7/24/2023 0 Comments Sticky note origami pdf![]() Here we investigate the determinants for designing the sequences of synthetic DNA origami scaffolds and the structures of DNA origami objects, so that genes encoded within DNA molecules in these objects become expressible by mammalian cells. 44 recently demonstrated delivery and integration of template DNA for gene editing structured within a DNA origami, bound together with Cas9 ribonuclear proteins (RNPs). Gene therapeutic applications have been approached thus far with DNA origami hybrid objects featuring additional RNA or proteins, which bypasses the need for expression from nucleic acids 41, 42, 43, 44. With the development of techniques for building fully sequence programmable synthetic scaffolds for DNA origami 40, however, these limitations can be overcome and designing synthetic mammalian-cell expressible gene cassettes and folding them into custom DNA origami objects has become more accessible. DNA origami objects are also often designed around generic single-stranded “scaffolds” derived from bacteriophage genomes that are not suited for gene expression in mammalian cells. Commonly, the sequences of the DNA molecules in DNA origami are designed for purely structural purposes, that is, to enable folding of the target object and for positioning guest molecules via site-specific DNA hybridization reactions. The capability for designing shapes and controlling their conformations and functionalization, and the option for positioning non-DNA components with nanometer-scale precision has popularized DNA origami and led to the pursuit of applications in a variety of fields 25 including drug delivery 30, 31, 32, immunotherapy 33, 34, 35, sensing and imaging 36, 37, 38, 39. DNA origami methods also allow the rational design of a great variety of stimuli-dependent reconfigurable assemblies 16, 25, that is, objects where the type of conformational change such as opening/closing of a cavity, piston-like actuation, rotation, and also the type of stimulus (hydrophobicity 26, ionic strength and temperature changes 16, pH changes 27, RNA detection 28, antigen detection 29) can be defined by the user. SNARE-protein-based fusion of solid-supported lipid membranes with lipid vesicles has been induced in vitro with a DNA origami platform 24. DNA nanostructures can also deform lipid membranes 20, 21, they can form channels in lipid membranes 22, and they can be enveloped within lipid membranes 23. DNA origami objects can be site-selectively functionalized with proteins such as antibodies, aptamers, and organic small molecules but also with inorganic particles 18, 19. DNA origami methodologies also allow making well-defined higher-order macromolecular objects with dimensions up to the size of viruses 14, 16, 17. ![]() ![]() DNA origami objects can comprise hundreds of unique DNA strands and thousands of DNA base pairs, and can form with high-yield and high-quality 12, 13, 14, 15. DNA origami is a popular design approach 9, 10, 11, in which a long template DNA single strand termed “scaffold” is folded into custom shapes by sets of DNA oligonucleotides with designed sequences (termed “staples”). The methods of DNA nanotechnology enable the rational design of custom-shaped objects that self-assemble in solution from sets of DNA molecules 8. Additional challenges arise for the simultaneous delivery and expression of multiple custom genes to cells, which could be desirable for yet more advanced genome or epigenome editing applications, for transcriptional modulation, and/or the programming of new genetic circuits 3, 4, 5, 6, 7. ![]() Packaging, delivering, and expressing the desired nucleic acids often must be addressed in an application-specific manner 2. The successful delivery of genetic material to specific cells or tissues continues to be a significant challenge. The delivery and expression of custom genes in cells drive major scientific discoveries and underpin a growing number of medical and technical applications 1.
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