This study presents the development and evaluation of molecularly self-assembled DNA tetrahedral nanoparticles (ONPs) for targeted in vivo delivery of small interfering RNAs (siRNAs). The ONPs are composed of six single-stranded DNA fragments and six double-stranded siRNAs, which self-assemble into a well-defined tetrahedral structure. This structure allows for precise control over the size, shape, and surface chemistry of the nanoparticles, as well as the spatial orientation and density of targeting ligands such as folate. In vitro studies demonstrated that these ONPs effectively deliver siRNAs to human cancer cells, with optimal gene silencing observed when at least three folate molecules were present per nanoparticle and the ligands were in the appropriate spatial orientation. In vivo experiments in nude mice bearing KB xenograft tumors showed that the ONPs had a longer blood circulation time (t1/2 ~ 24.2 min) compared to free siRNA (t1/2 ~ 6 min), and they accumulated preferentially in the tumor and kidney. Systemic and intra-tumor injections of ONPs resulted in approximately 60% decrease in bioluminescent intensity, confirming their therapeutic potential. The study highlights the advantages of using molecularly self-assembled ONPs for targeted siRNA delivery, offering a promising approach for cancer treatment.This study presents the development and evaluation of molecularly self-assembled DNA tetrahedral nanoparticles (ONPs) for targeted in vivo delivery of small interfering RNAs (siRNAs). The ONPs are composed of six single-stranded DNA fragments and six double-stranded siRNAs, which self-assemble into a well-defined tetrahedral structure. This structure allows for precise control over the size, shape, and surface chemistry of the nanoparticles, as well as the spatial orientation and density of targeting ligands such as folate. In vitro studies demonstrated that these ONPs effectively deliver siRNAs to human cancer cells, with optimal gene silencing observed when at least three folate molecules were present per nanoparticle and the ligands were in the appropriate spatial orientation. In vivo experiments in nude mice bearing KB xenograft tumors showed that the ONPs had a longer blood circulation time (t1/2 ~ 24.2 min) compared to free siRNA (t1/2 ~ 6 min), and they accumulated preferentially in the tumor and kidney. Systemic and intra-tumor injections of ONPs resulted in approximately 60% decrease in bioluminescent intensity, confirming their therapeutic potential. The study highlights the advantages of using molecularly self-assembled ONPs for targeted siRNA delivery, offering a promising approach for cancer treatment.