Generation of Functional-RNA Arrays by In Vitro Transcription and In Situ RNA Capture for the Detection of RNA-RNA Interactions.

RNA performs a wide variety of vital cellular functions. These functions typically require interactions with other biological macromolecules, often as part of an intricate communication network. High-throughput techniques capable of analyzing RNA-based interactions are therefore essential. Functional-RNA arrays address this need, providing the capability of performing hundreds of miniature assays in parallel. Here we describe a method to generate functional-RNA arrays using in vitro transcription of a DNA template array and in situ RNA capture. We also suggest how functional-RNA arrays could be applied to investigating RNA-RNA interactions.

Identification of small RNAs associated with RNA chaperone Hfq reveals a new stress response regulator in Actinobacillus pleuropneumoniae.

The RNA chaperone Hfq promotes the association of small RNAs (sRNAs) with cognate mRNAs, controlling the expression of bacterial phenotype. Actinobacillus pleuropneumoniae hfq mutants strains are attenuated for virulence in pigs, impaired in the ability to form biofilms, and more susceptible to stress, but knowledge of the extent of sRNA involvement is limited. Here, using A. pleuropneumoniae strain MIDG2331 (serovar 8), 14 sRNAs were identified by co-immunoprecipitation with Hfq and the expression of eight, identified as trans-acting sRNAs, were confirmed by Northern blotting. We focused on one of these sRNAs, named Rna01, containing a putative promoter for RpoE (stress regulon) recognition. Knockout mutants of rna01 and a double knockout mutant of rna01 and hfq, both had decreased biofilm formation and hemolytic activity, attenuation for virulence in Galleria mellonella, altered stress susceptibility, and an altered outer membrane protein profile. Rna01 affected extracellular vesicle production, size and toxicity in G. mellonella. qRT-PCR analysis of rna01 and putative cognate mRNA targets indicated that Rna01 is associated with the extracytoplasmic stress response. This work increases our understanding of the multilayered and complex nature of the influence of Hfq-dependent sRNAs on the physiology and virulence of A. pleuropneumoniae.

An Investigation into the Potential of Targeting Escherichia coli rne mRNA with Locked Nucleic Acid (LNA) Gapmers as an Antibacterial Strategy.

The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides.

Reprogramming Gene Expression by Targeting RNA-Based Interactions: A Novel Pipeline Utilizing RNA Array Technology.

Regulatory RNA-based interactions are critical for coordinating gene expression and are increasingly being targeted in synthetic biology, antimicrobial, and therapeutic fields. Bacterial trans-encoded small RNAs (sRNAs) regulate the translation and/or stability of mRNA targets through base-pairing interactions. These interactions are often integral to complex gene circuits which coordinate critical bacterial processes. The ability to predictably modulate these gene circuits has potential for reprogramming gene expression for synthetic biology and antibacterial purposes. Here, we present a novel pipeline for targeting such RNA-based interactions with antisense oligonucleotides (ASOs) in order to reprogram gene expression. As proof-of-concept, we selected sRNA-mRNA interactions that are central to the Vibrio cholerae quorum sensing pathway, required for V. cholerae pathogenesis, as a regulatory RNA-based interaction input. We rationally designed anti-sRNA ASOs to target the sRNAs and synthesized them as peptide nucleic acids (PNAs). Next, we devised an RNA array-based interaction assay to allow screening of the anti-sRNA ASOs in vitro. Finally, an Escherichia coli-based gene expression reporter assay was developed and used to validate anti-sRNA ASO regulatory activity in a cellular environment. The output from the pipeline was an anti-sRNA ASO that targets sRNAs to inhibit sRNA-mRNA interactions and modulate gene expression. This anti-sRNA ASO has potential for reprogramming gene expression for synthetic biology and/or antibacterial purposes. We anticipate that this pipeline will find widespread use in fields targeting RNA-based interactions as modulators of gene expression.

Identification of Novel Inhibitors of Escherichia coli DNA Ligase (LigA).

Present in all organisms, DNA ligases catalyse the formation of a phosphodiester bond between a 3' hydroxyl and a 5' phosphate, a reaction that is essential for maintaining genome integrity during replication and repair. Eubacterial DNA ligases use NAD+ as a cofactor and possess low sequence and structural homology relative to eukaryotic DNA ligases which use ATP as a cofactor. These key differences enable specific targeting of bacterial DNA ligases as an antibacterial strategy. In this study, four small molecule accessible sites within functionally important regions of Escherichia coli ligase (EC-LigA) were identified using in silico methods. Molecular docking was then used to screen for small molecules predicted to bind to these sites. Eight candidate inhibitors were then screened for inhibitory activity in an in vitro ligase assay. Five of these (geneticin, chlorhexidine, glutathione (reduced), imidazolidinyl urea and 2-(aminomethyl)imidazole) showed dose-dependent inhibition of EC-LigA with half maximal inhibitory concentrations (IC50) in the micromolar to millimolar range (11-2600 µM). Two (geneticin and chlorhexidine) were predicted to bind to a region of EC-LigA that has not been directly investigated previously, raising the possibility that there may be amino acids within this region that are important for EC-LigA activity or that the function of essential residues proximal to this region are impacted by inhibitor interactions with this region. We anticipate that the identified small molecule binding sites and inhibitors could be pursued as part of an antibacterial strategy targeting bacterial DNA ligases.

Identification and analysis of novel small molecule inhibitors of RNase E: Implications for antibacterial targeting and regulation of RNase E.

Increasing resistance of bacteria to antibiotics is a serious global challenge and there is a need to unlock the potential of novel antibacterial targets. One such target is the essential prokaryotic endoribonuclease RNase E. Using a combination of in silico high-throughput screening and in vitro validation we have identified three novel small molecule inhibitors of RNase E that are active against RNase E from Escherichia coli, Francisella tularensis and Acinetobacter baumannii. Two of the inhibitors are non-natural small molecules that could be suitable as lead compounds for the development of broad-spectrum antibiotics targeting RNase E. The third small molecule inhibitor is glucosamine-6-phosphate, a precursor of bacterial cell envelope peptidoglycans and lipopolysaccharides, hinting at a novel metabolite-mediated mechanism of regulation of RNase E.

A structural and biochemical comparison of Ribonuclease E homologues from pathogenic bacteria highlights species-specific properties.

Regulation of gene expression through processing and turnover of RNA is a key mechanism that allows bacteria to rapidly adapt to changing environmental conditions. Consequently, RNA degrading enzymes (ribonucleases; RNases) such as the endoribonuclease RNase E, frequently play critical roles in pathogenic bacterial virulence and are potential antibacterial targets. RNase E consists of a highly conserved catalytic domain and a variable non-catalytic domain that functions as the structural scaffold for the multienzyme degradosome complex. Despite conservation of the catalytic domain, a recent study identified differences in the response of RNase E homologues from different species to the same inhibitory compound(s). While RNase E from Escherichia coli has been well-characterised, far less is known about RNase E homologues from other bacterial species. In this study, we structurally and biochemically characterise the RNase E catalytic domains from four pathogenic bacteria: Yersinia pestis, Francisella tularensis, Burkholderia pseudomallei and Acinetobacter baumannii, with a view to exploiting RNase E as an antibacterial target. Bioinformatics, small-angle x-ray scattering and biochemical RNA cleavage assays reveal globally similar structural and catalytic properties. Surprisingly, subtle species-specific differences in both structure and substrate specificity were also identified that may be important for the development of effective antibacterial drugs targeting RNase E.

Generation of small molecule-binding RNA arrays and their application to fluorogen-binding RNA aptamers.

The discovery and engineering of more and more functions of RNA has highlighted the utility of RNA-targeting small molecules. Recently, several fluorogen-binding RNA aptamers have been developed that have been applied to live cell imaging of RNA and metabolites as RNA tags or biosensors, respectively. Although the design and application of these fluorogen-binding RNA aptamer-based devices is straightforward in theory, in practice, careful optimisation is required. For this reason, high throughput in vitro screening techniques, capable of quantifying fluorogen-RNA aptamer interactions, would be beneficial. We recently developed a method for generating functional-RNA arrays and demonstrated that they could be used to detect fluorogen-RNA aptamer interactions. Specifically, we were able to visualise the interaction between malachite green and the malachite green-binding aptamer. Here we expand this study to demonstrate that functional-RNA arrays can be used to quantify fluorogen-aptamer interactions. As proof-of-concept, we provide detailed protocols for the production of malachite green-binding RNA aptamer and DFHBI-binding Spinach RNA aptamer arrays. Furthermore, we discuss the potential utility of the technology to fluorogen-binding RNA aptamers, including application as a molecular biosensor platform. We anticipate that functional-RNA array technology will be beneficial for a wide variety of biological disciplines.

Application of mRNA Arrays for the Production of mCherry Reporter-Protein Arrays for Quantitative Gene Expression Analysis.

The development of programmable regulators that precisely and predictably control gene expression is a major goal of synthetic biology. Consequently, rapid high-throughput biochemical methods capable of quantitatively analyzing all components of gene expression would be of value in the characterization and optimization of regulator performance. In this study we demonstrate a novel application of RNA arrays, involving the production of reporter-protein arrays, to gene expression analysis. This method enables simultaneous quantification of both the transcription and post-transcription/translation components of gene expression, and it also allows the assessment of the orthogonality of multiple regulators. We use our method to directly compare the performance of a series of previously characterized synthetic post-transcriptional riboregulators, thus demonstrating its utility in the development of synthetic regulatory modules and evaluation of gene expression regulation in general.

High-density functional-RNA arrays as a versatile platform for studying RNA-based interactions.

We are just beginning to unravel the myriad of interactions in which non-coding RNAs participate. The intricate RNA interactome is the foundation of many biological processes, including bacterial virulence and human disease, and represents unexploited resources for the development of potential therapeutic interventions. However, identifying specific associations of a given RNA from the multitude of possible binding partners within the cell requires robust high-throughput systems for their rapid screening. Here, we present the first demonstration of functional-RNA arrays as a novel platform technology designed for the study of such interactions using immobilized, active RNAs. We have generated high-density RNA arrays by an innovative method involving surface-capture of in vitro transcribed RNAs. This approach has significant advantages over existing technologies, particularly in its versatility in regards to binding partner character. Indeed, proof-of-principle application of RNA arrays to both RNA-small molecule and RNA-RNA pairings is demonstrated, highlighting their potential as a platform technology for mapping RNA-based networks and for pharmaceutical screening. Furthermore, the simplicity of the method supports greater user-accessibility over currently available technologies. We anticipate that functional-RNA arrays will find broad utility in the expanding field of RNA characterization.