Release-Independent Transcription from Covalently Cross-Linked Hierarchically Folded DNA Condensates.

Ordered DNA condensates assembled with a block copolymer of PEG-poly(l-lysine) hierarchically fold the supercoiled plasmids into anisotropic "nanoworms" that maintain duplex continuity and display sequence-periodic, single-stranded hinges. After irreversible chemical cross-linking to preclude the potential DNA release, these architecturally preserved nanoscale condensates sustain sequence-specific transcription and coupled luciferase translation at efficiencies equaling or surpassing naked DNA, whereas jetPEI-derived disordered spheres remain transcriptionally silent. In both live cells and animals, eGFP-encoding nanoworm-like DNA condensates elicit robust cytosolic expression without requiring structural disassembly for liberation of the DNA payloads, thereby circumventing the obligatory "uncoat-and-release" step and eliminating the potential insertional-mutagenesis risk. These crystalline, nonviral DNA assemblies are postulated to offer a genome-integration-free platform for safer gene therapy.