Short modified oligonucleotides targeted at bacterial DNA or RNA could serve as antibacterial agents provided that they are efficiently taken up by bacterial cells. However, the uptake of such oligonucleotides is hindered by the bacterial cell wall. To overcome this problem, oligomers have been attached to cell-penetrating peptides, but the efficiency of delivery remains poor. Thus, we have investigated the ability of vitamin B₁₂ to transport peptide nucleic acid (PNA) oligomers into cells of Escherichia coli and Salmonella Typhimurium. Vitamin B₁₂ was covalently linked to a PNA oligomer targeted at the mRNA of a reporter gene expressing Red Fluorescent Protein. Cu-catalyzed 1,3-dipolar cycloaddition was employed for the synthesis of PNA-vitamin B₁₂ conjugates; namely the vitamin B₁₂ azide was reacted with PNA possessing the terminal alkyne group. Different types of linkers and spacers between vitamin B₁₂ and PNA were tested, including a disulfide bond. We found that vitamin B₁₂ transports antisense PNA into E. coli cells more efficiently than the most widely used cell-penetrating peptide (KFF)₃K. We also determined that the structure of the linker impacts the antisense effect. The results of this study provide the foundation for developing vitamin B₁₂ as a carrier of PNA oligonucleotides into bacterial cells.

Antibiotic resistance is an escalating, worldwide problem. Due to excessive use of antibiotics, multidrug-resistant bacteria have become a serious threat and a major global healthcare problem of the 21st century. This fact creates an urgent need for new and effective antimicrobials. The common strategies for antibiotic discovery are based on either modifying existing antibiotics or screening compound libraries, but these strategies have not been successful in recent decades. An alternative approach could be to use gene-specific oligonucleotides, such as peptide nucleic acid (PNA) oligomers, that can specifically target any single pathogen. This approach broadens the range of potential targets to any gene with a known sequence in any bacterium, and could significantly reduce the time required to discover new antimicrobials or their redesign, if resistance arises. We review the potential of PNA as an antibacterial molecule. First, we describe the physicochemical properties of PNA and modifications of the PNA backbone and nucleobases. Second, we review the carriers used to transport PNA to bacterial cells. Furthermore, we discuss the PNA targets in antibacterial studies focusing on antisense PNA targeting bacterial mRNA and rRNA.

Endolysins, bacteriophage-encoded peptidoglycan hydrolases, offer promising potential in antibacterial therapy, including treatments targeting gram-negative bacteria. While these enzymes naturally act primarily on gram-positive bacteria, their application against gram-negative pathogens is more challenging due to the presence of a dual-layer cell membrane, which acts as a protective barrier. However, innovative approaches, such as fusing endolysins with antimicrobial peptides (AMPs), have demonstrated increased efficacy against gram-negative bacteria. Modifying endolysins by introducing hydrophobic properties or positive charges or combining them with agents that disrupt the outer membrane enhances their bactericidal activity. Moreover, phage endolysins that exhibit activity against gram-negative bacteria are a promising source of membrane-active peptides. Identifying new peptide sequences derived from endolysins capable of penetrating the bacterial cell membrane represents a novel and increasingly explored research direction. Studying these innovative strategies had yielded promising results, though the field remains under active investigation and development. Ongoing efforts aim to optimize these approaches to improve their effectiveness against antibiotic-resistant gram-negative bacterial strains, which are particularly difficult to treat with conventional antibiotics. This review summarizes the latest advancements and solutions in the field, highlighting the potential of endolysins and membrane-active peptides as next-generation antibacterial agents.

The widespread emergence of bacterial resistance to existing antibiotics forces the development of new therapeutic agents. The use of short modified oligonucleotides, such as peptide nucleic acids (PNAs), seems a promising strategy. However, the uptake of such oligonucleotides is limited by the bacterial cell wall and is species-dependent. Therefore, new carriers for PNAs should be extensively explored. In this study, we examined the antibacterial activity of vitamin B₁₂–PNA conjugates. Vitamin B₁₂ was covalently linked to a PNA oligomer targeted at the mRNA of an essential acpP gene encoding acyl carrier protein in Escherichia coli. PNA–vitamin B₁₂ conjugates were synthesized using the Cu(I)-catalyzed 1,3-dipolar cycloaddition. We examined two types of linkers between vitamin B₁₂ and PNA, including a cleavable disulfide bond. As a positive control for PNA uptake, we used PNA conjugated to the most widely used cell-penetrating peptide (KFF)₃K. We found that vitamin B₁₂–PNA conjugates inhibit E. coli growth at a concentration of 5 μM, similar as (KFF)₃K–PNA. We also showed that vitamin B₁₂–PNA conjugates are stable in the presence of biological media. This study provides the foundation for improving and developing PNAs conjugated to vitamin B₁₂ as antibacterials.