The development of approaches to the design of two- and three-dimensional self-assembled DNA-based nanostructures with a controlled shape and size is an essential task for applied nanotechnology, therapy, biosensing, and bioimaging. We conducted a comprehensive study on the formation of various complexes from a pair of oligonucleotides with two transposed complementary blocks that can be linked through a nucleotide or non-nucleotide linker. A methodology is proposed to prove the formation of a self-limited complex and to determine its molecularity. It is based on the "opening" of a self-limited complex with an oligonucleotide that effectively binds to a duplex-forming block. The complexes assembled from a pair of oligonucleotides with different block length and different linker sizes and types were investigated by theoretical analysis, several experimental methods (a gel shift assay, atomic force microscopy, and ultraviolet melting analysis), and molecular dynamics simulations. The results showed a variety of complexes formed by only a pair of oligonucleotides. Self-limited associates, concatemer complexes, or mixtures thereof can arise if we change the length of a duplex and loop-forming blocks in oligonucleotides or via introduction of overhangs and chemical modifications. We postulated basic principles of rational design of native self-limited DNA complexes of desired structure, shape, and molecularity. Our foundation makes self-limited complexes useful tools for nanotechnology, biological studies, and therapeutics.
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