Transmembrane-spanning nanopores have emerged as powerful tools in a wide range of technological applications, particularly in single-molecule sensing. This review explores recent advancements in creating synthetic, membrane-spanning nanopores constructed from nucleic acids, focusing on DNA nanopores. These self-assembled nanochannels offer a highly programmable and versatile alternative to traditional protein-based nanopores. We summarize the rational design principles and examine advantages and disadvantages of diverse architectures ranging from subnanometer channels for selective ion translocation to customizable geometries for the transport of macromolecules. Key aspects of this emerging field are discussed, including methods for membrane anchoring, the influence of lipid rearrangements on ionic conductance, and the dynamic control of nanopore function. Nucleic acid nanopores are further highlighted as functional components for synthetic cell signaling, single-molecule detection, and cellular manipulation. This review concludes with an outlook on the field, focusing in particular on the unique opportunities of RNA origami for creating genetically encodable nanopores for bottom-up synthetic biology.