As foreshadowed by the COVID-19 pandemic, research on nucleic acids has recently gained renewed attention in the pharmaceutical landscape. Due to their versatile nature and relatively accessible manufacturing, nucleic acids promise to tackle the undruggable, as envisioned for gene therapies and RNA drugs. Thanks to their internal dynamics, these polymers populate the drug discovery process as targets, mediators of cellular responses, and therapeutic entities. Along this line, their pharmacological characterization has developed from different perspectives: from the molecular evaluation of nucleic acid-ligand interactions, through the comprehensive analysis of RNA-mediated cellular responses (i.e., with sequencing-based omics), to the research and development of nucleic acid-based therapeutics. This collection offers an outlook on current technologies and approaches for the bottom-up to a top-down analysis of nucleic acids in drug discovery and development.