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 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.

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.

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.

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.

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.

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. Newly discovered metabolite-sensing riboswitches have revealed that cellular processes extensively make use of RNA structural modulation to regulate gene expression in response to subtle changes in metabolite concentrations. Riboswitches are involved at various regulation levels of gene expression, such as transcription attenuation, translation initiation, mRNA splicing and mRNA processing. Riboswitches are found in the three kingdoms of life, and in various cases, are involved in the regulation of essential genes, which makes their regulation an essential part of cell survival. Because riboswitches operate without the assistance of accessory proteins, they are believed to be remnants of an ancient time, when gene regulation was strictly based on RNA, from which are left numerous "living molecular fossils", as exemplified by ribozymes, and more spectacularly, by the ribosome. Due to their nature, riboswitches hold high expectations for the manipulation of gene expression and the detection of small metabolites, and also offer an unprecedented potential for the discovery of novel classes of antimicrobial agents.

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.

Carboxymethyl high amylose starch for F4 fimbriae gastro-resistant oral formulation.

The carboxymethyl high amylose starch (CM-HAS) was proposed as excipient able to protect F4 fimbriae oral vaccine against gastric acidity and pepsin, allowing its subsequent liberation in the intestinal fluid. Thus, F4 fimbriae formulated with CM-HAS as tablets displayed a markedly higher stability after 2h of incubation in simulated gastric fluid (containing pepsin) than the free, non-protected F4 fimbriae, which, in these conditions, were almost completely digested after 120 min. In the presence of pancreatin (with alpha-amylase, lipase and proteolytic activities) in simulated intestinal conditions, the F4 fimbriae were liberated from CM-HAS tablets over a period of up to 5 h. The presence of pancreatin in intestinal medium did not affect the structural stability of the F4 fimbriae major subunits. Thus, F4 fimbriae formulated with CM-HAS would retain their receptor binding activity essential for the induction of an intestinal mucosal immune response.

Mucoadhesive properties of cross-linked high amylose starch derivatives.

Acetate (Ac-), aminoethyl (AE-) and carboxymethyl (CM-)derivatives of cross-linked high amylose starch (HASCL-6) were previously shown to control, over more than 20h, the release of drugs from highly loaded (up to 60% drug) monolithic tablets. It was now of interest to evaluate their mucoadhesive characteristics in view of further utilization in buccal or vaginal transmucosal delivery. The present study shows that ionic AE-HASCL-6 and CM-HASCL-6 derivatives exhibit higher mucoadhesive properties than neutral HASCL-6 and Ac-HASCL-6, suggesting that the ionic groups introduced on cross-linked starch chains play a role in the bioadhesion process. The adhesiveness seemed related to capillary attraction forces. Surface adhesion parameters were calculated for slabs based on the mentioned polymers and corroborated with their swelling behavior at various pH changes. The positively charged AE-derivatives presented a higher adhesion at acidic pH, being thus recommended for vaginal delivery, whereas the negatively charged derivatives (CM-HASCL-6) exhibited a better adhesion at neutral pH, being thus more appropriate for buccal delivery.

Cross-linked high amylose starch derivatives for drug release III. Diffusion properties.

Acetate (Ac-), aminoethyl (AE-) and carboxymethyl (CM-) derivatives of cross-linked high amylose starch (HASCL-6) were previously shown to control the release of drugs over 20 h from highly loaded (up to 60% drug) monolithic tablets. This report presents a diffusion analysis, aimed to facilitate a better understanding of the mechanisms involved in the control of the drug release from these hydrogels. The diffusion was found to depend on the molecular weight of the diffusant, whereas the partition coefficient depended on the affinities of the diffusant for the polymers and for the dissolution media via attractive or repulsive ionic interactions. The diffusion was also affected by the swelling of CM-HASCL-6, which, unexpectedly, increased with the decrease of the ionic strength. This diffusion analysis completes the swelling studies of HASCL-6 and of its derivatives, allowing the prediction of release kinetics of various active agents.

Carboxymethyl high amylose starch (CM-HAS) as excipient for Escherichia coli oral formulations.

Carboxymethyl high amylose starch (CM-HAS) is proposed as a novel excipient for oral tablet formulation of bioactive agents ensuring their protection in the stomach and delivery in the intestine. Three variants of CM-HAS, with different degrees of substitution, were synthesized by starch treatment with various amounts of monochloroacetic acid. The products were dried in powder form and tablets were obtained by direct compression of mixed powders of polymeric excipient and lyophilized Escherichia coli (E. coli) bacteria. Dosage forms of CM-HAS are unswollen and compact in acidic medium, ensuring protection of active agents against acidity. Release of bacteria from CM-HAS tablets is based on the fast swelling of the tablets during the passage from gastric acidity to alkaline intestinal medium, enzymatic hydrolysis triggering their rapid, almost total dissolution. The bacteria thus formulated displayed higher survival rates in acidic gastric conditions and for longer periods than the free bacteria or than the bacteria formulated with the non-derivatized starch. The CM-HAS selected matrix also assured a good viability of bacteria after 6 months under refrigeration.

Cross-linked high amylose starch derivatives for drug release. II. Swelling properties and mechanistic study.

Acetate (Ac-), aminoethyl (AE-), and carboxymethyl (CM-) high amylose starch cross-linked 6 (HASCL-6) derivatives were previously shown to control the release of drugs over 20 h from monolithic tablets highly loaded (up to 60% drug). This report describes the swelling characteristics, which allow a better understanding of the mechanisms involved in the control of the drug release from the said polymeric matrices. The tablet swelling of HASCL-6, Ac-HASCL-6, and AE-HASCL-6 was found to not be affected by the ionic strength and by the pH between 1.2 (gastric) and 7 (intestinal), whereas the swelling of CM-HASCL-6 was shown to depend on both ionic strength and pH of the release medium. For all the studied polymers the drug loading did not change the equilibrium swelling ratio but affected the initial swelling velocity, seemingly due to the competition between drug and polymer for water uptake, a phenomenon probably influenced by the loading and the drug solubility. It was also shown that the increase of ionic strength would slightly increase the drug release time probably by decreasing the amount of free water still available to solubilize the drug present into the matrix.

Modified high amylose starch for immobilization of uricase for therapeutic application.

Urate oxidase (uricase) was immobilized on carboxymethyl high amylose starch cross-linked 35 (CM-HASCL-35), on aminoethyl high amylose starch cross-linked 35, as well as on commercial supports, CNBr-activated Sepharose and diaminodipropylamine agarose. The N -ethyl-5-phenylisoxazolium-3'-sulphonate (Woodward reagent K) gave a high binding but totally inhibited the enzyme activity. Best results were obtained with CM-HASCL-35 using 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide as a coupling agent. The immobilized enzyme retained 88% of its initial limit rate [V (max)(app)=16 EU/mg for immobilized uricase versus V (max)=18 EU/mg for the free enzyme], with an apparent decrease of affinity for urate substrate [K (m)(app)=0.17 mM versus K (m)=0.03 mM for the free enzyme]. The coupling yield was 60% and the modified uricase was found more resistant to proteolysis than the free enzyme. The immobilized uricase retained 25% of its initial activity after 60 min in pancreatic proteolysis medium (pancreatin), whereas the free enzyme retained only 5% of its initial activity. The best immobilization yield was obtained with the polymeric support based on CM-HASCL-35 (53%), which gave better results than commercial supports based on agarose.