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1. Hybrid DNA/RNA nanostructures with 2′-5′ linkages

   https://doi.org/10.1039/d0nr05846g  

   Chandrasekaran, Arun Richard; Mathivanan, Johnsi; Ebrahimi, Parisa; Vilcapoma, Javier; Chen, Alan A.; Halvorsen, Ken; Sheng, Jia (November 2020, Nanoscale)

   null (Ed.)

   Nucleic acid nanostructures with different chemical compositions have shown utility in biological applications as they provide additional assembly parameters and enhanced stability. The naturally occurring 2′-5′ linkage in RNA is thought to be a prebiotic analogue and has potential use in antisense therapeutics. Here, we report the first instance of DNA/RNA motifs containing 2′-5′ linkages. We synthesized and incorporated RNA strands with 2′-5′ linkages into different DNA motifs with varying number of branch points (a duplex, four arm junction, double crossover motif and tensegrity triangle motif). Using experimental characterization and molecular dynamics simulations, we show that hybrid DNA/RNA nanostructures can accommodate interspersed 2′-5′ linkages with relatively minor effect on the formation of these structures. Further, the modified nanostructures showed improved resistance to ribonuclease cleavage, indicating their potential use in the construction of robust drug delivery vehicles with prolonged stability in physiological conditions. 

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2. Design, optimization and analysis of large DNA and RNA nanostructures through interactive visualization, editing and molecular simulation

   https://doi.org/10.1093/nar/gkaa417  

   Poppleton, Erik; Bohlin, Joakim; Matthies, Michael; Sharma, Shuchi; Zhang, Fei; Šulc, Petr (May 2020, Nucleic Acids Research)

   Abstract This work seeks to remedy two deficiencies in the current nucleic acid nanotechnology software environment: the lack of both a fast and user-friendly visualization tool and a standard for structural analyses of simulated systems. We introduce here oxView, a web browser-based visualizer that can load structures with over 1 million nucleotides, create videos from simulation trajectories, and allow users to perform basic edits to DNA and RNA designs. We additionally introduce open-source software tools for extracting common structural parameters to characterize large DNA/RNA nanostructures simulated using the coarse-grained modeling tool, oxDNA, which has grown in popularity in recent years and is frequently used to prototype new nucleic acid nanostructural designs, model biophysics of DNA/RNA processes, and rationalize experimental results. The newly introduced software tools facilitate the computational characterization of DNA/RNA designs by providing multiple analysis scripts, including mean structures and structure flexibility characterization, hydrogen bond fraying, and interduplex angles. The output of these tools can be loaded into oxView, allowing users to interact with the simulated structure in a 3D graphical environment and modify the structures to achieve the required properties. We demonstrate these newly developed tools by applying them to design and analysis of a range of DNA/RNA nanostructures. 

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3. Modular, Articulated Models of DNA and Peptide Nucleic Acids for Nanotechnology Education

   https://doi.org/10.35459/tbp.2022.000225  

   Goodwin-Schoen, Caleigh M.; Taylor, Rebecca E. (April 2023, The Biophysicist)

   ABSTRACT Dynamic and flexible nucleic acid models can provide current and future scientists with physical intuition for the structure of DNA and the ways that DNA and its synthetic mimics can be used to build self-assembling structures and advanced nanomachines. As more research labs and classrooms dive into the field of structural nucleic acid nanotechnology, students and researchers need access to interactive, dynamic, handheld models. Here, we present a 3D-printable kit for the construction of DNA and peptide nucleic acid (PNA). We have engineered a previous modular DNA kit to reduce costs while improving ease of assembly, flexibility, and robustness. We have also expanded the scope of available snap-together models by creating the first 3D-printable models of γPNA, an emerging material for nuclease- and protease-resistance nanotechnology. Building on previous research, representative nucleic acid duplexes were split into logical monomer segments, and atomic coordinates were used to create solid models for 3D printing. We used a human factors approach to customize 3 types of articulated snap-together connectors that allow for physically relevant motion characteristic of each interface in the model. Modules are easy to connect and separate manually but stay together when the model is manipulated. To greatly reduce cost, we bundled these segments for printing, and we created a miniaturized version that uses less than half the printing material to build. Our novel 3D-printed articulated snap-together models capture the flexibility and robustness of DNA and γPNA nanostructures. Resulting handheld helical models replicate the geometries in published structures and can now flex to form crossovers and allow biologically relevant zipping and unzipping to allow complex demonstrations of nanomachines undergoing strand displacement reactions. Finally, the same tools used to create these models can be readily applied to other types of backbones and nucleobases for endless research and education possibilities. 

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4. Evaluation of Thermodynamic Stabilities of in silico Designed Nucleic Acid 3WJ Motifs

   Abby Coffman, Megan Teter (March 2023, ACS SPRING 2023)

   Nucleic Acid (NA) nanotechnology is a rapidly emerging field demonstrating application of polynucleotides as a versatile biopolymer to fabricate nanostructures of various dimensions and shapes in a programmable and highly predictable way. The folding of DNA or RNA strands into a stable double helix configuration mainly relies on the Watson-Crick (Canonical) base pair composition (G=C and A-T or A-U in the case of RNA), base stacking, and metal ion concentrations. The thermodynamic parameters of DNA B-form helix formation and A-form helix of RNA can be computed using empirically defined sets of nearest neighboring parameters encompassed within the 2D structure predicting programs for example mfold, NUPAC. However, these programs are lacking parameters for a hybrid DNA/RNA base pairing and non-canonical base interactions. In this report, we focused our study to evaluate thermodynamic parameters of several in silico designed three-way junction (3WJ) DNA and hybrid DNA-RNA structural elements. The designed 3WJ motifs contain three helical stems linked with 4,3,2,1, and 0 single stranded Thymidine (T) or Uridine (U) nucleotides. We will report assembly efficiency of the 3WJs investigated by gel shift assay and thermodynamic parameters measured by UV-melting technique. Our experiments reveal that the amount of Ts and Us linkages in the three-way junction dictate the stability of the overall 3WJ conformations. This study is important as we expect it will contribute to the existing set of parameters used for NA structure prediction algorithms as well as provide a guidance for rational design of NA nanostructures. 

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5. Functionalizing DNA nanostructures for therapeutic applications

   https://doi.org/10.1002/wnan.1729  

   Henry, Skylar J. W.; Stephanopoulos, Nicholas (May 2021, WIREs Nanomedicine and Nanobiotechnology)

   Abstract Recent advances in nanotechnology have enabled rapid progress in many areas of biomedical research, including drug delivery, targeted therapies, imaging, and sensing. The emerging field of DNA nanotechnology, in which oligonucleotides are designed to self‐assemble into programmable 2D and 3D nanostructures, offers great promise for further advancements in biomedicine. DNA nanostructures present highly addressable and functionally diverse platforms for biological applications due to their ease of construction, controllable architecture and size/shape, and multiple avenues for chemical modification. Both supramolecular and covalent modification with small molecules and polymers have been shown to expand or enhance the functions of DNA nanostructures in biological contexts. These alterations include the addition of small molecule, protein, or nucleic acid moieties that enable structural stability under physiological conditions, more efficient cellular uptake and targeting, delivery of various molecular cargos, stimulus‐responsive behaviors, or modulation of a host immune response. Herein, various types of DNA nanostructure modifications and their functional consequences are examined, followed by a brief discussion of the future opportunities for functionalized DNA nanostructures as well as the barriers that must be overcome before their translational use. This article is categorized under:Nanotechnology Approaches to Biology > Nanoscale Systems in BiologyTherapeutic Approaches and Drug Discovery > Emerging TechnologiesBiology‐Inspired Nanomaterials > Nucleic Acid‐Based Structures 

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