Alternative nucleic acid structures in microbial biology: regulatory functions and antimicrobial opportunities

The rapid emergence and global dissemination of antimicrobial resistance (AMR) pose a critical threat to public health, necessitating the identification of unconventional biological targets and regulatory mechanisms in microbial pathogens. Beyond the canonical Watson-Crick double helix, nucleic acids can adopt a wide range of alternative DNA and RNA structures, including G-quadruplexes, Z-DNA, cruciforms, triplex DNA, R-loops, and selected RNA structures, including RNA G-quadruplexes and RNA-DNA hybrids. These structures are increasingly recognized as dynamic and functional elements in microbial genomes and transcriptomes, where they influence gene regulation, genome stability, stress adaptation, and pathogenicity. Importantly, many of these processes are directly linked to antibiotic tolerance, persistence, and resistance evolution. This review provides a comprehensive and critical overview of alternative nucleic acid structures in bacteria, archaea, and microbial eukaryotes, with a particular emphasis on their relevance to antibiotic resistance and antimicrobial drug development. We summarize updated knowledge of their formation, biological roles, and regulatory mechanisms, discuss emerging methodologies for their detection in vivo, and highlight their potential as novel antimicrobial targets. By integrating structural nucleic acid biology with infection microbiology, this review aims to frame non-canonical nucleic acid architectures as an underexploited layer of microbial regulation with significant translational potential.