A　major　impediment　to　the　clinical　translation　of　DNA　tiling　nanostructures　is　a　technical　bottleneck　for　the　programmable　assembly　of　DNA　architectures　with　well-defined　local　geometry　due　to　the　inability　to　achieve　both　sufficient　structural　rigidity　and　a　large　framework.　In　this　work,　a　Y-backbone　was　inserted　into　each　face　to　construct　a　superlarge,　sufficiently　rigidified　tetrahedral　DNA　nanostructure　(called　RDT)　with　extremely　high　efficiency.　In　RDT,　the　spatial　size　increased　by　6.86-fold,　and　the　structural　rigidity　was　enhanced　at　least　4-fold,　contributing　to　an　∼350-fold　improvement　in　the　resistance　to　nucleolytic　degradation　even　without　a　protective　coating.　RDT　can　be　mounted　onto　an　artificial　lipid-bilayer　membrane　with　molecular-level　precision　and　well-defined　spatial　orientation　that　can　be　validated　using　the　fluorescence　resonance　energy　transfer　(FRET)　assay.　The　spatial　orientation　of　Y-shaped　backbone-rigidified　RDT　is　unachievable　for　conventional　DNA　polyhedrons　and　ensures　a　high　level　of　precision　in　the　geometric　positioning　of　diverse　biomolecules　with　an　approximately　homogeneous　environment.　In　tests　of　RDT,　surface-confined　horseradish　peroxidase　(HRP)　exhibited　nearly　100%　catalytic　activity　and　targeting　aptamer-immobilized　gold　nanoparticles　showed　5.3-fold　enhanced　cellular　internalization.　Significantly,　RDT　exhibited　a　27.5-fold　enhanced　structural　stability　in　a　bodily　environment　and　did　not　induce　detectable　systemic　toxicity.　©　2024　American　Chemical　Society