Design and Characterization of Compact, Programmable, Multistranded Nonimmunostimulatory Nucleic Acid Nanoparticles Suitable for Biomedical Applications.

We report a thorough investigation of the role of single-stranded thymidine (ssT) linkers in the stability and flexibility of minimal, multistranded DNA nanostructures. We systematically explore the impact of varying the number of ssTs in three-way junction motifs (3WJs) on their formation and properties. Through various UV melting experiments and molecular dynamics simulations, we demonstrate that while the number of ssTs minimally affects thermodynamic stability, the increasing ssT regions significantly enhance the structural flexibility of 3WJs. Utilizing this knowledge, we design triangular DNA nanoparticles with varying ssTs, all showing exceptional assembly efficiency except for the 0T triangle. All triangles demonstrate enhanced stability in blood serum and are nonimmunostimulatory and nontoxic in mammalian cell lines. The 4T 3WJ is chosen as the building block for constructing other polygons due to its enhanced flexibility and favorable physicochemical characteristics, making it a versatile choice for creating cost-effective, stable, and functional DNA nanostructures that can be stored in the dehydrated forms while retaining their structures. Our study provides valuable insights into the design and application of nucleic acid nanostructures, emphasizing the importance of understanding stability and flexibility in the realm of nucleic acid nanotechnology. Our findings suggest the intricate connection between these ssTs and the structural adaptability of DNA 3WJs, paving the way for more precise design and engineering of nucleic acid nanosystems suitable for broad biomedical applications.

Thermodynamic Characterization of Nucleic Acid Nanoparticles Hybridization by UV Melting.

The advances in nucleic acid nanotechnology have given rise to various elegantly designed structural complexes fabricated from DNA, RNA, chemically modified RNA strands, and their mixtures. The structural properties of NA nanoparticles (NANP) generally dictate and significantly impact biological function; and thus, it is critical to extract information regarding relative stabilities of the different structural forms. The adequate stability assessment requires knowledge of thermodynamic parameters that can be empirically derived using conventional UV-melting technique. The focus of this chapter is to describe methodology to evaluate thermodynamic data of NANPs complexation based on DNA 12 base-pair (bp) duplex formation as an example.