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.

PCR-Based Assembly of Gene Sequences by Thermodynamically Balanced Inside-Out (TBIO) Gene Synthesis.

The ability to enzymatically assemble DNA oligonucleotides into longer DNA duplexes in a process known as gene synthesis has wide-ranging applications in the fields of genetic engineering and synthetic biology. Thermodynamically balanced inside-out (TBIO) gene synthesis is one of several PCR-based primer extension gene synthesis protocols that have been developed. In TBIO gene synthesis, overlapping primers with equivalent melting temperatures (Ts) are designed so that the 5' half of the DNA is encoded by sense primers and the 3' half of the DNA molecule is encoded by antisense primers.

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 -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.

An Investigation into the Potential of Targeting 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 gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As 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 mRNA.

Identification of Novel Inhibitors of 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.

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 high-throughput screening and validation we have identified three novel small molecule inhibitors of RNase E that are active against RNase E from , and .

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.

What Works? the Influence of Changing Wastewater Treatment Type, Including Tertiary Granular Activated Charcoal, on Downstream Macroinvertebrate Biodiversity Over Time.

The present study reviewed the impacts of wastewater on macroinvertebrates over 4 decades in a United Kingdom lowland river. This involved examining changes in chemicals, temperature, flow, and macroinvertebrate diversity from the 1970s until 2017 for a wastewater-dominated river downstream of Swindon in the United Kingdom (population ~ 220 000). When the wastewater treatment process changed from trickling filter to activated sludge in 1991, biological oxygen demand was nearly halved (90th percentile from 8.

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.

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.

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.

Web-based integrated bipolar parenting intervention for parents with bipolar disorder: a randomised controlled pilot trial.

Background: People with bipolar disorder (BD) experience additional parenting challenges associated with mood driven fluctuations in communication, impulse control and motivation. This paper describes a novel web-based self-management approach (Integrated Bipolar Parenting Intervention; IBPI) to support parents with BD.

Method: Parents with BD with children aged 3-10 years randomised to IBPI plus treatment as usual (TAU) or waitlist control (WL).

Inhibition of homologous phosphorolytic ribonucleases by citrate may represent an evolutionarily conserved communicative link between RNA degradation and central metabolism.

Ribonucleases play essential roles in all aspects of RNA metabolism, including the coordination of post-transcriptional gene regulation that allows organisms to respond to internal changes and environmental stimuli. However, as inherently destructive enzymes, their activity must be carefully controlled. Recent research exemplifies the repertoire of regulatory strategies employed by ribonucleases.

Rational Re-engineering of a Transcriptional Silencing PreQ1 Riboswitch.

Re-engineered riboswitches that no longer respond to cellular metabolites, but that instead can be controlled by synthetic molecules, are potentially useful gene regulatory tools for use in synthetic biology and biotechnology fields. Previously, extensive genetic selection and screening approaches were employed to re-engineer a natural adenine riboswitch to create orthogonal ON-switches, enabling translational control of target gene expression in response to synthetic ligands. Here, we describe how a rational targeted approach was used to re-engineer the PreQ1 riboswitch from Bacillus subtilis into an orthogonal OFF-switch.

An exploratory randomised controlled trial of a web-based integrated bipolar parenting intervention (IBPI) for bipolar parents of young children (aged 3-10).

Background: Communication, impulse control and motivation can all be affected by Bipolar Disorder (BD) making consistent parenting more difficult than for parents without mental health problems. Children of parents with BD (CPB) are at significantly increased risk of a range of mental health issues including Attention Deficit Hyperactivity Disorder (ADHD), anxiety, depression, substance use, and sleep disorders. Furthermore, CPB are also at elevated risk for BD compared to the general population.

The first small-molecule inhibitors of members of the ribonuclease E family.

The Escherichia coli endoribonuclease RNase E is central to the processing and degradation of all types of RNA and as such is a pleotropic regulator of gene expression. It is essential for growth and was one of the first examples of an endonuclease that can recognise the 5'-monophosphorylated ends of RNA thereby increasing the efficiency of many cleavages. Homologues of RNase E can be found in many bacterial families including important pathogens, but no homologues have been identified in humans or animals.

Modular riboswitch toolsets for synthetic genetic control in diverse bacterial species.

Ligand-dependent control of gene expression is essential for gene functional analysis, target validation, protein production, and metabolic engineering. However, the expression tools currently available are difficult to transfer between species and exhibit limited mechanistic diversity. Here we demonstrate how the modular architecture of purine riboswitches can be exploited to develop orthogonal and chimeric switches that are transferable across diverse bacterial species, modulating either transcription or translation, to provide tunable activation or repression of target gene expression, in response to synthetic non-natural effector molecules.

Generation of orthogonally selective bacterial riboswitches by targeted mutagenesis and in vivo screening.

Riboswitches are naturally occurring RNA-based genetic switches that control gene expression in response to the binding of small-molecule ligands, typically through modulation of transcription or translation. Their simple mechanism of action and the expanding diversity of riboswitch classes make them attractive targets for the development of novel gene expression tools. The essential first step in realizing this potential is to generate artificial riboswitches that respond to nonnatural, synthetic ligands, thereby avoiding disruption of normal cellular function.

An improved method for surface immobilisation of RNA: application to small non-coding RNA-mRNA pairing.

Characterisation of RNA and its intermolecular interactions is increasing in importance as the inventory of known RNA functions continues to expand. RNA-RNA interactions are central to post-transcriptional gene regulation mechanisms in bacteria, and the interactions of bacterial small non-coding RNAs (sRNAs) with their mRNA targets are the subject of much current research. The technology of surface plasmon resonance (SPR) is an attractive approach to studying these interactions since it is highly sensitive, and allows interaction measurements to be recorded in real-time.

Hfq binding changes the structure of small noncoding RNAs OxyS and RprA, which are involved in the riboregulation of .

OxyS and RprA are two small noncoding RNAs (sRNAs) that modulate the expression of rpoS, encoding an alternative sigma factor that activates transcription of multiple Escherichia coli stress-response genes. While RprA activates rpoS for translation, OxyS down-regulates the transcript. Crucially, the RNA binding protein Hfq is required for both sRNAs to function, although the specific role played by Hfq remains unclear.

Characterization of MicA interactions suggests a potential novel means of gene regulation by small non-coding RNAs.

MicA is a small non-coding RNA that regulates ompA mRNA translation in Escherichia coli. MicA has an inhibitory function, base pairing to the translation initiation region of target mRNAs through short sequences of complementarity, blocking their ribosome-binding sites. The MicA structure contains two stem loops, which impede its interaction with target mRNAs, and it is thought that the RNA chaperone protein Hfq, known to be involved in MicA regulation of ompA, may structurally remodel MicA to reveal the ompA-binding site for cognate pairing.

The low-resolution solution structure of Hfq in complex with Qrr1 sRNA.

In , the RNA binding protein and chaperone Hfq (VcHfq) facilitates the pairing of the quorum regulatory RNA (Qrr) small regulatory RNAs (sRNAs) to the 5' untranslated regions of the mRNAs for a number of global regulators that modulate the expression of virulence genes. This Qrr-mediated sRNA circuit is an attractive antimicrobial target, but characterization at the molecular level is required for this to be realized. Here, we investigate the interactions between VcHfq and the Qrr sRNAs using a variety of biochemical and biophysical techniques.

Characterization of Vibrio cholerae Hfq provides novel insights into the role of the Hfq C-terminal region.

Hfq is a bacterial RNA binding protein that facilitates small RNA-mediated posttranscriptional gene regulation. In Vibrio cholerae, Hfq and four Hfq-dependent small RNAs are essential for the expression of virulence genes, but little is known about this mechanism at the molecular level. To better understand V.

Polynucleotide phosphorylase activity may be modulated by metabolites in Escherichia coli.

RNA turnover is an essential element of cellular homeostasis and response to environmental change. Whether the ribonucleases that mediate RNA turnover can respond to cellular metabolic status is an unresolved question. Here we present evidence that the Krebs cycle metabolite citrate affects the activity of Escherichia coli polynucleotide phosphorylase (PNPase) and, conversely, that cellular metabolism is affected widely by PNPase activity.