Noncanonical Nucleic Acids under High Pressure and Crowding: Insights from Single-Molecule-FRET

Noncanonical nucleic acid conformations like G-quadruplexes and i-motifs are key regulators of gene function, genome stability, and drug action. Their structural dynamics in extreme conditions of high pressure and molecular crowding are therefore necessary to determine for a clear understanding of their biological role. Single-molecule Förster resonance energy transfer (sm-FRET) has proven to be an invaluable tool to examine these dynamic structures at heterogeneous environments. In this review, we discuss recent advancements in single molecule studies that investigate the impact of high pressure and crowding on noncanonical nucleic acids. We examine how these conditions influence the structural stability, folding kinetics, and conformational transitions, shedding light on their behavior in the crowded and heterogeneous intracellular environment. Through integration of existing knowledge, we provide insights into the broader implications of noncanonical nucleic acid structures in biological systems and their potential applications in biotechnology and medicine. This review underscores the necessity of studying nucleic acids beyond standard conditions to better grasp their functional relevance in vivo.