Various ground-breaking and promising technologies such as RNA interference (RNAi), CRISPR/Cas genome editing, gene including suicide gene therapy, somatic cell reprogramming, CAR T-cell therapy, and genetic vaccination, depend on efficient delivery of functional nucleic acids into target cells. Our lab has been specializing on the computational design of functional RNA and on technologies to overcome the cellular, nuclear and mitochondrial membrane barriers. Functional RNAs can be selected in silico from large sequence and structure spaces based on molecular signatures that correlate with activity. Using this approach, we could improve various RNA technologies including antisense, RNAi, RNA trans-splicing and CRISPR/Cas by orders of magnitude. For cellular and nuclear delivery, we developed non-viral, non-integrating dumbbell-shaped DNA minimal vectors which are not silenced in primary cells, and which can be featured with helper functions such as GalNAc3 residues or aptamers for targeted delivery. Our advanced dumbbell vectors exhibit 6- or 95-fold facilitated nuclear targeting and up to 60- or 160-fold enhanced gene expression compared with conventional dumbbell vectors (also called doggybones, ceDNA or hpDNA) or plasmids. We explore such dumbbells to deliver trans-splicing-based suicide RNAs for suicide gene therapy targeting cancer and virus infection. Our suicide vectors have been tested in domestic pigs and are currently tested in different mouse models. To overcome the mitochondrial double-membrane, we developed a novel RNA based vector that can deliver functional RNA and/or DNA into the mitochondria of human cells without any cargo size limitation. Using this vector, we could trigger or knockdown mitochondrial gene expression in vitro. We are exploring this technology for mitochondrial gene therapy which is currently hampered by the lack of an efficient mitochondrial delivery vector.