Architectures and complex functions of tandem riboswitches.

Riboswitch architectures that involve the binding of a single ligand to a single RNA aptamer domain result in ordinary dose-response curves that require approximately a 100-fold change in ligand concentration to cover nearly the full dynamic range for gene regulation. However, by using multiple riboswitches or aptamer domains in tandem, these ligand-sensing structures can produce additional, complex gene control outcomes. In the current study, we have computationally searched for tandem riboswitch architectures in bacteria to provide a more complete understanding of the diverse biological and biochemical functions of gene control elements that are made exclusively of RNA. Numerous different arrangements of tandem homologous riboswitch architectures are exploited by bacteria to create more 'digital' gene control devices, which operate over a narrower ligand concentration range. Also, two heterologous riboswitch aptamers are sometimes employed to create two-input Boolean logic gates with various types of genetic outputs. These findings illustrate the sophisticated genetic decisions that can be made by using molecular sensors and switches based only on RNA.

Employing a ZTP Riboswitch to Detect Bacterial Folate Biosynthesis Inhibitors in a Small Molecule High-Throughput Screen.

Various riboswitch classes are being discovered that precisely monitor the status of important biological processes, including metabolic pathway function, signaling for physiological adaptations, and responses to toxic agents. Biochemical components for some of these processes might make excellent targets for the development of novel antibacterial molecules, which can be broadly sought by using phenotypic drug discovery (PDD) methods. However, PDD data do not normally provide clues regarding the target for each hit compound. We have developed and validated a robust fluorescent reporter system based on a ZTP riboswitch that identifies numerous folate biosynthesis inhibitors with high sensitivity and precision. The utility of the riboswitch-based PDD strategy was evaluated using Escherichia coli bacteria by conducting a 128 310-compound high-throughput screen, which identified 78 sulfanilamide derivatives among the many initial hits. Similarly, representatives of other riboswitch classes could be employed to rapidly match antibacterial hits with the biological processes they target.

Rare variants of the FMN riboswitch class in Clostridium difficile and other bacteria exhibit altered ligand specificity.

Many bacteria use flavin mononucleotide (FMN) riboswitches to control the expression of genes responsible for the biosynthesis and transport of this enzyme cofactor or its precursor, riboflavin. Rare variants of FMN riboswitches found in strains of Clostridium difficile and some other bacteria typically control the expression of proteins annotated as transporters, including multidrug efflux pumps. These RNAs no longer recognize FMN, and differ from the original riboswitch consensus sequence at nucleotide positions normally involved in binding of the ribityl and phosphate moieties of the cofactor. Representatives of one of the two variant subtypes were found to bind the FMN precursor riboflavin and the FMN degradation products lumiflavin and lumichrome. Although the biologically relevant ligand sensed by these variant FMN riboswitches remains uncertain, our findings suggest that many strains of C. difficile might use rare riboswitches to sense flavin degradation products and activate transporters for their detoxification.

Challenges of ligand identification for the second wave of orphan riboswitch candidates.

Orphan riboswitch candidates are noncoding RNA motifs whose representatives are believed to function as genetic regulatory elements, but whose target ligands have yet to be identified. The study of certain orphans, particularly classes that have resisted experimental validation for many years, has led to the discovery of important biological pathways and processes once their ligands were identified. Previously, we highlighted details for four of the most common and intriguing orphan riboswitch candidates. This facilitated the validation of riboswitches for the signaling molecules c-di-AMP, ZTP, and ppGpp, the metal ion Mn2+, and the metabolites guanidine and PRPP. Such studies also yield useful linkages between the ligands sensed by the riboswitches and numerous biochemical pathways. In the current report, we describe the known characteristics of 30 distinct classes of orphan riboswitch candidates - some of which have remained unsolved for over a decade. We also discuss the prospects for uncovering novel biological insights via focused studies on these RNAs. Lastly, we make recommendations for experimental objectives along the path to finding ligands for these mysterious RNAs.

Detection of 224 candidate structured RNAs by comparative analysis of specific subsets of intergenic regions.

The discovery of structured non-coding RNAs (ncRNAs) in bacteria can reveal new facets of biology and biochemistry. Comparative genomics analyses executed by powerful computer algorithms have successfully been used to uncover many novel bacterial ncRNA classes in recent years. However, this general search strategy favors the discovery of more common ncRNA classes, whereas progressively rarer classes are correspondingly more difficult to identify. In the current study, we confront this problem by devising several methods to select subsets of intergenic regions that can concentrate these rare RNA classes, thereby increasing the probability that comparative sequence analysis approaches will reveal their existence. By implementing these methods, we discovered 224 novel ncRNA classes, which include ROOL RNA, an RNA class averaging 581 nt and present in multiple phyla, several highly conserved and widespread ncRNA classes with properties that suggest sophisticated biochemical functions and a multitude of putative cis-regulatory RNA classes involved in a variety of biological processes. We expect that further research on these newly found RNA classes will reveal additional aspects of novel biology, and allow for greater insights into the biochemistry performed by ncRNAs.

Thermoresponsive, in situ cross-linkable hydrogels based on N-isopropylacrylamide: fabrication, characterization and mesenchymal stem cell encapsulation.

Hydrogels that solidify in response to a dual, physical and chemical, mechanism upon temperature increase were fabricated and characterized. The hydrogels were based on N-isopropylacrylamide, which renders them thermoresponsive, and contained covalently cross-linkable moieties in the macromers. The effects of the macromer end group, acrylate or methacrylate, and the fabrication conditions on the degradative and swelling properties of the hydrogels were investigated. The hydrogels exhibited higher swelling below their lower critical solution temperature (LCST). When immersed in cell culture medium at physiological temperature, which was above their LCST, hydrogels showed constant swelling and no degradation over 8 weeks, with the methacrylated hydrogels showing greater swelling than their acrylated analogs. In addition, hydrogels immersed in cell culture medium under the same conditions showed lower swelling compared with phosphate-buffered saline. The interplay between chemical cross-linking and thermally induced phase separation affected the swelling characteristics of the hydrogels in different media. Mesenchymal stem cells encapsulated in the hydrogels in vitro were viable over 3 weeks and markers of osteogenic differentiation were detected when the cells were cultured with osteogenic supplements. Hydrogel mineralization in the absence of cells was observed in cell culture medium with the addition of fetal bovine serum and ß-glycerol phosphate. The results suggest that these hydrogels may be suitable as carriers for cell delivery in tissue engineering.