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      Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation

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          Abstract

          DNA nanostructures have evoked great interest as potential therapeutics and diagnostics due to ease and robustness of programming their shapes, site-specific functionalizations and responsive behaviours. However, their utility in biological fluids can be compromised through denaturation induced by physiological salt concentrations and degradation mediated by nucleases. Here we demonstrate that DNA nanostructures coated by oligolysines to 0.5:1 N:P (ratio of nitrogen in lysine to phosphorus in DNA), are stable in low salt and up to tenfold more resistant to DNase I digestion than when uncoated. Higher N:P ratios can lead to aggregation, but this can be circumvented by coating instead with an oligolysine-PEG copolymer, enabling up to a 1,000-fold protection against digestion by serum nucleases. Oligolysine-PEG-stabilized DNA nanostructures survive uptake into endosomal compartments and, in a mouse model, exhibit a modest increase in pharmacokinetic bioavailability. Thus, oligolysine-PEG is a one-step, structure-independent approach that provides low-cost and effective protection of DNA nanostructures for in vivo applications.

          Abstract

          The instability of DNA nanostructures in physiological environments has hampered their use as therapeutics and diagnostic agents in in vivo applications. Here, the authors show that coating DNA origami with oligolysine-PEG moieties improves their pharmacokinetic properties in mouse models.

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          Most cited references29

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          Self-assembly of DNA into nanoscale three-dimensional shapes

          Molecular self-assembly offers a ‘bottom-up’ route to fabrication with subnanometre precision of complex structures from simple components1. DNA has proven a versatile building block2–5 for programmable construction of such objects, including two-dimensional crystals6, nanotubes7–11, and three-dimensional wireframe nanopolyhedra12–17. Templated self-assembly of DNA18 into custom two-dimensional shapes on the megadalton scale has been demonstrated previously with a multiple-kilobase ‘scaffold strand’ that is folded into a flat array of antiparallel helices by interactions with hundreds of oligonucleotide ‘staple strands’19, 20. Here we extend this method to building custom three-dimensional shapes formed as pleated layers of helices constrained to a honeycomb lattice. We demonstrate the design and assembly of nanostructures approximating six shapes — monolith, square nut, railed bridge, genie bottle, stacked cross, slotted cross — with precisely controlled dimensions ranging from 10 to 100 nm. We also show hierarchical assembly of structures such as homomultimeric linear tracks and of heterotrimeric wireframe icosahedra. Proper assembly requires week-long folding times and calibrated monovalent and divalent cation concentrations. We anticipate that our strategy for self-assembling custom three-dimensional shapes will provide a general route to the manufacture of sophisticated devices bearing features on the nanometer scale.
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            A logic-gated nanorobot for targeted transport of molecular payloads.

            We describe an autonomous DNA nanorobot capable of transporting molecular payloads to cells, sensing cell surface inputs for conditional, triggered activation, and reconfiguring its structure for payload delivery. The device can be loaded with a variety of materials in a highly organized fashion and is controlled by an aptamer-encoded logic gate, enabling it to respond to a wide array of cues. We implemented several different logical AND gates and demonstrate their efficacy in selective regulation of nanorobot function. As a proof of principle, nanorobots loaded with combinations of antibody fragments were used in two different types of cell-signaling stimulation in tissue culture. Our prototype could inspire new designs with different selectivities and biologically active payloads for cell-targeting tasks.
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              Self-assembly of a nanoscale DNA box with a controllable lid.

              The unique structural motifs and self-recognition properties of DNA can be exploited to generate self-assembling DNA nanostructures of specific shapes using a 'bottom-up' approach. Several assembly strategies have been developed for building complex three-dimensional (3D) DNA nanostructures. Recently, the DNA 'origami' method was used to build two-dimensional addressable DNA structures of arbitrary shape that can be used as platforms to arrange nanomaterials with high precision and specificity. A long-term goal of this field has been to construct fully addressable 3D DNA nanostructures. Here we extend the DNA origami method into three dimensions by creating an addressable DNA box 42 x 36 x 36 nm(3) in size that can be opened in the presence of externally supplied DNA 'keys'. We thoroughly characterize the structure of this DNA box using cryogenic transmission electron microscopy, small-angle X-ray scattering and atomic force microscopy, and use fluorescence resonance energy transfer to optically monitor the opening of the lid. Controlled access to the interior compartment of this DNA nanocontainer could yield several interesting applications, for example as a logic sensor for multiple-sequence signals or for the controlled release of nanocargos.
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group
                2041-1723
                31 May 2017
                2017
                : 8
                : 15654
                Affiliations
                [1 ]Department of Cancer Biology, Dana-Farber Cancer Institute , 450 Brookline Avenue, Boston, Massachusetts 02215, USA
                [2 ]Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School , Boston, Massachusetts 02115, USA
                [3 ]Wyss Institute for Biologically Inspired Engineering at Harvard , Boston, Massachusetts 02115, USA
                [4 ]Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology , Seoul 02792, Republic of Korea
                [5 ]Centre for DNA Nanotechnology, Interdisciplinary Nanoscience Center, iNANO, Aarhus University, Gustav Wieds Vej 14 , 8000 Aarhus C, Denmark
                [6 ]School of Engineering and Applied Sciences, Harvard University , Cambridge Massachusetts 02138, USA
                Author notes
                Article
                ncomms15654
                10.1038/ncomms15654
                5460023
                28561045
                22d8e98c-f01a-4822-925d-d91b4d38672b
                Copyright © 2017, The Author(s)

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 08 September 2016
                : 13 April 2017
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