To deal with the growing threat of AR, it is important to cut down the use of antibiotics to the very minimum to diminish the risk of unknown drug-resistant bacteria and increase antibacterial vaccination programs. Furthermore, it is important to develop new classes of antibiotics that can deal with multidrug-resistant bacterial pathogens.

Due to the steady rise of multidrug-resistant pathogenic bacteria worldwide, it is critical to develop novel antibacterial drugs. This article presents chimeric antisense oligonucleotides that inhibit the bacterial growth of Staphylococcus aureus, one of the most frequent causes of hospital-acquired infections. The chimeric antisense oligonucleotides have a combination of first- and second-generation chemical modification. To deliver the antisense oligonucleotides into a cell, we apply a cell-penetrating oligopeptide attached to them. We have performed complete bioinformatics analyses of the glmS ribozyme present in S. aureus and its essential role in the biochemical pathway of glucosamine-6-phosphate synthesis. Besides, we have analyzed the bacteria for alternative metabolic pathways, such as the nagA gene. The first antisense oligonucleotide explicitly targets the glmS riboswitch, while the second explicitly targets the nagA mRNA. We have evaluated that combined, the antisense oligonucleotides block the synthesis of glucosamine-6-phosphate entirely and inhibit the bacterial growth of S. aureus. However, the glmS riboswitch targeting the antisense oligonucleotide is sufficient to inhibit the growth of S. aureus with a MIC80 of 5 μg/mL. The glmS ribozyme is a very suitable target for antibacterial drug development with antisense oligonucleotides.

In the past several decades, antibiotic drug resistance has emerged as a significant challenge in modern medicine due to the rise of many bacterial pathogenic strains resistant to all known antibiotics. At the same time, riboswitches have emerged as novel targets for antibacterial drug discovery.Here for the first time, we describe the design and applications of antisense oligonucleotides as antibacterial agents that target a riboswitch. The antisense oligonucleotides are covalently coupled with two different cell-penetrating peptides, penetrating Grampositive and Gram-negative bacterial cells. We specifically target Flavin MonoNucleotide (FMN) riboswitches in Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli that control both synthesis and import of FMN precursors. We have established an average antibiotic dosage by antisense oligonucleotides that inhibit 80% of bacterial growth at 700 nM (4.5 μg/mL). Furthermore, the antisense oligonucleotides do not exhibit toxicity in human cell lines at this concentration. The results demonstrate that riboswitches are suitable targets in antisense technology for antibacterial drug development.

Nowadays, the emergence and the transmission of multidrug-resistant pathogenic bacteria are a severe menace mounting a lot of pressure on the healthcare systems worldwide. Many severe outbreaks of bacterial infections have been reported worldwide in recent years. Thus, there is an immediate demand to develop antibiotics. Some riboswitches are potential targets for overcoming bacterial resistance. This paper demonstrates the bacteriostatic effect of an antisense oligonucleotide (ASO) engineered to suppress the growth of pathogenic bacteria such as Listeria monocytogenes by targeting the Thiamine Pyrophosphate (TPP) riboswitch. It does not inhibit the growth of the conditional pathogenic bacteria Escherichia coli, as it lacks the TPP riboswitch, showing the specificity of action of our ASO. It is covalently bonded with the cell-penetrating protein pVEC. We did bioinformatics analyses of the thiamine pyrophosphate riboswitch regarding its role in synthesizing the metabolite thiamine pyrophosphate, which is essential for bacteria. L. monocytogenes is intrinsically resistant to cephalosporins and usually is treated with ampicillin. A dosage of ASO has been established that inhibits 80% of bacterial growth at 700 nM (4.5 μg/mL). Thus, the TPP riboswitch is a valuable antibacterial target.

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With the discovery of antibiotics, a productive period of antibacterial drug innovation and application in healthcare systems and agriculture resulted in saving millions of lives. Unfortunately, the misusage of antibiotics led to the emergence of many resistant pathogenic strains. Some riboswitches have risen as promising targets for developing antibacterial drugs. Here, we describe the design and applications of the chimeric antisense oligonucleotide (ASO) as a novel antibacterial agent. The pVEC-ASO-1 consists of a cell-penetrating oligopeptide known as pVEC attached to an oligonucleotide part with modifications of the first and the second generations. This combination of modifications enables specific mRNA degradation under multiple turnover conditions via RNase H. The pVEC-ASO targets the S-adenosyl methionine (SAM)-I riboswitch found in the genome of many Gram-positive bacteria. The SAM-I riboswitch controls not only the biosynthesis but also the transport of SAM. We have established an antibiotic dosage of 700 nM (4.5 µg/mL) of pVEC-ASO that inhibits 80% of the growth of Staphylococcus aureus and Listeria monocytogenes. The pVEC-ASO-1 does not show any toxicity in the human cell line at MIC80’s concentration. We have proven that the SAM-I riboswitch is a suitable target for antibacterial drug development based on ASO. The approach is rational and easily adapted to other bacterial RNA targets.

Nanobiotechnology and synthetic biology are emerging as novel fields that integrate research from science and technology to create novel organisms with new desired properties. We present here the new revolutionary methods of synthetic biology that enable us to engineer gene control circuits, edit genomes, and create de novo whole genomes. The creation of new genomes that function in the cell means that we can create new organisms that are different from those observed in nature. The synthetic genomes can contain novel combinations of genes that offer the opportunities to create novel biological species that possess predefined combination of properties. Therefore, the synthetic genomes can be regarded as a new kind of materials. The methods for whole genome assemble applied so far combined several in vitro and in vivo steps that possess certain technical limitations and shortcomings. In this chapter, we discuss all technical aspects of assembling novel genomes and their current limitations. The genome editing technologies that have been developed over the last several years based on the CRISPR-Cas system is also discussed. In addition, we present major RNA-based methods for design of gene control circuits both in prokaryotes and eukaryotes, including humans.

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