Synthetic small RNAs: Current status, challenges, and opportunities.

Small regulatory RNAs act at the levels of transcription, posttranscription, and translation, with numerous roles that include binding to protein targets, protein modification, binding to messenger RNA targets, and regulation of gene expression. We discuss the development of a number of riboregulators and riboswitches, highlighting their use in metabolic engineering and genetic control. Riboregulators and riboswitches are self-assembled RNA molecules that can dynamically change their conformation, acting as regulatory switches that affect biological changes. They have currently been designed, characterized, and implemented in a wide range of organisms and cell types, including bacteria, yeast, and mammalian cells. We have identified and examined the recent advances in RNA synthetic biology, underlining the potential future development of their use and capabilities, noting how these can be ultimately expanded and improved into novel biotechnological, biomedical, and industrial applications.

Recent Advances in CRISPR-Cas9 Genome Editing Technology for Biological and Biomedical Investigations.

The Type II CRISPR-Cas9 system is a simple, efficient, and versatile tool for targeted genome editing in a wide range of organisms and cell types. It continues to gain more scientific interest and has established itself as an extremely powerful technology within our synthetic biology toolkit. It works upon a targeted site and generates a double strand breaks that become repaired by either the NHEJ or the HDR pathway, modifying or permanently replacing the genomic target sequences of interest. These can include viral targets, single-mutation genetic diseases, and multiple-site corrections for wide scale disease states, offering the potential to manage and cure some of mankind's most persistent biomedical menaces. Here, we present the developing progress and future potential of CRISPR-Cas9 in biological and biomedical investigations, toward numerous therapeutic, biomedical, and biotechnological applications, as well as some of the challenges within. J. Cell. Biochem. 119: 81-94, 2018. © 2017 Wiley Periodicals, Inc.

Isoguanine and 5-methyl-isocytosine bases, in vitro and in vivo.

The synthesis, base-pairing properties and in vitro and in vivo characteristics of 5-methyl-isocytosine (isoC(Me) ) and isoguanine (isoG) nucleosides, incorporated in an HNA(h) (hexitol nucleic acid)-DNA(d) mosaic backbone, are described. The required h-isoG phosphoramidite was prepared by a selective deamination as a key step. As demonstrated by Tm measurements the hexitol sugar showed slightly better mismatch discrimination against dT. The d-isoG base mispairing follows the order T>G>C while the h-isoG base mispairing follows the order G>C>T. The h- and d-isoC(Me) bases mainly mispair with G. Enzymatic incorporation experiments show that the hexitol backbone has a variable effect on selectivity. In the enzymatic assays, isoG misincorporates mainly with T, and isoC(Me) misincorporates mainly with A. Further analysis in vivo confirmed the patterns of base-pair interpretation for the deoxyribose and hexitol isoC(Me) /isoG bases in a cellular context, through incorporation of the bases into plasmidic DNA. Results in vivo demonstrated that mispairing and misincorporation was dependent on the backbone scaffold of the base, which indicates rational advances towards orthogonality.

Observation of the time-course for peptidoglycan lipid intermediate II polymerization by Staphylococcus aureus monofunctional transglycosylase.

The polymerization of lipid intermediate II by the transglycosylase activity of penicillin-binding proteins (PBPs) represents an important target for antibacterial action, but limited methods are available for quantitative assay of this reaction, or screening potential inhibitors. A new labelling method for lipid II polymerization products using Sanger's reagent (fluoro-2,4-dinitrobenzene), followed by gel permeation HPLC analysis, has permitted the observation of intermediate polymerization products for Staphylococcus aureus monofunctional transglycosylase MGT. Peak formation is inhibited by 6 µM ramoplanin or enduracidin. Characterization by mass spectrometry indicates the formation of tetrasaccharide and octasaccharide intermediates, but not a hexasaccharide intermediate, suggesting a dimerization of a lipid-linked tetrasaccharide. Numerical modelling of the time-course data supports a kinetic model involving addition to lipid-linked tetrasaccharide of either lipid II or lipid-linked tetrasaccharide. Observation of free octasaccharide suggests that hydrolysis of the undecaprenyl diphosphate lipid carrier occurs at this stage in peptidoglycan transglycosylation.

In vitro biosynthesis of bacterial peptidoglycan using D-Cys-containing precursors: fluorescent detection of transglycosylation and transpeptidation.

Peptidoglycan precursors containing D-Cys at position 4 were polymerised to form a synthetic peptidoglycan layer, which could be fluorescently labelled, providing a new method to monitor peptidoglycan transglycosylation and transpeptidation.

Engineering Strategies in Microorganisms for the Enhanced Production of Squalene: Advances, Challenges and Opportunities.

The triterpene squalene is a natural compound that has demonstrated an extraordinary diversity of uses in pharmaceutical, nutraceutical, and personal care industries. Emboldened by this range of uses, novel applications that can gain profit from the benefits of squalene as an additive or supplement are expanding, resulting in its increasing demand. Ever since its discovery, the primary source has been the deep-sea shark liver, although recent declines in their populations and justified animal conservation and protection regulations have encouraged researchers to identify a novel route for squalene biosynthesis. This renewed scientific interest has profited from immense developments in synthetic biology, which now allows fine-tuning of a wider range of plants, fungi, and microorganisms for improved squalene production. There are numerous naturally squalene producing species and strains; although they generally do not make commercially viable yields as primary shark liver sources can deliver. The recent advances made toward improving squalene output from natural and engineered species have inspired this review. Accordingly, it will cover in-depth knowledge offered by the studies of the natural sources, and various engineering-based strategies that have been used to drive the improvements in the pathways toward large-scale production. The wide uses of squalene are also discussed, including the notable developments in anti-cancer applications and in augmenting influenza vaccines for greater efficacy.

Corrigendum: Engineering Strategies in Microorganisms for the Enhanced Production of Squalene: Advances, Challenges and Opportunities.

[This corrects the article DOI: 10.3389/fbioe.2019.00050.].

Exploring the Potential of Cell-Free Protein Synthesis for Extending the Abilities of Biological Systems.

Cell-free protein synthesis (CFPS) system is a simple, rapid, and sensitive tool that is devoid of membrane-bound barriers, yet contains all the mandatory substrates, biomolecules, and machineries required for the synthesis of the desired proteins. It has the potential to overcome loopholes in the current in vivo production systems and is a promising tool in both basic and applied scientific research. It facilitates a simplified organization of desired experiments with a variety of reaction conditions, making CFPS a powerful tool in biological research. It has been used for the expansion of genetic code, assembly of viruses, and in metabolic engineering for production of toxic and complex proteins. Subsequently, CFPS systems have emerged as potent technology for high-throughput production of membrane proteins, enzymes, and therapeutics. The present review highlights the recent advances and uses of CFPS systems in biomedical, therapeutic, and biotechnological applications. Additionally, we highlight possible solutions to the potential biosafety issues that may be encountered while using CFPS technology.

Exploring the potential of genome editing CRISPR-Cas9 technology.

CRISPR-Cas9 is an RNA-mediated adaptive immune system that protects bacteria and archaea from viruses or plasmids. Herein we discuss the recent development of CRISPR-Cas9 into a key technology for genome editing, targeting, and regulation in a wide range of organisms and cell types. It requires a custom designed single guide-RNA (sgRNA), a Cas9 endonuclease, and PAM sequences in the target region. The sgRNA-Cas9 complex binds to its target and creates a double-strand break (DSB) that can be repaired by non-homologous end joining (NHEJ) or by the homology-directed repair (HDR) pathway, modifying or permanently replacing the genomic target sequence. Additionally, we highlight recent advances in the repurposing of CRISPR-Cas9 for repression, activation, and loci imaging. In this review, we underline the current progress and the future potential of the CRISPR-Cas9 system towards biomedical, therapeutic, industrial, and biotechnological applications.

Correction: Base pairing involving artificial bases in vitro and in vivo.

[This corrects the article DOI: 10.1039/C5SC03474D.].

Base pairing involving artificial bases in vitro and in vivo.

Herein we report the synthesis of N8-glycosylated 8-aza-deoxyguanosine (N8-8-aza-dG) and 8-aza-9-deaza-deoxyguanosine (N8-8-aza-9-deaza-dG) nucleotides and their base pairing properties with 5-methyl-isocytosine (d-isoCMe), 8-amino-deoxyinosine (8-NH2-dI), 1-N-methyl-8-amino-deoxyinosine (1-Me-8-NH2-dI), 7,8-dihydro-8-oxo-deoxyinosine (8-Oxo-dI), 7,8-dihydro-8-oxo-deoxyadenosine (8-Oxo-dA), and 7,8-dihydro-8-oxo-deoxyguanosine (8-Oxo-dG), in comparison with the d-isoCMe:d-isoG artificial genetic system. As demonstrated by Tm measurements, the N8-8-aza-dG:d-isoCMe base pair formed less stable duplexes as the C:G and d-isoCMe:d-isoG pairs. Incorporation of 8-NH2-dI versus the N8-8-aza-dG nucleoside resulted in a greater reduction in Tm stability, compared to d-isoCMe:d-isoG. Insertion of the methyl group at the N1 position of 8-NH2-dI did not affect duplex stability with N8-8-aza-dG, thus suggesting that the base paring takes place through Hoogsteen base pairing. The cellular interpretation of the nucleosides was studied, whereby a lack of recognition or mispairing of the incorporated nucleotides with the canonical DNA bases indicated the extent of orthogonality in vivo. The most biologically orthogonal nucleosides identified included the 8-amino-deoxyinosines (1-Me-8-NH2-dI and 8-NH2-dI) and N8-8-aza-9-deaza-dG. The 8-oxo modifications mimic oxidative damage ahead of cancer development, and the impact of the MutM mediated recognition of these 8-oxo-deoxynucleosides was studied, finding no significant impact in their in vivo assay.

Recent advances and versatility of MAGE towards industrial applications.

The genome engineering toolkit has expanded significantly in recent years, allowing us to study the functions of genes in cellular networks and assist in over-production of proteins, drugs, chemicals and biofuels. Multiplex automated genome engineering (MAGE) has been recently developed and gained more scientific interest towards strain engineering. MAGE is a simple, rapid and efficient tool for manipulating genes simultaneously in multiple loci, assigning genetic codes and integrating non-natural amino acids. MAGE can be further expanded towards the engineering of fast, robust and over-producing strains for chemicals, drugs and biofuels at industrial scales.

Bacterial cell wall assembly: still an attractive antibacterial target.

The development of new antibacterial agents to combat worsening antibiotic resistance is still a priority area in anti-infectives research, but in the post-genomic era it has been more difficult than expected to identify new lead compounds from high-throughput screening, and very challenging to obtain antibacterial activity for lead compounds. Bacterial cell-wall peptidoglycan biosynthesis is a well-established target for antibacterial chemotherapy, and recent developments enable the entire biosynthetic pathway to be reconstituted for detailed biochemical study and high-throughput inhibitor screening. This review article discusses recent developments in the availability of peptidoglycan biosynthetic intermediates, the identification of lead compounds for both the earlier cytoplasmic steps and the later lipid-linked steps, and the application of new methods such as structure-based drug design, phage display and surface science.

Structure-activity relationships for organometallic osmium arene phenylazopyridine complexes with potent anticancer activity.

We report the synthesis and characterisation of 32 half sandwich phenylazopyridine Os(II) arene complexes [Os(η(6)-arene)(phenylazopyridine)X](+) in which X is chloride or iodide, the arene is p-cymene or biphenyl and the pyridine and phenyl rings contain a variety of substituents (F, Cl, Br, I, CF(3), OH or NO(2)). Ten X-ray crystal structures have been determined. Cytotoxicity towards A2780 human ovarian cancer cells ranges from high potency at nanomolar concentrations to inactivity. In general the introduction of an electron-withdrawing group (e.g. F, Cl, Br or I) at specific positions on the pyridine ring significantly increases cytotoxic activity and aqueous solubility. Changing the arene from p-cymene to biphenyl and the monodentate ligand X from chloride to iodide also increases the activity significantly. Activation by hydrolysis and DNA binding appears not to be the major mechanism of action since both the highly active complex [Os(η(6)-bip)(2-F-azpy)I]PF(6) (9) and the moderately active complex [Os(η(6)-bip)(3-Cl-azpy)I]PF(6) (23) are very stable and inert towards aquation. Studies of octanol-water partition coefficients (log P) and subcellular distributions of osmium in A2780 human ovarian cancer cells suggested that cell uptake and targeting to cellular organelles play important roles in determining activity. Although complex 9 induced the production of reactive oxygen species (ROS) in A2780 cells, the ROS level did not appear to play a role in the mechanism of anticancer activity. This class of organometallic osmium complexes has new and unusual features worthy of further exploration for the design of novel anticancer drugs.