Precise Coating of a Wide Range of DNA Templates by a Protein Polymer with a DNA Binding Domain.

Emerging DNA-based nanotechnologies would benefit from the ability to modulate the properties (e.g., solubility, melting temperature, chemical stability) of diverse DNA templates (single molecules or origami nanostructures) through controlled, self-assembling coatings. We here introduce a DNA coating agent, called C8-BSso7d, which binds to and coats with high specificity and affinity, individual DNA molecules as well as folded origami nanostructures. C8-BSso7d coats and protects without condensing, collapsing or destroying the spatial structure of the underlying DNA template. C8-BSso7d combines the specific nonelectrostatic DNA binding affinity of an archeal-derived DNA binding domain (Sso7d, 7 kDa) with a long hydrophilic random coil polypeptide (C8, 73 kDa), which provides colloidal stability (solubility) through formation of polymer brushes around the DNA templates. C8-BSso7d is produced recombinantly in yeast and has a precise (but engineerable) amino acid sequence of precise length. Using electrophoresis, AFM, and fluorescence microscopy we demonstrate protein coat formation with stiffening of one-dimensional templates (linear dsDNA, supercoiled dsDNA and circular ssDNA), as well as coat formation without any structural distortion or disruption of two-dimensional DNA origami template. Combining the programmability of DNA with the nonperturbing precise coating capability of the engineered protein C8-BSso7d holds promise for future applications such as the creation of DNA-protein hybrid networks, or the efficient transfection of individual DNA nanostructures into cells.

[1]  Thomas H. LaBean,et al.  Constructing novel materials with DNA , 2007 .

[2]  Stefan Knapp,et al.  Architecture of nonspecific protein–DNA interactions in the Sso7d–DNA complex , 1998, Nature Structural Biology.

[3]  L. Lim,et al.  The crystal structure of the hyperthermophile chromosomal protein Sso7d bound to DNA , 1998, Nature Structural Biology.

[4]  S Povey,et al.  Dynamic molecular combing: stretching the whole human genome for high-resolution studies. , 1997, Science.

[5]  B. Kundukad,et al.  Control of the flow properties of DNA by topoisomerase II and its targeting inhibitor. , 2010, Biophysical journal.

[6]  P. S. Ho,et al.  The Holliday junction in an inverted repeat DNA sequence: sequence effects on the structure of four-way junctions. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[7]  A Bensimon,et al.  Alignment and sensitive detection of DNA by a moving interface. , 1994, Science.

[8]  P. Bergquist,et al.  Solid-binding peptides: smart tools for nanobiotechnology. , 2015, Trends in biotechnology.

[9]  F. D. de Wolf,et al.  Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris. , 2001, Protein engineering.

[10]  R. Carbonell,et al.  Design of pH Sensitive Binding Proteins from the Hyperthermophilic Sso7d Scaffold , 2012, PloS one.

[11]  P. Kwok,et al.  Genome mapping on nanochannel arrays for structural variation analysis and sequence assembly , 2012, Nature Biotechnology.

[12]  R. C. Wright,et al.  Highly stable binding proteins derived from the hyperthermophilic Sso7d scaffold. , 2011, Journal of molecular biology.

[13]  M. C. Stuart,et al.  Coating of single DNA molecules by genetically engineered protein diblock copolymers. , 2012, Small.

[14]  Cees Dekker,et al.  Electrophoretic force on a protein-coated DNA molecule in a solid-state nanopore. , 2009, Nano letters.

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

[16]  B. Kundukad,et al.  Viscoelasticity of entangled lambda-phage DNA solutions. , 2008, The Journal of chemical physics.

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

[18]  S. Edmondson,et al.  The hyperthermophile chromosomal protein Sac7d sharply kinks DNA , 1998, Nature.

[19]  J. A. van Kan,et al.  Amplified stretch of bottlebrush-coated DNA in nanofluidic channels , 2013, Nucleic acids research.

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

[21]  U Deva Priyakumar,et al.  Crenarchaeal chromatin proteins Cren7 and Sul7 compact DNA by inducing rigid bends , 2012, Nucleic acids research.

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

[23]  M. C. Stuart,et al.  Pearl-necklace complexes of flexible polyanions with neutral–cationic diblock copolymers , 2013 .

[24]  H. Kumar,et al.  Biopolymers in nanopores: challenges and opportunities , 2011 .

[25]  P. B. Vander Horn,et al.  A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. , 2004, Nucleic acids research.

[26]  S. Knapp,et al.  DNA-binding surface of the Sso7d protein from Sulfolobus solfataricus. , 1995, Journal of molecular biology.

[27]  David Pastré,et al.  High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein—DNA complexes , 2007, Nucleic acids research.

[28]  P. Forterre,et al.  DNA bending, compaction and negative supercoiling by the architectural protein Sso7d of Sulfolobus solfataricus. , 2002, Nucleic acids research.

[29]  Patrick K. Martin,et al.  Biochemical characterization of DNA-binding proteins from Pyrobaculum aerophilum and Aeropyrum pernix , 2008, Extremophiles.

[30]  K. Brown,et al.  Combinatorial peptide libraries: mining for cell-binding peptides. , 2014, Chemical reviews.

[31]  Leslie Y Yeo,et al.  Microfluidic devices for bioapplications. , 2011, Small.

[32]  S. Edmondson,et al.  DNA binding proteins Sac7d and Sso7d from Sulfolobus. , 2001, Methods in enzymology.

[33]  S. Knapp,et al.  Solution structure and DNA-binding properties of a thermostable protein from the archaeon Sulfolobus solfataricus , 1994, Nature Structural Biology.