Protein Coating of DNA Nanostructures for Enhanced Stability and Immunocompatibility

Fully addressable DNA nanostructures, especially DNA origami, possess huge potential to serve as inherently biocompatible and versatile molecular platforms. However, their use as delivery vehicles in therapeutics is compromised by their low stability and poor transfection rates. This study shows that DNA origami can be coated by precisely defined one-to-one protein-dendron conjugates to tackle the aforementioned issues. The dendron part of the conjugate serves as a cationic binding domain that attaches to the negatively charged DNA origami surface via electrostatic interactions. The protein is attached to dendron through cysteine-maleimide bond, making the modular approach highly versatile. This work demonstrates the coating using two different proteins: bovine serum albumin (BSA) and class II hydrophobin (HFBI). The results reveal that BSA-coating significantly improves the origami stability against endonucleases (DNase I) and enhances the transfection into human embryonic kidney (HEK293) cells. Importantly, it is observed that BSA-coating attenuates the activation of immune response in mouse primary splenocytes. Serum albumin is the most abundant protein in the blood with a long circulation half-life and has already found clinically approved applications in drug delivery. It is therefore envisioned that the proposed system can open up further opportunities to tune the properties of DNA nanostructures in biological environment, and enable their use in various delivery applications.

[1]  Veikko Linko,et al.  Automated design of DNA origami , 2016, Nature Biotechnology.

[2]  Hao Yan,et al.  DNA origami as a carrier for circumvention of drug resistance. , 2012, Journal of the American Chemical Society.

[3]  Erik Winfree,et al.  Self-assembly of carbon nanotubes into two-dimensional geometries using DNA origami templates. , 2010, Nature nanotechnology.

[4]  Hanadi F Sleiman,et al.  Development of DNA Nanostructures for High-Affinity Binding to Human Serum Albumin. , 2017, Journal of the American Chemical Society.

[5]  Yamuna Krishnan,et al.  Designing DNA nanodevices for compatibility with the immune system of higher organisms. , 2015, Nature nanotechnology.

[6]  F. Simmel,et al.  DNA-based self-assembly of chiral plasmonic nanostructures with tailored optical response , 2011, Nature.

[7]  Shawn M. Douglas,et al.  A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads , 2012, Science.

[8]  William M. Shih,et al.  Virus-Inspired Membrane Encapsulation of DNA Nanostructures To Achieve In Vivo Stability , 2014, ACS nano.

[9]  J. Kjems,et al.  Quantification of cellular uptake of DNA nanostructures by qPCR. , 2014, Methods.

[10]  William M. Shih,et al.  Addressing the Instability of DNA Nanostructures in Tissue Culture , 2014, ACS nano.

[11]  J. Fréchet,et al.  Convergent dendrons and dendrimers: from synthesis to applications. , 2001, Chemical reviews.

[12]  Hao Yan,et al.  DNA Origami with Complex Curvatures in Three-Dimensional Space , 2011, Science.

[13]  David J. Mooney,et al.  Oligolysine-based coating protects DNA nanostructures from low-salt denaturation and nuclease degradation , 2017, Nature Communications.

[14]  Peng Yin,et al.  Casting inorganic structures with DNA molds , 2014, Science.

[15]  Patrick D. Halley,et al.  Daunorubicin-Loaded DNA Origami Nanostructures Circumvent Drug-Resistance Mechanisms in a Leukemia Model. , 2016, Small.

[16]  Olli Ikkala,et al.  Precisely defined protein-polymer conjugates: construction of synthetic DNA binding domains on proteins by using multivalent dendrons. , 2007, ACS nano.

[17]  Hao Yan,et al.  DNA Gridiron Nanostructures Based on Four-Arm Junctions , 2013, Science.

[18]  E. W. Meijer,et al.  About Dendrimers: Structure, Physical Properties, and Applications. , 1999, Chemical reviews.

[19]  M. R. Imam,et al.  Dendron-mediated self-assembly, disassembly, and self-organization of complex systems. , 2009, Chemical reviews.

[20]  Sampo Tuukkanen,et al.  One-step large-scale deposition of salt-free DNA origami nanostructures , 2015, Scientific Reports.

[21]  Shawn M. Douglas,et al.  Self-assembly of DNA into nanoscale three-dimensional shapes , 2009, Nature.

[22]  Friedrich C Simmel,et al.  Electrotransfection of Polyamine Folded DNA Origami Structures. , 2016, Nano letters.

[23]  Shawn M. Douglas,et al.  Folding DNA into Twisted and Curved Nanoscale Shapes , 2009, Science.

[24]  Daniel G. Anderson,et al.  Molecularly Self-Assembled Nucleic Acid Nanoparticles for Targeted In Vivo siRNA Delivery , 2012, Nature nanotechnology.

[25]  P. Rothemund,et al.  Engineering and mapping nanocavity emission via precision placement of DNA origami , 2016, Nature.

[26]  Pekka Orponen,et al.  DNA rendering of polyhedral meshes at the nanoscale , 2015, Nature.

[27]  Hao Yan,et al.  Structural DNA Nanotechnology: State of the Art and Future Perspective , 2014, Journal of the American Chemical Society.

[28]  Sébastien Perrier,et al.  Smart hybrid materials by conjugation of responsive polymers to biomacromolecules. , 2015, Nature materials.

[29]  Hélder A Santos,et al.  Cellular delivery of enzyme-loaded DNA origami. , 2016, Chemical communications.

[30]  Stephen Mann,et al.  Interfacial assembly of protein–polymer nano-conjugates into stimulus-responsive biomimetic protocells , 2013, Nature Communications.

[31]  J. Kjems,et al.  Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin-Receptor Internalization Pathway. , 2016, Small.

[32]  Veikko Linko,et al.  Custom-shaped metal nanostructures based on DNA origami silhouettes. , 2015, Nanoscale.

[33]  Qiao Jiang,et al.  DNA origami as an in vivo drug delivery vehicle for cancer therapy. , 2014, ACS nano.

[34]  Tim Liedl,et al.  Cellular immunostimulation by CpG-sequence-coated DNA origami structures. , 2011, ACS nano.

[35]  H. Wagner,et al.  All is not Toll: new pathways in DNA recognition , 2006, The Journal of experimental medicine.

[36]  C. Niemeyer,et al.  Designed Intercalators for Modification of DNA Origami Surface Properties. , 2015, Chemistry.

[37]  John M. Hoffman,et al.  A helical flow, circular microreactor for separating and enriching "smart" polymer-antibody capture reagents. , 2010, Lab on a chip.

[38]  Veikko Linko,et al.  Cationic polymers for DNA origami coating - examining their binding efficiency and tuning the enzymatic reaction rates. , 2016, Nanoscale.

[39]  Veikko Linko,et al.  DNA-Based Enzyme Reactors and Systems , 2016, Nanomaterials.

[40]  Jiye Shi,et al.  Smart Drug Delivery Nanocarriers with Self‐Assembled DNA Nanostructures , 2013, Advanced materials.

[41]  E. Meijer,et al.  A synthetic "tour de force": well-defined multivalent and multimodal dendritic structures for biomedical applications. , 2011, Angewandte Chemie.

[42]  Antti-Pekka Eskelinen,et al.  Virus-encapsulated DNA origami nanostructures for cellular delivery. , 2014, Nano letters.

[43]  Lars Röglin,et al.  Ein Meisterstück in der Synthese: wohldefinierte, multivalente und multimodale dendritische Architekturen für biomedizinische Anwendungen , 2011 .

[44]  T. G. Martin,et al.  Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures , 2012, Science.

[45]  N. Seeman,et al.  Programmable materials and the nature of the DNA bond , 2015, Science.

[46]  Luvena L. Ong,et al.  Three-Dimensional Structures Self-Assembled from DNA Bricks , 2012, Science.

[47]  V. Linko,et al.  The enabled state of DNA nanotechnology. , 2013, Current opinion in biotechnology.

[48]  En-Wei Lin,et al.  Therapeutic protein-polymer conjugates: advancing beyond PEGylation. , 2014, Journal of the American Chemical Society.

[49]  Baoquan Ding,et al.  Observation of intracellular interactions between DNA origami and lysosomes by the fluorescence localization method. , 2016, Chemical communications.

[50]  Lei Liu,et al.  Routing of individual polymers in designed patterns. , 2015, Nature nanotechnology.

[51]  Adrian Keller,et al.  Regular Nanoscale Protein Patterns via Directed Adsorption through Self-Assembled DNA Origami Masks. , 2016, ACS applied materials & interfaces.

[52]  Michael Matthies,et al.  Block Copolymer Micellization as a Protection Strategy for DNA Origami. , 2017, Angewandte Chemie.

[53]  W. Chiu,et al.  Designer nanoscale DNA assemblies programmed from the top down , 2016, Science.

[54]  J. Kjems,et al.  Self-assembly of a nanoscale DNA box with a controllable lid , 2009, Nature.

[55]  Frank A. Veliz,et al.  Serum albumin 'camouflage' of plant virus based nanoparticles prevents their antibody recognition and enhances pharmacokinetics. , 2016, Biomaterials.

[56]  Ralf Seidel,et al.  Shape-controlled synthesis of gold nanostructures using DNA origami molds. , 2014, Nano letters.

[57]  T. G. Martin,et al.  Facile and Scalable Preparation of Pure and Dense DNA Origami Solutions** , 2014, Angewandte Chemie.

[58]  P. Rothemund.  Folding DNA to create nanoscale shapes and patterns , 2006, Nature.

[59]  Björn Högberg,et al.  DNA origami delivery system for cancer therapy with tunable release properties. , 2012, ACS nano.

[60]  Veikko Linko,et al.  DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices. , 2015, Trends in biotechnology.

[61]  O. Ikkala,et al.  Multivalent dendrons for high-affinity adhesion of proteins to DNA. , 2006, Angewandte Chemie.

[62]  Richard A. Muscat,et al.  DNA nanotechnology from the test tube to the cell. , 2015, Nature nanotechnology.

[63]  H. Dietz,et al.  Placing molecules with Bohr radius resolution using DNA origami. , 2016, Nature nanotechnology.

[64]  A. Turberfield,et al.  DNA-templated protein arrays for single-molecule imaging. , 2011, Nano letters.

[65]  Mark Bathe,et al.  A primer to scaffolded DNA origami , 2011, Nature Methods.