect on bacterial growth, including a counter-screen to discriminate against riboswitch-independent antibacterial effects. Altogether, our results suggest that improvements in riboswitch binding affinity in vitro do not necessarily translate into improved antibacterial activity in bacteria, despite the fact that some structure-activity relationship was observed at least with respect to binding affinity.