WO2010019725A2 - Polypeptides nœuds pour un ensemble nanostructure - Google Patents

Polypeptides nœuds pour un ensemble nanostructure Download PDF

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Publication number
WO2010019725A2
WO2010019725A2 PCT/US2009/053628 US2009053628W WO2010019725A2 WO 2010019725 A2 WO2010019725 A2 WO 2010019725A2 US 2009053628 W US2009053628 W US 2009053628W WO 2010019725 A2 WO2010019725 A2 WO 2010019725A2
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Prior art keywords
node
nanostructure
streptavidin
protein
amino acid
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PCT/US2009/053628
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English (en)
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WO2010019725A8 (fr
WO2010019725A3 (fr
WO2010019725A9 (fr
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Francis R. Salemme
Mark A. Rould
Patricia C. Weber
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Salemme Francis R
Rould Mark A
Weber Patricia C
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Application filed by Salemme Francis R, Rould Mark A, Weber Patricia C filed Critical Salemme Francis R
Publication of WO2010019725A2 publication Critical patent/WO2010019725A2/fr
Publication of WO2010019725A8 publication Critical patent/WO2010019725A8/fr
Publication of WO2010019725A3 publication Critical patent/WO2010019725A3/fr
Priority to US12/892,911 priority Critical patent/US9102526B2/en
Publication of WO2010019725A9 publication Critical patent/WO2010019725A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • Cambrios uses virus structures for material sciences applications (www.cambrios.com).
  • a method of making a nanostructure node by operating on the 3-dimensional structure of a member of a list of multimeric node template proteins derived from thermostable organisms, to define the amino sequence of nodes that can form nanoassemblies incorporating multimeric nodes and streptavidin or streptavidin-incorporating struts attached with defined stoichiometry and orientation.
  • a method of making a nanostructure node by operating on the 3-dimensional structure of a member of a list of multimeric node template proteins with Cn symmetry derived from thermostable organisms, to define the amino sequence of nodes that can form planar nanoassemblies incorporating Cn planar nodes and streptavidin or streptavidin-incorporating struts attached with defined stoichiometry and orientation.
  • a method of making a nanostructure node by operating on the 3-dimensional structure of a member of a list of multimeric node template proteins with Cn symmetry derived from thermostable organisms, using an aligned search procedure with a relative rotational increment of between 0.001 and 5 degrees to define the amino sequence of nodes that can form planar nanoassemblies incorporating Cn planar nodes and streptavidin or streptavidin- incorporating struts attached with defined stoichiometry and orientation.
  • a method of making an optimal nanostructure node by operating on the 3- dimensional structure of a member of a list of multimeric node template proteins with Cn symmetry derived from thermostable organisms to define the amino sequence of nodes that can form planar nanoassemblies incorporating Cn planar nodes and streptavidin or streptavidin- incorporating struts attached with defined stoichiometry and orientation.
  • a method of making an optimal nanostructure node that is produced through expression in an E, coli bacterium or another heterologous protein expression system [0021] A method of making an optimal planar nanostructure node by using computer graphics, mathematical, or experimental methods of improving the interface interactions between a Cn polyhedral node and streptavidin resulting in modified node protein amino acid sequences.
  • a method of making a nanostructure node by operating on the 3-dimensional structure of a member of a list of multimeric proteins with Cn symmetry derived from thermostable organisms, to define the amino sequence of nodes that can form polyhedral nanoassemblies incorporating streptavidin or streptavidin-incorporating struts connected to nodes with geometry and stoichiometry corresponding to the apex of a regular polyhedron.
  • a method of making an optimal nanostructure node by operating on the 3- dimensional structure of a member of a list of multimeric proteins with Cn symmetry derived from thermostable organisms, to define the amino sequence of nodes that can form polyhedral nanoassemblies incorporating streptavidin or streptavidin-incorporating struts connected to nodes with geometry and stoichiometry corresponding to the apex of a regular polyhedron.
  • a method of making an optimal polyhedral nanostructure node by using computer graphics, mathematical, or experimental methods of improving the interface interactions between a Cn polyhedral node and streptavidin resulting in modified node protein amino acid sequences.
  • a method of making nanostructure nodes by operating on the 3-dimensional structure of a member of a list of multimeric proteins with Dn or higher symmetry derived from thermostable organisms, to define the amino sequence of nanostructure nodes that can form nanoassemblies incorporating streptavidin or streptavidin-incorporating struts connected to nodes with defined geometry and stoichiometry along node dyad symmetry axes.
  • a method of making optimal nanostructure nodes by operating on the 3- dimensional structure of a member of a list of multimeric proteins with Dn or higher symmetry derived from thermostable organisms, to define the optimal amino sequence of nodes that can form nanoassemblies incorporating streptavidin or streptavidin-incorporating struts connected to nodes with defined geometry and stoichiometry along node dyad symmetry axes.
  • a nanostructure node generated from a template multimeric protein with Cn, Dn, or higher symmetry, derived from a list of known three-dimensional protein structures with corresponding symmetry as determined from X-ray crystallography, that incorporates specific attachment sites for nanostructure struts with predefined stoichiometry and orientation, and is derived from a thermophilic microorganism, as a nanostructure node.
  • a nanostructure node generated from a template multimeric protein with Cn, Dn, or higher symmetry, derived from a protein that is homologous to one derived from a list of known three-dimensional protein structures with corresponding symmetry as determined from X-ray crystallography, that incorporates specific attachment sites for nanostructure struts with predefined stoichiometry and orientation, and is derived from a thermophilic microorganism.
  • a protein node where at least one subunit polypeptide chain has been modified through reaction with a bifunctional reagent to incorporate additional binding or other functionality into the node polypeptide chain.
  • a protein node where at least one subunit polypeptide chain has been modified through covalent incorporation of a polypeptide chain sequence coding for protein binding or other functionality.
  • a protein node with subunit polypeptide chains related by Cn a protein node with subunit polypeptide chains related by Cn
  • a protein node with subunit polypeptide chains related by Cn a protein node with subunit polypeptide chains related by Cn
  • Dn or higher symmetry where some subunits have been covalently interconnected to form a protein multimer with a reduced number of polypeptide chains, and modified to incorporate specific binding sites for chemical modification leading to the covalent attachment of biotin groups.
  • a protein node with subunit polypeptide chains related by Cn a protein node with subunit polypeptide chains related by Cn
  • a protein node with subunit polypeptide chains related by Cn symetry where two of the subunits have been covalently interconnected to form a protein multimer with a reduced number of polypeptide chains, and modified to incorporate specific binding sites for chemical modification leading to the covalent attachment of biotin groups.
  • a planar C3 node based on the pdb code:lthj trimer whose subunits have been interconnected using a short polypeptide linker to form a single polypeptide chain or homologues therof.
  • a planar C3 node based on amino acid sequences that are homologous to the pdb code:lthj trimer whose subunits have been interconnected using a short polypeptide linker to form a single polypeptide chain.
  • a planar protein node based on the template protein pdb code a planar protein node based on the template protein pdb code:
  • Ij5s incorporating three subunit polypeptide chains related by C3 symmetry, and incorporating cysteine amino acid residues as reactive sites for the covalent attachment of biotin groups, subsequently allowing C3 symmetric interconnection with 3 streptavidin tetramers in a planar orientation.
  • a planar protein node based on the template protein pdb code: lvcg incorporating four subunit polypeptide chains related by C4 symmetry, where each subunit incorporates cysteine amino acid residues as reactive sites for the covalent attachment of biotin groups, subsequently allowing C4 symmetric interconnection with 4 streptavidin tetramers in a planar orientation.
  • a planar protein node based on the template protein pdb code:2cuO incorporating four subunit polypeptide chains related by C4 symmetry, where each subunit has been modified according to a computer graphical or mathematical method to define and incorporate two cysteine amino residues as reactive sites for the covalent attachment of biotin groups, subsequently allowing C4 symmetric interconnection with 4 streptavidin tetramers in a planar orientation.
  • a planar protein node based on the template node protein pdb code: lvdh that incorporates five subunit polypeptide chains related by C5 symmetry, and where each subunit incorporates two cysteine amino acid residues, as determined using a computer graphics or mathematical method, as reactive sites for the covalent attachment of biotin groups, subsequently allowing C5 symmetric interconnection with 5 streptavidin tetramers in a planar orientation.
  • a planar protein node based on the template node sequence pdb code: 2ekd that incorporates six subunit polypeptide chains related by C6 symmetry, and where each subunit incorporates two cysteine amino acid residues, as determined using a computer graphics or mathematical method, as reactive sites for the covalent attachment of biotin groups, subsequently allowing C6 symmetric interconnection with 6 streptavidin tetramers in a planar orientation.
  • a polyhedral protein node incorporating three subunit polypeptide chains related by C3 symmetry, based on the template protein pdb code: Iv4n, and incorporating specific binding sites for chemical modification leading to the covalent attachment of biotin groups, subsequently allowing interconnection with 3 streptavidin tetramers in an orientation corresponding to the apex of a dodecahedron.
  • a polyhedral protein node incorporating three subunit polypeptide chains related by C3 symmetry, based on the template protein pdb code: Iv4n, and incorporating specific binding sites for chemical modification leading to the covalent attachment of biotin groups, subsequently allowing interconnection with 3 streptavidin tetramers in an orientation corresponding to the apex of a truncated icosahedron or "bucky ball" structure.
  • a polyhedral protein node incorporating five subunit polypeptide chains related by C5 symmetry, based on the template protein pdb code:lvdh, and incorporating specific binding sites for chemical modification leading to the covalent attachment of biotin groups, subsequently allowing interconnection with 5 streptavidin tetramers in an orientation corresponding to the apex of an icosahedron.
  • a protein node based on the hexameric D3-symmetric node template pdb code:lb4b.
  • Such nodes have utility in the formation of 2-dimensional and 3- dimensional hexagonal lattices.
  • a protein node based on the hexameric D3-symmetric node template pdb code:lhyb. Such nodes have utility in the formation of 2-dimensional and 3- dimensional hexagonal lattices.
  • a protein node based on the hexameric D3-symmetric node template pdb code:2prd. Such nodes have utility in the formation of 2-dimensional and 3- dimensional hexagonal lattices.
  • a protein node based on the octameric D4-symmetric node template pdb code:lo4v.
  • Such nodes have utility in the formation of 2-dimensional and 3- dimensional lattices with tetragonal node symmetry.
  • a protein node based on the octameric D4-symmetric node template pdb code:2h2i.
  • Such nodes have utility in the formation of 2-dimensional and 3- dimensional lattices with tetragonal node symmetry.
  • a protein node based on the octameric D4-symmetric node template pdb code:2iel.
  • Such nodes have utility in the formation of 2-dimensional and 3- dimensional lattices with tetragonal node symmetry.
  • a protein node based on the dodecameric tetrahedral T23- symmetric node template pdb code:lpw.
  • Such nodes have utility in the formation of 3- dimensional lattices with cubic symmetry.
  • modified forms of the D2-symmetric, tetrameric protein streptavidin (pdb code:lstp), where cysteine residues have been introduced along tetramer dyad axes to protect biotin binding sites or allow subsequent in situ functionalization of nanostructures incorporating streptavidin struts.
  • an extended strut composed of a protein node based on a tetrameric D2-symmetric node template pdb code: lmal complexed with two streptavidin tetramers to form an extended nanostructure strut.
  • Composition of Matter Assemblies with a Nanostructure Node
  • a nanostructure assembly geometry incorporating Cn- symmetric or Dn symmetric nodes and streptavidin or streptavidin-incorporating (or streptavidin derivative, or avidin, or avidin derivative) struts.
  • a nanostructure assembly incorporating streptavidin or streptavidin-incorporating struts together with Cn-symmetric or Dn symmetric nodes based on node templates derived from a list of known three-dimensional protein structures with corresponding symmetry as determined from X-ray crystallography, and that incorporate specific attachment sites for nanostructure struts with predefined stoichiometry and orientation, and are derived from thermophilic microorganisms.
  • a nanostructure assembly incorporating streptavidin or streptavidin-incorporating struts together with Cn-symmetric or Dn symmetric nodes based on templates that are amino acid sequence homologs of structures derived from a list of known three-dimensional protein structures with corresponding symmetry as determined from X-ray crystallography, and that incorporate specific attachment sites for nanostructure struts with predefined stoichiometry and orientation, and are derived from thermophilic microorganisms.
  • a nanostructure assembly incorporating streptavidin or streptavidin-incorporating struts together with D2 symmetric nodes that are based on a modified forms of streptavidin that incorporate specific attachment sites for nanostructure struts with predefined stoichiometry and orientation.
  • a nanostructure assembly incorporating Cn-symmetric or Dn symmetric nodes and streptavidin or streptavidin-incorporating struts.
  • the nanostructure may be functionalized through the incorporation of node constructs that have been modified either through reaction with a bifunctional reagent to incorporate additional binding or other functionality into the node polypeptide chain, or where node subunits have been modified through covalent incorporation of a polypeptide chain sequence coding for protein binding or other functionality.
  • a nanostructure assembly incorporating Cn-symmetric or Dn symmetric nodes and streptavidin or streptavidin-incorporating struts taking the geometrical form of a radial planar array.
  • a nanostructure with 2-dimensional polygonal geometry incorporating Cn-symmetric nodes and streptavidin or streptavidin-incorporating struts.
  • a nanostructure with 2-dimensional polygonal geometry incorporating single-chain Cn-symmetric nodes and streptavidin or streptavidin-incorporating struts.
  • a nanostructure with 2-dimensional hexagonal polygonal geometry incorporating single-chain C3-symmetric nodes and streptavidin or streptavidin- incorporating struts is provided.
  • a nanostructure with 2-dimensional square polygonal geometry incorporating single-chain C4-symmetric nodes and streptavidin or streptavidin- incorporating struts is provided.
  • a 2-dimensional lattice incorporating Cn-symmetric nodes and streptavidin or streptavidin-incorporating struts [0080] In an embodiment, a 2-dimensional lattice incorporating Cn-symmetric nodes and streptavidin or streptavidin-incorporating struts. [0081] In an embodiment, a 2-dimensional lattice incorporating Dn-symmetric nodes and streptavidin or streptavidin-incorporating struts.
  • a 2-dimensional hexagonal lattice incorporating C3- symmetric nodes and streptavidin or streptavidin-incorporating struts.
  • a 2-dimensional square lattice incorporating C4-symmetric nodes homologous to node template sequences corresponding to the pdb coderlvcg protein tetramer and streptavidin or streptavidin-incorporating struts.
  • a 3-dimensional radial nanostructure incorporating a node derived from thermophilic node templates with Dn, tetrahedral (T23), cubeoctahedral (432), or with icosahedral/dodecahedral (532) symmetry derived from a thermophilic organism, and Dn symmetric nodes and streptavidin or streptavidin-incorporating struts.
  • a 3-dimensional radial nanostructure incorporating a node that is homologous to thermophilic node templates with Dn, tetrahedral (T23), cubeoctahedral (432), or with icosahedral/dodecahedral (532) symmetry derived from a thermophilic organism, and Dn symmetric nodes and streptavidin or streptavidin-incorporating struts
  • T23 tetrahedral
  • a 3-dimensional radial nanostructure incorporating a node with cubeoctahedral symmetry based on a 24-subunit node template derived from a list of known three-dimensional protein structures with corresponding symmetry as determined from
  • a 3-dimensional radial nanostructure incorporating a node with icosahedral/dodecahedral 532 symmetry based on a 60-subunit node template derived from a thermophilic organism and Dn symmetric nodes and streptavidin or streptavidin-incorporating struts.
  • a 3-dimensional radial nanostructure incorporating a node with icosahedral/dodecahedral 532 symmetry based on a 60-subunit node template derived from a list of known three-dimensional protein structures with corresponding symmetry as determined from X-ray crystallography, and streptavidin or streptavidin-incorporating struts.
  • a 3-dimensional polyhedron formed of streptavidin or streptavidin-incorporating struts, and nodes with Cn symmetry incorporating binding interactions corresponding to the apex geometry of a polyhedron .
  • a 3-dimensional dodecahedron formed of streptavidin or streptavidin-incorporating struts, and nodes with C3 symmetry, incorporating binding interactions corresponding to the apex geometry of a dodecahedron.
  • a 3-dimensional dodecahedron formed of streptavidin or streptavidin-incorporating struts, and nodes with C3 symmetry, based on the pdb code:lv4n node protein, incorporating binding interactions corresponding to the apex geometry of a dodecahedron.
  • a 3-dimensional "bucky" polyhedron formed of streptavidin or streptavidin-incorporating struts, and nodes with C3 symmetry, incorporating binding interactions corresponding to the apex geometry of a truncated icosahedron.
  • a 3 -dimensional "bucky” polyhedron formed of streptavidin or streptavidin-incorporating struts, and nodes with C3 symmetry, based on the pdb code:lv4n node protein, incorporating binding interactions corresponding to the apex geometry of a truncated icosahedron.
  • a 3-dimensional icosahedron formed of streptavidin or streptavidin-incorporating struts, and nodes with C5 symmetry, incorporating binding interactions corresponding to the apex geometry of an icosahedron.
  • a 3-dimensional icosahedron formed of streptavidin or streptavidin-incorporating struts, and nodes with C5 symmetry, based on the pdb code:lvdh node protein, incorporating binding interactions corresponding to the apex geometry of an icosahedron.
  • a 3-dimensional, three-connected lattice formed of streptavidin or streptavidin-incorporating struts, and nodes with D3 symmetry derived from a list of known three-dimensional protein structures with corresponding symmetry as determined from X-ray crystallography, the same D3 templates being alternatively modified to allow binding to streptavidin in two orientations.
  • a 3-dimensional, three-connected lattice formed of streptavidin or streptavidin-incorporating struts, and nodes with D3 symmetry based on the pdb coderlhyb node protein template, alternatively modified to allow binding to streptavidin in two orientations.
  • a nanostructure comprising a 3-dimensional, four-connected, cubic pattern lattice formed of nodes with D4 symmetry and streptavidin or streptavidin- incorporating struts.
  • a nanostructure comprising a 3 -dimensional, four-connected, cubic pattern lattice formed of streptavidin or streptavidin-incorporating struts, and nodes with
  • D4 symmetry derived from a list of known three-dimensional protein structures with corresponding symmetry as determined from X-ray crystallography, alternatively modified to allow binding to streptavidin in two orientations.
  • a 3 -dimensional, four-connected lattice formed of streptavidin or streptavidin-incorporating struts, and nodes with D4 symmetry based on the pdb code:2h2i node protein template, alternatively modified to allow binding to streptavidin in two orientations.
  • a 3-dimensional, six-connected cubic lattice formed of streptavidin or streptavidin-incorporating struts.
  • a 3-dimensional, six-connected cubic lattice formed of streptavidin or streptavidin-incorporating struts, and nodes with tetrahedral symmetry.
  • a 3-dimensional, six-connected cubic lattice formed of streptavidin or streptavidin-incorporating struts, and nodes with T23 symmetry derived from a list of known three-dimensional protein structures with corresponding symmetry as determined from X-ray crystallography.
  • a 3-dimensional, six-connected cubic lattice formed of streptavidin or streptavidin-incorporating struts, and nodes with tetrahedral symmetry based on the pdb code:lpw node protein template.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits and specific binding functionality.
  • a nanostructure node incorporating 3, 5, or 6 subunits.
  • a nanostructure node incorporating 3, 5, or 6 subunits, where the subunits are related by rotational symmetry.
  • a nanostructure node multimeric protein incorporating multiple polypeptide subunits related by tetrahedral, octahedral, or icosahedral symmetry.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits, each with a specific binding functionality.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits, where at least one subunit lacks a specific binding functionality.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising 4 polypeptide chain subunits, each with a specific binding functionality and related by 4-fold symmetry.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising 3,4, or 6 polypeptide chain subunits incorporating specific binding functionality that lie in a plane.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising 3,4, or 6 polypeptide chain subunits, each with a specific binding functionality and related by rotational symmetry.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising 4 polypeptide chain subunits, each with a specific binding functionality , and where at least one specific binding site does not lie within the same plane as the other specific binding sites.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits and wherein a first subunit is covalently bonded to a second subunit.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising at least 3 polypeptide chain subunits and wherein at least three subunits are covalently bonded to form a single polypeptide chain.
  • thermostable nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits and specific binding functionality.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits and specific binding functionality and where the amino acid sequence of at least one subunit is different from the amino acid sequence of another subunit.
  • a nanostructure node protein with at least 80% sequence homology with a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits and specific binding functionality.
  • a trimeric C3-symmetric nanostructure node multimeric protein where the amino acid sequence of each polypeptide subunit has at least 80% sequence identity with an amino acid sequence of the uronate isomerase TM0064 from Thermotoga maritime (pdb code:lj5s).
  • a tetrameric C4-symmetric nanostructure where the amino acid sequence of each polypeptide subunit has at least 80% amino acid sequence identity with an amino acid sequence of the isopentenyl-diphosphate delta-isomerase (pdbcode: lvcg) from
  • thermophilus Thermus thermophilus.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits and specific binding functionality, wherein each specific binding site incorporates a specific amino acid residue separated from the other specific amino acid residue by a distance of about
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits and specific binding functionality, wherein each specific binding site incorporating a specific amino acid residue is separated from the other specific amino acid residue by a distance such that with biotin groups bound to the specific amino acid residues, the biotin groups are positioned to bind with a pair of binding sites on streptavidin.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits and specific binding functionality, where at least one subunit incorporates a polypeptide extension of from 5 to 1000 amino acid residues linked with a peptide bond to the designated amino and/or carboxy terminus.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits and specific binding functionality, where at least one subunit incorporates a polypeptide extension of from 5 to 1000 amino acid residues linked with a peptide bond to the designated amino and/or carboxy terminus that comprises a binding function for a protein or a metallic or other solid surface.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits and specific binding functionality, where at least one subunit incorporates a polypeptide extension of from 5 to 1000 amino acid residues linked with a peptide bond to the designated amino and/or carboxy terminus that comprises an amino acid subsequence that is a substrate for an enzyme.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits and specific binding functionality, where at least one subunit incorporates a polypeptide extension of from 5 to 1000 amino acid residues linked with a peptide bond to the designated amino and/or carboxy terminus that comprises a polypeptide subsequence selected from the group consisting of an immunoglobulin polypeptide, a polyhistidine, a streptavidin binding polypeptide, StrepTag, an antibody binding polypeptide, staphylococcus Protein A, staphylococcus Protein G, an antigenic polypeptide, and a hapten-binding polypeptide.
  • a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits and specific binding functionality, where at least one subunit incorporates a polypeptide extension of from 5 to 1000 amino acid residues linked with a peptide bond to the designated amino and/or carboxy terminus that comprises an antibody binding polypeptide subsequence together with a bound antibody.
  • a nanostructure assembly incorporating a multimeric nanostructure node protein together with a specifically bound nanostructure strut.
  • a nanostructure node comprising three subunits where two subunits incorporate specific binding sites and one subunit does not. In its C3 symmetric form, the nanostructure node functions as a 120 degree linker between two nanostructure struts.
  • a nanostructure node comprising three subunits where one subunit incorporates a specific binding site and two subunits do not. The nanostructure node functions as a cap or terminator for a nanostructure struts.
  • a nanostructure node comprising four subunits where three subunits incorporate specific binding sites and one subunit does not. In its C4 symmetric form, the nanostructure node functions as a "T" linker between three nanostructure struts. [00143] In an embodiment, a nanostructure node comprising four subunits where two subunits incorporate specific binding sites and two subunits do not. In its C4 symmetric form, and where two subunits are related by a 180 degree rotation about the C4 axis, the nanostructure node functions as a linear linker between two nanostructure struts.
  • a nanostructure node comprising four subunits where two subunits incorporate specific binding sites and two subunits do not.
  • the nanostructure node In its C4 symmetric form, and where two subunits are related by a 90 degree rotation about the C4 axis, the nanostructure node functions as a right angle "L" linker between two nanostructure struts.
  • a nanostructure node comprising four subunits where one subunit incorporates a specific binding site and three subunits do not.
  • the nanostructure node functions as a cap or terminator for a nanostructure struts.
  • a protein superstructure comprising a multisubunit nanostructure node with specifically bound strut components.
  • a protein superstructure comprising a multisubunit nanostructure node with specifically bound strut components, where the struts are comprised of streptavidin and are bound to the node via biotin groups covalently bound to the specific amino acid residues on the node.
  • a protein superstructure comprising a multisubunit nanostructure node, specifically bound to a surface-immobilized strut component, where the strut is comprised of streptavidin and is bound to the node via biotin groups covalently coupled to the specific amino acid residues on the node.
  • a protein superstructure comprising a multisubunit nanostructure node with specifically bound strut components, where the struts are comprised of streptavidin together with an adaptor protein that is linked to streptavidin through a bifunctional biotin-ATP crosslinking agent.
  • a protein superstructure comprising a multisubunit nanostructure node with specifically bound strut components, where the strut component is an adaptor protein that is linked to the node via ATP derivative groups covalently coupled to specific amino acid residues on the node.
  • a protein superstructure comprising a multisubunit nanostructure node with specifically bound strut components, where the strut component is comprised of a complex of streptavidin and an adaptor protein, all associated through specific linkers.
  • kits comprising a nanostructure multisubunit node and a monostructure strut.
  • a kit comprising a nanostructure multisubunit node and a monostructure strut comprised of streptavidin.
  • thermostable nanostructure node multimeric protein that takes advantage of the thermostability in performing separation from the producing cells, optionally including isolating the thermostable nanostructure node multimeric protein in substantially pure form from the lysate.
  • thermostable nanostructure node multimeric protein that takes advantage of the thermostability in performing separation from the producing cells and uses recombinant DNA technology or site-specific modification techniques to modify a nucleotide sequence of a thermophilic organism for directing the expression of the nanostructure node multimeric protein.
  • thermostable nanostructure node multimeric protein that takes advantage of the thermostability in performing separation from the producing cells and uses a gene fusion technique to modify a nucleotide sequence of a thermophilic organism for directing the expression of the nanostructure node multimeric protein to have at least two subunits covalently interconnected with a polypeptide linker.
  • thermostable nanostructure node multimeric protein that takes advantage of the thermostability in performing separation from the producing cells and involves inserting the nucleotide sequence of a thermophilic organism or a modified nucleotide sequence of a thermophilic organism in the cell host to direct expression of the nanostructure node multimeric protein by the cell host.
  • a chromatographic or electrophoretic method of purifying nanostructure node multimeric proteins prepared by mixing combined subunits, some of which have a linker binding site and others of which do not have linker binding sites.
  • a chromatographic or electrophoretic method of purifying trimeric nanostructure node multimeric proteins prepared by mixing combined subunits, some of which have a linker binding site and others of which do not have linker binding sites.
  • a chromatographic or electrophoretic method of purifying tetrameric nanostructure node multimeric proteins prepared by mixing combined subunits, some of which have a linker binding site and others of which do not have linker binding sites.
  • a chromatographic or electrophoretic method of purifying 4-fold symmetric tetrameric nanostructure node multimeric proteins prepared by mixing combined subunits, some of which have a linker binding site and others of which do not have linker binding sites.
  • a chromatographic or electrophoretic method of purifying 4-fold symmetric tetrameric nanostructure node multimeric proteins prepared by mixing combined subunits, by separation into subtractions incorporating a variable number of subunits with linker binding sites
  • a chromatographic or electrophoretic method of purifying D2 or tetrahedrally symmetric tetrameric nanostructure node multimeric proteins prepared by mixing combined subunits, by separation into subtractions incorporating a variable number of subunits with linker binding sites
  • a method of making a protein nanostructure that includes a nanostructure node multimeric protein binding to a nanostructure strut.
  • a method of making a protein nanostructure that includes a nanostructure node multimeric protein binding to a nanostructure strut, that allows mixing and reaction of the binding components.
  • a method of making a protein nanostructure that includes a nanostructure node multimeric protein and nanostructure struts comprising streptavidin.
  • a method of making a protein nanostructure that includes a nanostructure node multimeric protein incorporating covalently bound iminobiotin groups and nanostructure struts comprising streptavidin.
  • a method of making a protein nanostructure that includes a nanostructure node multimeric protein incorporating covalently bound photo-ATP groups and nanostructure struts comprising adaptor molecules with ATP binding sites.
  • a method of using a 2-dimensional proteinaceous nanostructure assembly as a pattern for the fabrication of devices with sub-100 nanometer features is a method of using a 2-dimensional proteinaceous nanostructure assembly as a pattern for the fabrication of devices with sub-100 nanometer features.
  • [00175] A method of using a 3-dimensional proteinaceous nanostructure assembly as a patterning material for the fabrication of devices with sub- 100 nanometer features.
  • a method of using a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a soft lithography stamp for nanolithography is a method of using a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a soft lithography stamp for nanolithography.
  • a method of using a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a semiconductor device is a method of using a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a semiconductor device.
  • a method of using a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a microelectromechanical system (MEMS) device is described.
  • a method of using a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a nanofluidics device is described.
  • [00181] A method of making devices with sub- 100 nanometer features using a proteinaceous nanostructure assembly as a pattern or resist masking material.
  • [00182] A method of making devices with sub- 100 nanometer features using a 2- dimensional proteinaceous nanostructure assembly as a patterning material.
  • a method of making devices with sub- 100 nanometer features using a 2- dimensional proteinaceous nanostructure assembly as a method of patterning a resist material is a method of making devices with sub- 100 nanometer features using a 2- dimensional proteinaceous nanostructure assembly as a method of patterning a resist material.
  • a method of making devices with sub-100 nanometer, 3-dimensional channel features wherein the features form a negative image of a 3-dimensional proteinaceous nanostructure assembly, and binding a node protein or the node protein assembly to the resist layer surface at specific attachment sites through a chemical linkage.
  • a method of making devices with sub-100 nanometer features with 3- dimensional channel features wherein the features form a negative image of a 3-dimensional proteinaceous nanostructure assembly, and a substrate is composed of a metal (such as iron, gold, platinum, or silver), a noble metal (such as gold, platinum, or silver), a glass (such as silicon dioxide), a self-assembling monolayer, plastic, a polymer, an organic polymer (such as polytetrafluoroethylene), a ceramic, an organic material, or a semiconductor material (such as silicon or germanium).
  • a metal such as iron, gold, platinum, or silver
  • a noble metal such as gold, platinum, or silver
  • a glass such as silicon dioxide
  • a self-assembling monolayer plastic
  • a polymer such as polytetrafluoroethylene
  • ceramic such as polytetrafluoroethylene
  • an organic material such as silicon or germanium
  • a method of making devices with sub-100 nanometer features with 3- dimensional channel features wherein the features form a negative image of a 3-dimensional proteinaceous nanostructure assembly, and a matrix material comprises a metal (such as iron, gold, platinum, or silver), a noble metal (such as gold, platinum, or silver), a glass (such as silicon dioxide), a self-assembling monolayer, plastic, a polymer, an organic polymer (such as polytetrafluoroethylene) a ceramic, an organic material, or a semiconductor material (such as silicon or germanium).
  • a metal such as iron, gold, platinum, or silver
  • a noble metal such as gold, platinum, or silver
  • a glass such as silicon dioxide
  • a self-assembling monolayer plastic
  • a polymer such as polytetrafluoroethylene
  • ceramic such as polytetrafluoroethylene
  • an organic material such as silicon or germanium
  • [00193] A method of making devices with sub-100 nanometer, 3-dimensional channel features, wherein the 3-dimensional proteinaceous nanostructure assembly is assembled from engineered nodes derived from a list of thermostable multimers with known structure and optionally, streptavidin or streptavidin-incorporating struts.
  • [00194] A method of making devices with sub- 100 nanometer, 3-dimensional features, wherein the features form a replica image of a 3-dimensional proteinaceous nanostructure assembly.
  • the substrate composed of a metal (such as iron, gold, platinum, or silver), a noble metal (such as gold, platinum, or silver), a glass (such as silicon dioxide), a self-assembling monolayer, plastic, a polymer, an organic polymer (such as polytetrafluoroethylene), an organic material, a ceramic, or a semiconductor material (such as silicon or germanium).
  • a metal such as iron, gold, platinum, or silver
  • a noble metal such as gold, platinum, or silver
  • a glass such as silicon dioxide
  • a self-assembling monolayer plastic
  • a polymer such as polytetrafluoroethylene
  • an organic material such as polytetrafluoroethylene
  • a ceramic such as silicon or germanium
  • a method of making devices with sub-100 nanometer, 3-dimensional features wherein the features form a replica image of a 3-dimensional proteinaceous nanostructure assembly, optionally embedded in a matrix material composed of metal, glass, plastic, ceramic, or a semiconductor material.
  • a method of making devices with sub-100 nanometer, 3-dimensional features wherein the 3-dimensional proteinaceous nanostructure assembly forming the pattern to be replicated is assembled from engineered nodes derived from a list of thermostable multimers with known structure and optionally, streptavidin or streptavidin-incorporating struts.
  • a method of making a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a soft lithography stamp for nanolithography is a method of making a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a soft lithography stamp for nanolithography.
  • a method of making a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a semiconductor device is a method of making a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a semiconductor device.
  • a method of making a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a zero-mode waveguide is a method of making a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a zero-mode waveguide.
  • a method of making a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a microelectromechanical system (MEMS) device is also known.
  • MEMS microelectromechanical system
  • a method of making a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a nanofluidics device is also known.
  • a device that includes a substrate having a surface, a nucleation site on the substrate surface, and a nanostructure node coupled to the nucleation site [00206] A device that includes a substrate having a surface, a nucleation site on the substrate surface, and a nanostructure node coupled to the nucleation site, with more than one nucleation site on the substrate surface and with the nucleation sites arranged in a periodic, quasiperiodic, or nonperiodic pattern.
  • a device that includes a substrate having a surface, a nucleation site on the substrate surface, and a nanostructure node coupled to the nucleation site, the substrate comprising, for example, a metal (such as iron, gold, platinum, or silver), a noble metal (such as gold, platinum, or silver), a glass (such as silicon dioxide), a ceramic, a semiconductor (such as silicon or germanium), a carbon allotrope (such as diamond or graphite), a polymer, an organic polymer (such as tetrafluoroethylene), and/or an organic material and the nucleation site comprising, for example, a metal atom (such as iron, gold, platinum, or silver), a noble metal atom (such as a gold, platinum, silver, or copper), a metal and/or noble metal cluster, a chemically reactive molecule, and/or a patch of chemically reactive molecules.
  • a metal such as iron, gold, platinum, or silver
  • a noble metal such as gold, platinum, or silver
  • a glass
  • a device that includes a substrate having a surface, a nucleation site on the substrate surface, and a nanostructure node coupled to the nucleation site, the nanostructure node comprising a nanostructure node multimeric protein comprising at least one polypeptide chain.
  • the nanostructure node multimeric protein can have a known 3-dimensional structure, the nanostructure node multimeric protein can essentially have Cn, Dn, or higher symmetry with a number of subunits, the nanostructure node multimeric protein can be stable at a temperature of 70 0 C or greater, the nanostructure node multimeric protein can have an amino acid sequence not found in nature, the nanostructure node multimeric protein can include a specific binding site for the attachment of a nanostructure strut with predefined stoichiometry and orientation, the specific binding site can include at least two specific amino acid reactive residues, and each specific amino acid reactive residue can have a covalently attached biotin group.
  • the subunit can include an amino acid sequence having a designated amino and/or carboxy terminus and can include an amino acid (polypeptide) extension of from 5 to 1000 amino acid residues linked with a peptide bond to the designated amino and/or carboxy terminus, and the amino acid extension can include a binding function coupled to the nucleation site.
  • a nanostructure strut can be attached to the specific binding site.
  • a device includes a substrate having a surface with a node-occupied area and a node-unoccupied area.
  • a nanostructure node can be on the node-occupied area of the surface.
  • a coating can cover the nanostructure node and can cover the surface node-unoccupied area of the surface.
  • the coating can include a metal (such as iron, gold, platinum, or silver), a noble metal (such as gold, platinum, or silver), a glass (such as silicon dioxide), a ceramic, a semiconductor (such as silicon or germanium), a carbon allotrope (such as diamond or graphite), a polymer, an organic polymer (such as tetrafluoroethylene), and/or an organic material.
  • a device can include a substrate having a surface with a node-occupied area and a node-unoccupied area.
  • the surface can be coated with a resist layer.
  • a nanostructure node can be on the resist layer.
  • the node-occupied area of the surface of the substrate can be coated with the resist layer.
  • the node-unoccupied area of the surface of the substrate can be not coated with the resist layer.
  • the node-unoccupied area of the surface of the substrate can be lower than (recessed with respect to) the node-occupied area of the surface of the substrate.
  • a device can include a proteinaceous nanostructure assembly comprising a nanostructure node.
  • the device can include a substrate having a surface, and the proteinaceous nanostructure assembly can be coupled to the surface of the substrate.
  • the device can include a first matrix, and the first matrix can interpenetrate the proteinaceous nanostructure assembly.
  • the proteinaceous nanostructure assembly can have the form of a cubic lattice
  • the first matrix can have the form of a cubic lattice.
  • the first matrix can include a metal (such as iron, gold, platinum, or silver), a noble metal (such as gold, platinum, or silver), a glass (such as silicon dioxide), a ceramic, a semiconductor (such as silicon or germanium), a polymer, an organic polymer (such as tetrafluoroethylene), and/or an organic material.
  • a device can include a second matrix material having the same or similar form as a proteinaceous nanostructure assembly.
  • the device can include a second matrix that includes a metal (such as iron, gold, platinum, or silver), a noble metal (such as gold, platinum, or silver), a glass (such as silicon dioxide), a ceramic, a semiconductor (such as silicon or germanium), a polymer, an organic polymer (such as tetrafluoroethylene), and/or an organic material.
  • a metal such as iron, gold, platinum, or silver
  • a noble metal such as gold, platinum, or silver
  • a glass such as silicon dioxide
  • a ceramic such as silicon dioxide
  • a semiconductor such as silicon or germanium
  • a polymer such as silicon or germanium
  • organic polymer such as tetrafluoroethylene
  • Table IA lists template protein structures useful for the construction of nanostructure nodes having various symmetries.
  • Table IB provides the four character Protein Data Bank code, amino acid sequence (using the standard 1 -letter abbreviation for amino acid residues) as contained in the
  • Protein Data Bank database protein function, and organism from which the amino acid sequence is derived for template protein structures useful for the construction of nanostructure nodes.
  • Table 2 lists specifications and amino acid sequences for node embodiments with various symmetries.
  • Figure 1 shows schematic backbone and surface representations of the streptavidin strut molecule, a tetrameric protein with D2 symmetry, indicating geometry of biotin ligand binding sites and interaction geometry of node attachment sites.
  • Figure 2 shows the reaction of protein cysteine sulfhydryl groups with biotinylation reagents.
  • Figure 3 presents schematic illustrations of nodes with three-fold (C3) rotational symmetry and examples of corresponding protein multimers from thermostable microorganisms useful as node templates.
  • Figure 4 presents schematic illustrations of single-chain nodes based on protein multimers with three-fold rotational (C3) symmetry.
  • Figure 5 presents schematic illustrations of multiple and single-chain nodes based protein multimers with three-fold rotational (C3) symmetry, incorporating functional binding sites and fused protein domains.
  • Figure 6 shows the reaction of protein cysteine sulfhydryl groups with bifunctional crosslinking reagents.
  • Figure 7 presents schematic illustrations of nodes with four-fold (C4) rotational symmetry and examples of corresponding protein multimers from thermostable microorganisms useful as node templates.
  • Figure 8 presents schematic illustrations of single-chain nodes based on a protein multimers with four-fold rotational (C4) symmetry.
  • Figure 9 presents schematic illustrations of multiple and single-chain nodes with four-fold rotational (C4) symmetry, incorporating functional binding sites and fused protein domains.
  • Figure 10 presents schematic illustrations of nodes with C5, C6, and C7 rotational symmetry and representative examples of corresponding protein multimers from thermostable microorganisms useful as node templates.
  • Figure 11 presents schematic illustrations of 3-dimensional polyhedra incorporating nodes with C3 and C5 symmetry.
  • Figure 12 presents schematic illustrations of D2 symmetric nodes used as strut extenders.
  • Figure 13 presents illustrations of a D2 node used as a strut extender that introduces an axial rotation along the strut axis.
  • Figure 14 presents illustrations of D2 symmetric protein multimers from thermostable microorganisms useful as node templates.
  • Figure 15 presents schematic illustrations of a hexameric node with D3 symmetry and an octameric node with D4 symmetry.
  • Figure 16 presents schematic illustrations of a doubly-modified hexameric node with D3 symmetry and an doubly-modified octameric node with D4 symmetry.
  • Figure 17 presents illustrations of hexameric protein multimers with D3 symmetry from thermostable microorganisms useful as node templates.
  • Figure 18 presents illustrations of octameric protein multimers with D4 symmetry from thermostable microorganisms useful as node templates.
  • Figure 19 presents illustrations of regular polyhedra with dyad axes of symmetry.
  • Figure 20 presents illustrations of protein multimers from thermostable microorganisms having the symmetry properties of regular polyhedra and utility as templates for nanostructure node proteins.
  • Figure 22 illustrates methods used to determine the sites of surface amino acid substitution to transform multimeric node templates into nodes able to bind streptavidin with defined relative geometry.
  • Figure 23 presents a stereoscopic image of a D2 symmetric protein showing bounding boxes used to determine the sites of surface amino acid substitution to transform multimeric node templates into nodes able to bind streptavidin with defined relative geometry.
  • Figure 24 presents schematic illustrations and computer models of C3 and C4 symmetric nodes together with streptavidin tetramers oriented to allow linkages through biotin linkages.
  • Figure 25 presents a stereoscopic representation of an engineered single-chain
  • Figure 26 presents computer models of a C3 symmetric node complexed with three streptavidin tetramers with geometry suitable for the apex formation of a dodecahedron.
  • Figure 27 presents computer models of a C5 symmetric node complexed with five streptavidin tetramers with geometry suitable for the apex formation of an icosahedron.
  • Figure 28 presents schematic illustrations of D2 symmetric nodes engineered from streptavidin.
  • Figure 29 presents schematic illustrations and computer models of a D2 symmetric node oriented to allow linkages to streptavidin tetramers through biotin linkages along 3 dyad axes.
  • Figure 30 presents schematic illustrations and computer models of D2 symmetric nodes useful as a strut extender together with streptavidin tetramers oriented to allow biotin linkages along one dyad axis.
  • Figure 31 presents schematic illustrations and computer models of hexameric nodes with D3 symmetry with streptavidin tetramers oriented to allow linkages to streptavidin through biotin linkages along a dyad axis.
  • Figure 32 presents schematic illustrations and computer models of octameric nodes with D4 symmetry with streptavidin tetramers oriented to allow linkages to streptavidin through biotin linkages along dyad axes.
  • Figure 33 presents schematic illustrations and computer models of dodacameric nodes with tetrahedral symmetry with streptavidin tetramers oriented to allow linkages through biotin linkages along dyad axes.
  • Figure 34 presents schematic illustrations of complexes of streptavidin with linear strut connectors having D2 symmetry to produce struts of various lengths.
  • Figure 35 presents schematic illustrations of streptavidin-linked two-dimensional radial structures formed using variants of nodes with three-fold (C3) and four-fold (C4) and seven-fold (Cl) rotational symmetry.
  • Figure 36 presents schematic illustrations of streptavidin-linked two-dimensional lattices formed using nodes with three-fold (C3) and four-fold (C4) rotational symmetry.
  • Figure 37 presents schematic illustrations of streptavidin-linked two-dimensional polygonal structures formed using single-chain variants of nodes with three-fold and four-fold rotational symmetry.
  • Figure 38 presents a molecular illustration of two hexameric D3 nodes interconnected by streptavidin enabling formation of a three-connected three-dimensional lattice.
  • Figure 39 presents a molecular illustration of two octameric D4 nodes interconnected by streptavidin enabling formation of a four-connected three-dimensional lattice.
  • Figure 40 presents schematic illustrations of various three-dimensional lattices with different node connectivity.
  • Figure 41 presents a method of making a proteinaceous nanostructure pattern on a substrate surface.
  • Figure 42 presents a method of making a repetitively patterned proteinaceous nanostructure on a substrate surface.
  • Figure 43 presents a method of making a coated patterned nanostructure on a substrate surface.
  • Figure 44 presents a method of making a patterned structure using a proteinaceous nanostructure as a mask for a photoresist material.
  • Figure 45 presents a method of making a 3-dimensionally patterned structure in a solid matrix material or, in additional steps, a replica of a 3 -dimensional proteinaceous nanostructure assembly.
  • Figures 46A-46C present diagrams of the expression vectors EXP14Q3193C2,
  • Figure 47 presents a diagram of the expression vector EXP14Q3164.
  • Figure 48 presents images of electrophoretic analysis of NODE:SAV complexes.
  • indication of a protein having "80 percent or greater sequence identity" with the sequence of another protein is to be understood as including, as alternatives, proteins that are required to have a higher percentage of sequence identity with the other protein.
  • alternatives include proteins that have 90, 95, 98, 99, 99.5, or 99.9 percent or greater sequence identity with the sequence of the other protein.
  • One of skill in the art would understand that given a second amino acid sequence having 80 percent or greater sequence identity to a first amino acid sequence, the three-dimensional protein structure of the second amino acid sequence would be the same or similar to that of the first amino acid sequence.
  • "80 percent or greater sequence identity" can mean that the linear amino acid sequence of a second polypeptide, whether considered as a continuous sequence or as subsections of amino acid sequence of ten or more residues (the order of the subsections with respect to each other being preserved), has identical amino acid residues with a first polypeptide at 80 percent or greater of corresponding sequence positions.
  • a second polypeptide having 20 percent or less of the amino acid residues of a first polypeptide replaced by other amino acid residues would have "80 percent or greater sequence identity".
  • this document is to be considered to claim those protein sequences meeting the requirements of claim 1 of this document and having 80 percent or greater sequence identity to the amino acid sequences listed in Table IB.
  • certain residues can be more important to the structural integrity, symmetry, and reactivity of the proteins, and these must be more highly conserved, while other residues can be modified with less of an effect on the node protein.
  • proteins that are homologous or have sufficient sequence identity are those without changes that would detract from adequate structural integrity, reactivity, and symmetry.
  • amino acid sequences listed in Table IB and Table 2 represent template sequences upon which a nanostructure node multimeric protein, such as that defined by claim 1, can be based. Along with the sequence is listed is the 4 alphanumeric character Protein Data Bank code (pdb code) that contains the crystallographic structure corresponding to the amino acid sequence (in the case of Table IB, the name of the protein and the organism from which the amino acid sequence is derived is also listed). Note that each amino acid sequence itself listed in Table IB and Table 2 was derived from the electron density crystallographic structure data in the Protein Data Bank, rather than from, for example, chemical analysis.
  • pdb code Protein Data Bank code
  • amino acid sequences listed in Table IB and Table 2 can be expected to exhibit some differences from amino acid sequences that would be derived from chemical analysis.
  • certain residues near the terminal N or C residues or present in outlying loops of the 3-dimensional tertiary protein structure may not be represented in the amino acid sequences presented in Tables IB and 2.
  • residue numbers indicated in Table 2 correspond to the standard residue numbering assigned in the art to the sequence for the protein, and not necessarily to the number of the residue in the crystallographic-derived sequence presented in Table 2.
  • Described embodiments according to the present invention include molecular components that are extremely stable, easily manufactured and purified, and designed with high precision to enable the controlled assembly of a wide range of one-, two- and 3-dimensional protein-based nanostructure assemblies. Described embodiments according to the present invention include the design and manufacture of such molecular components.
  • the protein components of the nanostructure assembly are functional, as appropriate for the development of biological sensors, filters, materials, or bioelectronic devices where charge, spin, or optical properties are intrinsic properties of the protein or prosthetic groups that are bound to the protein structure.
  • the protein nanostructure assembly provides a means of high- resolution patterning of a silicon, glass, metal, or other substrate, either by using the protein nanostructure assembly directly as a means of patterning a substrate, or alternatively as a mask for a radiation-sensitive resist.
  • This approach can allow manufacture of microelectronic devices, devices incorporating zero-mode waveguides (Levene et. al, 2003) or microelectromechanical systems (MEMS) using conventional semiconductor fabrication (Widman et. al, 2000) and/or MEMS fabrication technology (Judy, 2001). Additional patterning applications include the generation of soft lithography stamps and molds (Xia & Whitesides 1998, Rogers & Nuzzo 2005) for MEMS and nanofluidic applications.
  • the biomolecular components can include molecular-scale “struts” and “nodes”.
  • Struts are components that basically function as linear structural elements or linear connectors, and typically have attachment points to nodes oriented in a linear arrangement. Different struts or arrays of strut extenders or adaptors can be used to establish predetermined distances in a structure. Nodes are connectors that can have either two attachment points with defined, for example, nonlinear, geometry, or more generally, multiple attachment points with defined geometry. Nodes can be linked together, for example, by struts, to establish the topology of a structure. Thus, with the struts and nodes, structures with 2-dimensional and 3-dimensional geometry can be constructed.
  • Structures organized in two dimensions can be finite to allow the formation of locally structured patterns of molecules arrayed on a surface, or alternatively form infinitely extensible 2-dimensional lattices.
  • the symmetry properties required of nodes suitable to build structures with the regular 2-dimensional geometry are well known from mathematics and crystallography (Williams 1979, Pearce 1979, Vainshtein, 1994).
  • Two-dimensional structures can have utility themselves and/or can be further functionalized through chemical modification or the incorporation of additional specific binding proteins.
  • Structures organized in three dimensions can also be usefully classified as finite or infinite.
  • finite structures potentially constructed using molecular strut and node architecture include dendritic structures as well as the Platonic and Archimedian polyhedra and their many variations (Pugh 1976, Pearce 1979).
  • the strut and node architecture also potentially allows the assembly of infinite 3-dimensional lattices.
  • the symmetry requirements for nodes that can form infinite 3-dimensional lattices have been described comprehensively by Wells and others (Wells 1977, Wells 1979, Williams 1979).
  • Three- dimensional structures can have utility themselves as materials and filters and/or can be further functionalized through chemical modification or the incorporation of additional specific binding proteins.
  • compositions and methods discussed herein apply the philosophies of interchangeable parts and mass production, which drove unprecedented economic expansion in the last two centuries, to the nanoscale.
  • Providing such a "parts box" of biomolecular components will allow users to experiment with a range of structures and thereby facilitate the development of a new generation of functional nanodevices, biosensors, and biomaterials, potentially finding broad application in areas as diverse as biomedical devices and nanoelectronic applications.
  • Proteins have a number of advantages for use as components and templates for biomolecular components, including, but not limited to the following. Proteins already exist in nature as functional polypeptide units with well-defined 3-dimenensional structures, so that effort can focus on tailoring them as building blocks for specific applications, rather than having to develop building blocks from scratch. A very large number of proteins exist, and the detailed atomic structure of many are known, so that there is an excellent chance of finding a protein that, with minimal tailoring, can perform as a desired building block.
  • Naturally occurring proteins have diverse and sophisticated functionality. They can show high interaction specificity and manifest catalytic properties. They can exhibit interesting and useful optical, magnetic, and redox properties, for example, by incorporating metal centers and a wide variety of prosthetic groups. Such metal centers and prosthetic groups can, as well as the polypeptide sequence itself, be tailored to produce a protein having a desired functionality.
  • DNA encodes a polypeptide sequence that spontaneously and reproducibly folds to form a predetermined 3 -dimensional protein of thousands of atoms of which each atom is precisely placed.
  • proteins as building blocks are reproducible and have precise configuration, they can be relied upon as components in the construction of extensive and complex structures.
  • Naturally occurring proteins frequently form cooperative hierarchical assemblies of great structural and functional complexity. These natural assemblies can be studied to derive assembly techniques and simplify the development of analogous artificial structures having an intended purpose.
  • Naturally occurring proteins can form highly stable multimeric structures that are symmetric and contain multiple copies of the individual polypeptide chains.
  • Symmetric multimeric structures are geometrically precise. If modification sites are introduced into a component polypeptide chain, then these are symmetrically arrayed in the multimeric structure with great geometrical precision; typically within errors of less than 1-2 Angstrom units (0.1 to 0.2 nM) from structure to structure.
  • Symmetric protein multimers are excellent template structures for the generation of macromolecular protein nodes.
  • a selected amino acid unit or subsequence (a plurality) of amino acid units of a natural protein can be substituted with a different natural amino acid, with an artificial amino acid, or with a different subsequence of natural and/or artificial amino acids to modify the natural protein.
  • a natural amino acid, artificial amino acid, or subsequence of natural and/or artificial amino acids can be inserted into the amino acid sequence of a natural protein to modify the natural protein.
  • An amino acid or a subsequence of amino acids can be removed (deleted) from the amino acid sequence of a natural protein to modify the natural protein.
  • a "parts box" of proteins may initially be applied to make devices that are analogous to or in some way emulate natural systems.
  • two- and 3-dimensional structures formed from struts and nodes, as described herein may be applied in the fields of biosensors and diagnostics.
  • the specific immobilization and precise geometric control facilitated by strut-node technology presented herein, along with the functionality inherent in proteins, can enable the development of new kinds of sensors incorporating, for example, multiple antibodies specifically immobilized in patterned arrays.
  • strut-node technology can be used in devices that couple directly to living systems, for example, that provide an interface between semiconductor substrates and living organisms and nanostructures. Such devices could, for example, be used as biocompatible materials for prostheses.
  • Applications of a "parts box" of proteins as biomolecular components are not limited to devices analogous to or for interacting with natural biological systems.
  • structures can be assembled that emulate the architecture and functions of silicon-based microprocessor architecture and computer memory or possess novel material properties.
  • the protein components of the nanostructure assembly can be functional, as appropriate for the development of biological sensors, filters, materials, or bioelectronic devices where charge, spin, or optical properties are intrinsic properties of the protein or prosthetic groups that are bound to the protein structure.
  • the protein nanostructure assembly can provide a means of high- resolution patterning of a silicon, glass, metal, or other substrate, so providing high resolution templates or resists that allow production of microelectronic devices, devices incorporating zero- mode waveguides (Levene et al, 2003), or microelectromechanical systems (MEMS) using conventional semiconductor fabrication (Widman et al., 2000) and/or MEMS fabrication technology (Judy, 2001).
  • the "parts box" strategy can be fundamentally exploited as a way of creating self-assembling or sequentially assembled structures where the nanometer size and designed-in precision of the interaction geometry between the protein molecular components can be used to create complex and highly precise structures in two and three dimensions.
  • These patterns can then be used as optical resists, molds, metallization substrates, or negatives for the fabrication of semiconductor, MEMS, soft lithography molds (Xia & Whitesides 1998, Rogers & Nuzzo 2005), or other devices where miniaturization at the sub- 100 nanometer scale is useful.
  • a reference to a protein or protein amino acid sequence is to be understood to also encompass variations of that protein or protein amino acid sequence including derived proteins or protein amino acid sequences derived from gene fusion techniques and/or circular or cyclic permutation techniques applied to that protein or protein amino acid sequence.
  • the C- terminal amino acid residue of a normally separate polypeptide chain can be spliced together with the N-terminal amino acid residue of another normally separate polypeptide chain (such a splicing can also encompass a splicing made when one or more amino acid residues normal present at the C-terminus or N-terminus are eliminated, and/or when one or more amino acid residues not normally present at the C-terminus or N-terminus are added, as when a linker sequences is used to splice together two normally separate polypeptide chains).
  • a gene fusion technique can be applied to covalently join polypeptide chains that are normally separate in a multimeric protein, such as a multimeric protein having Cn, Dn, or higher symmetry.
  • a single gene fused polypeptide chain formed from the N separate polypeptide chains of an N-mer can fold into the same or a similar three-dimensional tertiary protein structure as the N separate polypeptide chains.
  • the C-terminal and N-terminal amino acids of a polypeptide chain can be joined and other normally adjacent amino acid residues can be disjoined, so as to create new C-terminal and N-terminal amino acid ends.
  • N separate polypeptide chains of a native protein N-mer can be cyclically permuted, so that the amino acids at the N-terminus and C-terminus in the native protein are covalently joined, and two amino acid residues normally adjacent to each other and covalently bonded in the native protein are disjoined to become new N-terminal and C-terminal amino acid residues.
  • the new N-terminal amino acid residue of a polypeptide chain can be covalently joined to a new C-terminal amino acid residue of another polypeptide chain (that is normally separate in the native protein) through a gene fusion technique, with or without the addition of an intermediate linker sequence of amino acids and with or without the deletion of one or more amino acids.
  • thermophilic organisms that live in hot environments also have thermal stabilities in excess of 70 0 C, an environment not very dissimilar from the maximum operating temperatures for conventional semiconductor devices.
  • Evolutionary forces have allowed living organisms to exploit a wide range of habitats including environments that represent extremes of temperature, salinity, pH, specific mineral content, and/or pressure.
  • the organisms adapted to the most extreme environment like hot springs, thermal vents at the ocean bottom, high salt environments like the Dead Sea, etc. are termed extremeophiles and are generally microorganisms such as bacteria or algae.
  • thermophilic organisms also called microorganisms such as bacteria or algae
  • microorganisms such as bacteria or algae
  • mesophilic organisms or mesophiles Most plants and animals could not survive at such elevated temperatures because the basic molecules responsible for most of the biological functions of the organism, i.e. the polypeptide proteins encoded by the organism's genetic material or DNA, would become denatured.
  • Proteins are poly-amino acid polymers (or polypeptides) of defined sequence that fold to form highly organized 3-dimensional structures. Maintenance of the biological function of a protein as a chemical catalyst, receptor, channel, etc.
  • thermophilic organisms have evolved their amino acid sequences so that they are especially stable and can maintain their properly folded 3-dimensional structures and biological functions at high temperatures.
  • experimental approaches have been developed to improve the thermal stability of mesophilic proteins, these are laborious, costly and often ineffective, so that it is highly advantageous to use proteins from thermophilic organisms in situations where high protein stability is desired.
  • these applications have included industrial processes that use enzymes to carry out chemical reactions.
  • thermostable proteins for nanotechnology applications.
  • the use of engineered thermostable proteins for nanotechnology applications has many advantages.
  • thermostable proteins are much more stable than proteins in found in most bacteria (e.g. E. coli, B. subtilis, etc.), insect (e.g. sf9, etc.), or mammalian (e.g. CHO, HELA, etc.) cell lines typically used for recombinant expression of proteins. This greatly facilitates the isolation of these protein since once the thermostable protein has been expressed in the host cell line, it is often possible to gain a significant initial purification simply by treating the cells containing the thermostable protein to denaturing conditions (e.g.
  • thermophilic protein by heating or urea treatment) that cause most all of the mesophilic cell components to denature and become insoluble, leaving the thermophilic protein intact and in solution where it can be easily separated from the insoluble cell components by centrifugation, filtration, or a number of other methods. This substantially reduces the time and cost required to produce the materials required for nanotechnology applications.
  • thermostable proteins for nanotechnology applications.
  • thermostable proteins that will be used for nanotechnology applications will not be used in their native form as they are found in nature, but in some modified form.
  • modifications are expected to have a relatively small effect on the functional stability of a thermostable protein relative to a protein derived from a mesophilic organism.
  • thermostable protein can be achieved in two general ways.
  • the first approach involves the modification of the "native" protein amino acid sequence as it occurs in nature through manipulation of the DNA sequence that encodes the protein.
  • the manipulated DNA sequence can then be expressed in an expression system, for example, a bacterium, such as E. coli, to produce the desired modified amino acid sequence.
  • This process is generally termed protein engineering and is broadly used in the biotechnology industry.
  • the second general method involves reacting a protein composed of naturally occurring amino acids with chemical reagents or enzymes that post-process the protein to make a chemical derivative of the product encoded by the DNA sequence.
  • DNA technology is broadly used in biomedical research and is the basis of many pharmaceutical products.
  • Salemme & Weber (2007) no reports exist for using protein engineering for structural nanotechnology applications using thermostable proteins.
  • thermostable proteins intended for nanotechnology applications can be introduced using recombinant DNA technology to modify the DNA sequence that encodes the corresponding protein polypepetide sequence.
  • Useful modifications could include, for example:
  • the second type of modification which may often be combined with the gene modification strategies outlined above that alter the native protein sequence, involves the reaction of the modified protein with a chemical reagent or enzyme to produce a "chemically modified" protein.
  • chemical modifications include the formation of a covalent connection between the polypeptide structure and chemical groups with specific protein binding activity.
  • chemical reagents are known that can react covalently with the cysteine groups on the surface of proteins to covalently attach biotin.
  • Biotin is a vitamin that has very high and specific binding affinity for several proteins of the avidin family including streptavidin from Streptomyces avidinii and bird avidins.
  • proteins that are chemically modified through covalent attachment of biotin groups can form tight and specific interactions with streptavidin and avidin (and derivatives of streptavidin and avidin), and as a result have found wide application in biotechnology and diagnostic applications. Because all chemical reactions, including those that tend to spontaneously modify proteins (e.g. oxidation of sulfur containing amino acids and side chain deamidation of asparagine and glutamine residues) tend to occur more rapidly at high temperatures, proteins that are adapted to be stable at high temperature are also unusually stable to changes in chemical environment. This does not mean that modifications like the biotinylation reaction outlined above will not occur with thermostable proteins, but that there is less likelihood that undesirable side reactions will take place that could give rise to defective molecular structures with reduced assembly fidelity for self-assembling nanostructures.
  • thermostable proteins are ease of processing during the production and assembly of nanostructures.
  • the production of components for assembly of nanostructures incorporating thermostable proteins will often involve separation steps using chromatography, electrophoresis or other methods used to isolate biological macromolecules and complexes.
  • the enhanced stability of thermostable proteins relative to mesophilic proteins is an advantage that allows better separations of intermediate reaction products and/or molecular subassemblies using a wider range of separation conditions (e.g. solution pH, ionic strength, range of allowable solvents, presence of detergents, etc.).
  • thermostable proteins in nanodevices relates to the allowable range of practical operating conditions for devices incorporating engineered nanostructures. Many important applications for functional nanodevices will be in temperature environments that are not too much different from those normally tolerated by human beings - nominally 0 deg C to 50 deg C.
  • nanodevices designed for medical applications will have to operate at about 37 deg C, the temperature of the human body.
  • Even current semiconductor-based electronics typically do not operate reliably above ⁇ 70 deg C and typically require active cooling in applications like computers.
  • Many proteins from thermophilic organisms, as well as a small number of unusually stable proteins from mesophilic organisms like streptavidin from the microorganism Streptomyces avidinii remain stable above 70 deg C, whereas most proteins from mesophilic organisms denature in the range of 40 to 50 deg C making them less suitable for nanodevice applications.
  • Stability of a protein at a given temperature can refer to tertiary stability of the protein, i.e., the protein does not unfold from its three-dimensional folded structure into a disordered or random coiled polypeptide chain or into a structure having only secondary structure such as alpha- helices and beta-pleated sheets.
  • Stability of a protein at a given temperature can refer to quaternary stability of the protein, that is, the subunits of the protein retain their relative spatial arrangement, for example, the subunits of the protein do not disaggregate into individual tertiary structures (or less ordered secondary structures or primary structures (disordered or random coiled polypeptide chains)) and do not undergo a substantial relative spatial rearrangement.
  • a protein that is stable above 70 deg C will retain its tertiary structure and/or its quaternary structure above a temperature of 70 deg C.
  • thermostable proteins Most of the biomolecular components describe herein are based on proteins of thermostable microorganisms of known 3-dimensional crystal structure. As outlined above, the use of thermostable proteins provides us with several advantages in economical node production, handling and purification. [00306] The enzymatic binding sites of proteins used as nodes can provide additional sites for functionalization of the nanostructure through covalent binding of inhibitors linked to other chemical moieties or proteins.
  • struts Two fundamental nanoscale biomolecular components of a "parts box” from which a structure, for example, a device, can be assembled are “struts” and “nodes”. Struts are molecular components that function as linear connectors. Nodes connect struts and orient them with defined geometries.
  • a strut can be formed from streptavidin, a tetrameric protein of 60 kiloDalton molecular weight secreted by the bacterium Streptomyces avidinii.
  • Figure 1 shows molecular models and schematic illustrations of the streptavidin tetramer showing biotin ligand binding sites.
  • the streptavidin tetramer has D2 symmetry with 3 mutually perpendicular two-fold or dyad axes of symmetry relating the 4 subunits of the tetramer.
  • Dyad axes are labeled x,y, and z in Figure 1.
  • Figures la,b, and c show schematic backbone representations of the streptavidin tetramer viewed down the x, z, and y dyad axes of symmetry, respectively.
  • the bound biotin ligands are shown in space filling representation.
  • Figures 1 a through f is a "bounding box" aligned along the x dyad axis that defines the positions of the biotin ligands along the direction that they make bonded interactions with nodes.
  • Figures ld,e, and f show surface representations of the streptavidin tetramer viewed down the x, z, and y dyad axes of symmetry respectively.
  • Figures lg,h, and i show schematic representations used elsewhere in this document for illustrative purposes.
  • the 2 facing biotin binding sites are spaced approximately 20.5 Angstroms apart and aligned along a line that is inclined at a 72 degree angle relative to the z dyad axis of the streptavidin tetramer.
  • the streptavidin tetramer has dimensions of approximately 45 Angstroms (4.5 nanometers) along the x-axis, by 60 Angstroms (6 nanometers) on the y-axis, by 55 Angstroms (5.5 nanometers) on the z-axis.
  • the biotin-binding sites are arranged as pairs where the surface accessible valeric acid side chains of the biotin moieties are oriented along the verticals of an "H" in an orientation that facilitates specific pairwise binding.
  • the biotin binding sites are arranged with D2 symmetry.
  • the biotin molecules When bound to the streptavidin biotin-binding sites, the biotin molecules have their terminal valeric acid chains (which are the usual chemical modification sites for generating biotin conjugated reagents) in extended conformation and oriented approximately parallel to the x diad axis of the streptavidin tetramer.
  • the distance between the two closest and approximately parallel pair of bound biotin chain termini is about 20.5 Angstroms, which are aligned along a line that is inclined at a 72 degree angle relative to the z- dyad axis of the streptavidin tetramer (Fig 1).
  • a streptavidin tetramer can form be linked to other biomolecular components, such as nodes, at two sites through biotin molecules.
  • streptavidin Although the present descriptions refer specifically to streptavidin, several related proteins are known (e.g. egg white avidin) that have similar amino acid sequence, structure, and biotin binding properties as streptavidin. These proteins could be substituted for streptavidin in the applications described here.
  • thermostable strut templates with D2 symmetry are given in Table 1.
  • a node can connect two or more struts with predefined orientation of each strut with respect to the other connected struts.
  • a node can be a symmetric protein multimer.
  • a node can be an enzyme that has catalytic binding sites with high binding specificity for certain substrates and cofactors.
  • a naturally occurring protein can be used in its native state, or can be engineered, for example, using site-specific modification techniques, to render it suitable or optimal for an intended function as a node. Selection of a naturally occurring protein for use as a node can be made from the large number of X-ray crystal structures of stable protein multimers having different symmetries available.
  • selection can be made from protein sequences that have over 70% sequence homology with sequences with known X-ray structures, since it is known that homologous protein sequences also have similar 3-dimensional structures, and the multimeric state of a protein can be determined by physical methods like light scattering, electrophoresis, ultracentrifugation, gel exclusion chromatography, or other methods.
  • multimers serving as nodes can be interconnected by biomolecular components serving as struts (such as streptavidin) to create nano-scale structures with defined two- and 3-dimensional geometry.
  • suitable multimeric proteins with utility as node templates are known having 3-fold (C3), 4-fold (C4), 5-fold (C5), 6-fold (C6), 7-fold (C7), and other rotational symmetries.
  • multimeric proteins with utility as node templates are available with higher symmetry, including D2, D3, D4, tetrahedral, cubeoctahedral, icosahedral, and other symmetries. While nodes or node variants having Cn rotational symmetry are primarily suited to the assembly of 2-dimensional planar structures, nodes with higher fold symmetry more naturally lend themselves to the assembly of 3-dimensional structures and lattices.
  • the structures referenced in Table 1 of these and additional proteins that can serve as templates for nodes can be viewed at the Protein Data Bank (PDB) website http://www.rcsb.org/pdb/home/home.do (accessed Oct. 2, 2007) by entering the appropriate PDB Code as listed in Table IA.
  • the Protein Data Bank is a Federally supported, archival database that includes complete 3-dimensional structure coordinate data, amino acid sequence data, and links to relevant scientific literature.
  • the structures in the Protein Data Bank are hereby incorporated by reference. Proteins are labeled with their 4-letter protein Protein Data Bank identification code (pdb code) throughout this document. Amino acid sequences as stored in the Protein Data Bank for proteins identified by PDB Code are provided in Table IB.
  • site-specific modification techniques can be used to introduce surface cysteine residues at pairs of points on the surface of a multimer to function as a node.
  • Biotinylating reagents for example, a thiol-reactive biotinylating reagent, can be covalently bonded to such surface cysteine residues to introduce biotin groups at defined, for example, at symmetric points on multimeric node.
  • a node of defined geometry can be formed.
  • the pairs of biotin groups on the multimer functioning as a node can then be bound to the binding sites on streptavidin tetramers, which can act as struts, to form a two- or 3-dimensional nanostructure.
  • FIG. 2 Reactions of biotinylating reagents that can modify protein cysteine sulfhydryl groups are presented in Fig. 2.
  • Figure 2a shows a free sulfhydryl group on a protein.
  • Figure 2b shows the biotinylation reagent Sulfosuccinimidyl 2-(biotinamido)-ethyl-l,3-dithiopropionate (EZ-Link Sulfo-NHS-SS-Biotin: Pierce).
  • Figure 2c shows the reaction product after biotinylation.
  • Figure 2d shows an analogous reagent for the introduction of 2-imino biotin groups. The binding of imino-biotin to streptavidin is pH dependent.
  • FIG. 2e shows the imino-biotin reaction product.
  • Figure 2f shows the above reaction sequences schematically as used in schematic illustrations elsewhere in this application.
  • the reagents in Figure 2 show a specific linker length, biotinylation reagents are readily available with various linker lengths and custom ones are readily synthesized through incorporation of amino-alkyl-thiol coupling groups with variable alkyl or glycol chain lengths.
  • Nodes with Cn Symmetry The simplest symmetry that a multimeric note can have is Cn rotational symmetry. Since proteins are polymers composed of L-amino acids they are intrinsically asymmetric, and consequently nodes with Cn symmetry have polarity. As such nodes with Cn symmetry are well-suited to the assembly of 2-dimensional structures on surfaces where, for example, structural features on one polar face of the multimer (which is generally normal to the Cn symmetry axis), can be functionalized to provide the ability to bind to a planar substrate that can be a surface or self-assembling monolayer.
  • Figure 3a shows a schematic view of a three-fold symmetric multimer
  • Figs 3b and 3c shows representations of the uronate isomerase protein (TM0064) from Thermotoga maritima (Schwarzenbacher et al. 2003, pdb code: Ij 5s) in space filling and schematic backbone representations respectively.
  • Each chain of the trimer comprises 450 amino acid residues.
  • Figs 3d and 3e shows a representations of a carbonic anhydrase protein from Methanosarcina thermophila (Kisker, et al 1996, pdb codes: lthj & lqrf) in space filling representation and schematic backbone views respectively.
  • Each chain of the trimer comprises 213 amino acid residues. Additional C3 symmetric node templates are presented in Table IA.
  • Single chain constructs of a node protein can be formed.
  • these fused protein multimers can be constructed by incorporating a DNA sequence coding for a polypeptide linker connecting the C-terminus of a first multimer gene to the N-terminus of a second multimer, and so on, to create a single contiguous gene coding for the complete multimer.
  • This approach can allow for the subunits of a multimeric protein to be non-identical.
  • surface cysteine residues for biotinylation can be included in some subunits, but not in other subunits, so that struts can be attached at certain faces of the multimeric protein, but not at others.
  • the individual multimer subunits may be individually varied to introduce asymmetry into the node.
  • the individual multimer subunits have enzyme or cofactor binding sites that can serve as attachment points of additional inorganic, organic or biomolecules that can additionally functionalize the structure, these may be selectively eliminated using recombinant DNA technology to produce nodes where the only some of the binding sites remain intact.
  • methods of protein engineering may be used to introduce new binding functionality into the individual multimer subunits to produce single-chain multimeric nodes with asymmetric binding geometry.
  • Each node is composed of a trimeric protein where the subunits have been modified through site-specific mutagenesis to introduce surface amino acid residues that can be chemically modified to introduce pairs of biotin groups with geometry that is complementary to two of the binding sites on the streptavidin tetramer.
  • Figure 4a shows a node that is a trimer formed from three independent, identical chains that are not covalently connected. Two biotins are bound to each chain, so that a streptavidin strut can bind to each subunit. In this construct, the pairs of sites of surface biotinylation that are geometrically complementary to streptavidin are on different subunits.
  • Figure 4b shows a node that is a trimer as formed from three independent, identical chains that are not covalently connected.
  • FIG. 4c shows a node based on a protein trimer formed from a single chain construct, that is, with each subunit linked to another by a polypeptide linker. That is, the individual chains of the non-covalently associated trimer have been covalently connected together in a single continuous polypeptide chain.
  • Two biotins are bound to each chain, so that a streptavidin strut can bind to each subunit.
  • Figure 4d shows a node based on a protein trimer formed from a single chain construct. Two of the subunits of the trimer have bound biotin pairs, but the third does not. Thus, only two streptavidin struts can be linked to the trimer. As such, the trimer can serve as a connector between struts, but does not allow branching from one strut to two other struts.
  • Figure 4e shows a node based on a protein trimer formed from a single chain construct. Only one of the subunits of the trimer has a bound biotin pair; the other two do not. Thus, only one streptavidin strut can be linked to the trimer. As such, the trimer can serve as a terminator of a strut, and cannot serve as a connector or branch point between struts.
  • Nodes can be fimctionalized in at least two ways. Nodes may be selected that are enzymes that are characterized by the presence of specific substrate and cofactor binding sites.
  • An approach to functionalizing nodes uses bifunctional crosslinking reagents that specifically bind to binding sites on enzymes for substrates or cofactors (Fig 5a,b).
  • Bifunctional crosslinking reagents can incorporate an enzyme-specific reactive agent on one end and specific protein-reacting group (for example, a group able to react with cysteine side chain thiol group or a polypeptide chain terminal amine group) on the other end of the linker. For example, many enzymes use ATP as a specific cofactor.
  • Figure 6 shows reactions of a bifunctional crosslinking reagent incorporating an azido-ATP group on one end (which forms a covalent bond between the reagent and the protein upon ultraviolet light irradiation) and a thiol reactive reagent on the other end that will specifically react with a protein cysteine side chain.
  • Figure 6a shows a protein with a surface cysteine sulfhydryl group that can react with the sulfhydryl reactive reagent (Fig 6b) incorporating a 2-azidoadenosine 5'-triphosphate group to produce the reaction product in Fig 6c.
  • the 2-azido ATP modified protein (Fig 6c) can then bind to an ATP cofactor binding site on a node protein (Fig 6e).
  • the azido-ATP reacts with amino acid side chains of the node protein in the ATP binding site to form a covalent bond (Fig 6f).
  • Figure 6g presents the reaction sequence schematically using symbols used elsewhere in this application.
  • the bifunctional reagents in Figure 6 show a specific linker length, reagents with various linker lengths are readily synthesized through incorporation of amino-alkyl-thiol coupling groups with variable alkyl chain lengths.
  • the preceding and related linkers can be generated using commercially available reagents (Affinity Labeling Technologies, Lexington KY; Pierce, Rockford IL) or are compounds readily synthesized by one with skill in the art.
  • azido-ATP analog represents one example, but many additional examples can be envisioned where other biochemical cofactors such as flavins, vitamins, and other biochemical cofactors that bind specifically to proteins can be chemically modified so that they can be photo-crosslinked to protein molecules functioning as. either struts or nodes in assembled nanostructures.
  • biochemical cofactors such as flavins, vitamins, and other biochemical cofactors that bind specifically to proteins can be chemically modified so that they can be photo-crosslinked to protein molecules functioning as. either struts or nodes in assembled nanostructures.
  • binding sites that are specific for binding substrates and cofactors. In many cases, this binding specificity can be modified, eliminated, or new binding specificity created de novo from site-specific modification of the template protein sequence.
  • di- or multimeric strut or node proteins can potentially be modified forms of enzymes that carry out specific catalytic processes on biochemical substrates
  • many such nodes built on enzyme templates will incorporate active sites that bind substrates and catalyze reactions with great specificity.
  • covalent inhibitors or suicide substrates are known that irreversibly inhibit the enzyme activity by forming a highly specific covalent bond with the catalytic amino acid side chain groups in the enzyme's active site. These agents are generally termed suicide substrates or covalent inhibitors of enzyme activity.
  • These agents when connected to one end of a bifunctional crosslinking reagent as described above, can provide a means of specific immobilization of a protein to an underlying strut-node architecture.
  • FIG. 5b shows a schematic of a C3 symmetric node that is a trimer formed from three independent, identical subunits, where each subunit possesses additional specific binding functionality, and where proteins have been specifically linked to the node using a bifunctional crosslinking reagent.
  • Such functionalization can be used in nanostructures intended to serve in filters, diagnostics or biological sensing applications.
  • nucleotide sequence coding for the node or strut component is modified by a sequence insertion or extended (e.g., in the form of a polypeptide extension) at either the amino or carboxy terminus with nucleotide sequences coding for additional binding function.
  • sequence insertion or extended e.g., in the form of a polypeptide extension
  • Fig 5c shows a schematic of a C3 node composed of 3 independent chains, where each chain incorporates a covalently linked or fused protein domain.
  • the fused domains can have utility in both protein isolation and in creating protein assemblies.
  • fused domain binding sequences include immunoglobulin domains, polyhistidine sequences, polypeptide sequences that bind to streptavidin (StrepTag), Staphylococcus Protein-A, Staphylococcus Protein-G, an antibody binding polypeptide sequence to which an antibody can bind, an antigenic polypeptide sequence, a hapten polypeptide binding sequence, a binding function for a protein or a metallic surface, a polypeptide sequence that is a substrate for an enzyme, and others together with sequences designed to be linkers with greater or lesser conformational flexibility.
  • Figure 5e shows a single-chain construct of a C3 node where multimer subunits with different functionalities have been interconnected with polypeptide linkers creating an asymmetric multimeric node.
  • the first node subunit has no binding capability (e.g. enzyme active site groups removed through site-specific mutagenesis) or incorporated biotinylation sites
  • the second node subunit has also had binding capability removed but has incorporated biotinylation sites
  • the third subunit incorporates both a fused domain and a protein bound through a bifunctional crosslinking reagent.
  • Figures 5f and 5g show additional possibilities that generally illustrate the modularity and combinatorial flexibility of the approach in generating a wide variety of geometries and functionalized structures.
  • Figure 7a shows a schematic view of a four-fold (C4) symmetric multimer
  • Figures 7b and 7c show representations of the isopentenyl-diphosphate delta-isomerase from Thermus thermophilus (Wada et al. 2006, pdb code:lvcg) protein in space filling and schematic backbone representation respectively. Each chain of the tetramer incorporates 332 amino acid residues, and a non-covalently bound flavin mononucleotide cofactor.
  • Figures 7d and 7e show representations of the inosine-5'-monophosphate dehydrogenase protein from Pyrococcus horikoshii (Asada & Kunishima 2006, pdb code:2cuO) in space filling and schematic backbone representation respectively.
  • FIGS. 8a through 8g show schematic views of nodes based on a protein tetramer having four-fold (C4) rotational symmetry.
  • Each node is composed of a tetrameric protein where the subunits have been modified through site-specific mutagenesis to introduce surface amino acid residues that can be chemically modified to introduce pairs of biotin groups with geometry that is complementary to two of the binding sites on the streptavidin tetramer.
  • Figure 8a shows a node that is a tetramer as formed from four independent, identical chains that are not covalently connected. All of the subunits of the tetramer are symmetrically equivalent. Two biotins are bound to each chain, so that a streptavidin strut can bind to each subunit. In this construct, the pairs of sites of surface biotinylation that are geometrically complementary to streptavidin are on different subunits.
  • Figure 8b shows a node that is a tetramer as formed from four independent, identical chains that are not covalently connected. All of the subunits of the tetramer are symmetrically equivalent.
  • FIG. 8c shows a node based on a protein tetramer formed from a single chain construct, that is, with each subunit linked to another by a polypeptide linker. That is, in the structure shown in Figure 8c the individual chains of the non-covalently associated tetramer are covalently connected together in a single continuous polypeptide chain. Two biotins are bound to each chain, so that a streptavidin strut can bind to each subunit.
  • Figure 8d shows a node based on a protein tetramer formed from a single chain construct. Three of the subunits of the tetramer have bound biotin pairs, but the fourth does not. Thus, only three streptavidin struts can be linked to the tetramer. As such, the tetramer can serve as a branch point for three struts.
  • Figure 8e shows a node based on a protein tetramer formed from a single chain construct. Two adjacent subunits of the trimer have bound biotin pairs, but the third and fourth subunits do not. Thus, only two streptavidin struts can be linked to the tetramer.
  • the tetramer can serve as a connector between struts, but does not allow branching from one strut to two or more other struts.
  • the tetramer can serve, for example, to form a corner of a rectangular assembly.
  • Figure 8f shows a node based on a protein tetramer formed from a single chain construct. Two opposed subunits of the tetramer have bound biotin pairs; the first and third subunits do not. Because only two streptavidin struts can be linked to the tetramer, the tetramer can serve as a connector between struts, but does not allow branching from one strut to two or more other struts.
  • the tetramer can serve, for example, to form a connector between two struts oriented along the same axis.
  • Figure 8g shows a node based on a protein tetramer formed from a single chain construct. Only one of the subunits of the tetramer has a bound biotin pair; the other three do not. Thus, only one streptavidin strut can be linked to the tetramer. As such, the tetramer can serve as a terminator of a strut, and cannot serve as a connector or branch point between struts.
  • Figs. 8d through 8g show covalently connected tetramers of which the surface binding sites on some subunits have been deleted, creating nodes with various streptavidin binding geometry and valency.
  • Figure 9 shows variations of C4 symmetric nodes that have been functionalized and have various geometrical properties.
  • C4 nodes can be functionalized in at least two ways. Nodes may be selected that are enzymes that can be reacted with bifunctional crosslinking reagents that specifically bind to enzyme binding sites for substrates or cofactors.
  • Figure 9a shows a schematic of a C4 symmetric node that is a tetramer formed from four independent, identical subunits, where each subunit possesses additional specific binding functionality corresponding to an enzyme substrate and/or cofactor binding site.
  • Figure 9b shows a schematic of a C4 symmetric node formed from four independent, identical subunits, where proteins have been specifically linked to the node using a bifunctional crosslinking reagent.
  • multimer subunits of C4 nodes my also be modified by a sequence insertion or extended at either the amino or carboxy with nucleotide sequences coding for additional binding function.
  • Figure 9c shows a schematic of a C4 node composed of 4 independent chains, where each chain incorporates a covalently linked or "fused" protein domain.
  • Figure 9d shows a single-chain construct of a C4 node where multimer subunits with different functionalities have been interconnected with polypeptide linkers creating an asymmetric multimeric node.
  • the first node subunit incorporates a fused binding domain (any substrate or cofactor binding ability of the native template node having been removed through site-specific mutagenesis), the second subunit incorporates streptavidin binding capability, the third subunit incorporates a binding protein linked through a bifunctional linker , and the fourth subunit incorporates both a binding protein linked through a bifunctional linker and a fused binding domain.
  • fused domains could include immunoglobin binding domains such as Staphylococcus aureus Protein A, Streptococcal Protein G, nucleotide binding domains, or others while bound proteins could include immunoglobulins or other proteins.
  • the node show in Figure 9d can function as a strut terminator in nanostructures.
  • Figure 9e shows an analogous construct that can form a 90 degree corner in a 2-dimensional planar array.
  • Many additional constructs based on C4 symmetric templates are possible through combinations of the features outlined above, retaining all of the properties of modularity and combinatorial flexibility of the approach in generating a wide variety of geometries and functionalized structures.
  • nodes with higher symmetry do not generally include explicit descriptions of nodes functionalized through incorporation of fused domains or bound proteins, although it can be recognized that these approaches are equally applicable to node subunits forming complexes of higher symmetry.
  • nodes of higher symmetry may be formed using polypeptide chains where two or more of the polypeptide sequences comprising a multimer subunit in the node template structure, have been interconnected to form a single continuous polypeptide chain by interconnection through a polypeptide linker.
  • design of nodes of higher symmetry can incorporate all of the properties of modularity and combinatorial flexibility of the approach defined above in generating a wide variety of geometries and functionalized structures.
  • FIG. 10 presents schematic illustrations of biotinylated nodes with C5 (Fig 10a), C6 (Fig 10b), C7 (Fig 10c) symmetry together will illustrations of thermophile-derived proteins with corresponding symmetry.
  • the C5 symmetric protein shown in surface representation in Figure 10c is a pentameric heme-binding protein from Thermus thermophilus HB8 (Ebihara et. al. 2005, pdb code: lvdh). Each polypeptide chain of the pentamer has 249 amino acid residues.
  • Figure 1Oe shows a surface representation of the PH0250 protein from Pyrococcus horikoshii OTS (Asada and Kunishima 2007, pdb code:2ekd) with C6 symmetry. Each polypeptide chain of the hexamer has 207 amino acid residues.
  • Figure 1Of shows a surface representation of an heptameric RNA binding protein from Methanobacterium thermoautotrophicum (Collins et. al. 2001, pdb code:li81) with C7 symmetry. Each polypeptide chain of the heptamer has 83 amino acid residues. Additional Cn symmetric node templates are presented in Table IA.
  • Non-planar Cn Nodes In addition to Cn nodes with radial planar symmetry
  • Cn multimers with suitable geometrical features can be site-specifically modified to orient streptavidin tetramers at an angle ⁇ to the Cn multimer axis.
  • n 3
  • Nodes with Dn Symmetry Many multimeric structures with Dn symmetry are known from x-ray crystallography studies of proteins from thermophilic organisms (Table IA). Dn-symmetric structures arise through the combination of dyad symmetry and other rotational symmetry operations (Table 1). Nodes with Dn symmetry are particularly useful in the assembly of extended nanostructures since biotinylation sites can be introduced symmetrically across multimer dyad symmetry axes to precisely complement dyad-related biotin binding sites on streptavidin (Fig 1).
  • D n symmetry The simplest D n symmetry is D2, a symmetric tetramer where the multimer subunits are related by 3 mutually perpendicular dyad axes.
  • the streptavidin molecule is itself a tetramer with D2 symmetry.
  • tetrameric D2 symmetric nodes can potentially function as 3 -dimensional nodes in orthorhombic lattices, they are more practically utilized as strut extenders and/or to provide attachment points for additional functionalization.
  • Many tetrameric multimers with D2-symmetry that exhibit a wide range of geometrical features are known from thermophilic microorganisms (Table 1).
  • D2 nodes with suitable structural features can be used to control the relative geometrical orientation and rotational geometry of connected streptavidin struts.
  • Figures 12a and 12c schematically show projection views of a D2- symmetric node able to connect to two streptavidin tetramers through surface biotinylation sites that are introduced at the D2 node surface through site-specific modification of the template protein sequence, followed by a chemical biotinylation reaction (Fig 2).
  • biotinylation sites are schematically indicated by black circles (defining the corners of a rectangle) on the front surface of the D2 tetramer and as shaded circles on the rear surface of the tetramer. These biotinylation sites are geometrically complementary to the biotin binding sites on streptavidin (Fig. Ig).
  • Figures 12b and 12d schematically illustrate 2 streptavidin:node:streptavidin complexes, one incorporating a node of the sort shown in Figure 12a (Fig 12b), and a second (Fig 12d) incorporating the node of Figure 12c.
  • the difference between the complexes is a relative rotation of the streptavidin and node proteins by 90 degrees about the common x-axes of the complexes. In either case, the relative orientations of the terminal free biotin binding sites in the complex are preserved as if the complex were essentially a single streptavidin molecule elongated along the streptavidin x-axis (Fig 1).
  • Such extended struts are useful for the construction of nanostructures with defined dimensions between nodes as outlined below.
  • the ability to control node orientation in such struts is also a useful property allowing controlled of orientation of additional node functionalizing groups.
  • many D2 symmetric node structures will incorporate substrate or cofactor binding sites that can be utilized as linkage sites for the introduction of additional protein domains with binding or functional properties. These binding sites provide a means for introducing functional features into the strut components of the nanostructure.
  • FIG. 13a schematically shows a projection view of a nearly tetrahedral D2 node where the geometry allows symmetrically equivalent introduction of biotinylation sites in two bounding boxes that are oriented at an angle ⁇ to each other along the multimer node x-axis. This feature can introduce a twist in orientation of bound streptavidin tetramers around the common axis of a multimeric complex.
  • Extended struts that incorporate some degree of axis rotation of terminal streptavidin binding sites are useful for the geometrical placement of components in nanostructures as well as for construction of 3-dimensional nanostructures with defined dimensions between nodes as outlined below.
  • Figure 14 presents illustrations of some D2 symmetric protein multimers useful as node templates.
  • Figure 14a shows the iron superoxide dismutase protein from Methanobacterium thermoautotrophicum (Adams et al. 2002, pdb code:lmal) in schematic backbone representation and in surface representation (Fig 14b).
  • Figure 14c shows the alcohol dehydrogenase protein from Sulfolobus solfataricus (Esposito, et. al. 2003, pdb code: Into) in schematic backbone representation and in surface representation (Fig 14d).
  • Figure 14e shows the TenA homolog protein from Pyrococcus furiosus (Benach, et. al.
  • Figure 15 presents schematic illustrations of a hexameric node with D3 symmetry and an octameric node with D4 symmetry.
  • nodes with Dn symmetry are particularly useful in the assembly of extended nanostructures since biotinylation sites can be introduced symmetrically across multimer dyad symmetry axes to precisely complement dyad- related biotin binding sites on streptavidin (Fig 1).
  • this will generally involve introduction of a single site-specific modification in each polypeptide chain of the multimer to introduce a suitable biotinylation site.
  • Dn multimer structures are sufficiently large, it may be possible to introduce 2 biotinylation sites into each polypeptide chain of a Dn multimer (Figs 16 a,b and Figs 16 c,d) that are related by multimer dyad symmetry elements and complementary to the dyad symmetry of the biotin binding sites on streptavidin.
  • the resulting structures shown in Figure 16e (D3) and Figure 16f (D4) could bind 6 and 8 streptavidin strut elements, respectively.
  • Figure 17 presents illustrations of hexameric protein multimers with D3 symmetry and octameric proteins with D4 symmetry useful as node templates.
  • Figure 17 presents illustrations of some D3 symmetric protein hexamers useful as node templates.
  • Figure 17a shows the arginine repressor protein from Bacillus stearothermophilus. (Ni et. al. 1999, pdb code:lb4b) in schematic backbone representation and in surface representation (Fig 17b).
  • Figure 17c shows the adenylyltransferase protein from Methanobacterium thermoautotrophicum (Saridakis et. al 2001, pdb code:lhyb) in schematic backbone representation and in surface representation (Fig 17d).
  • Figure 17e shows the inorganic pyrophosphatase protein from Thermus thermophilus (Teplyakov et. al.
  • Figure 18 presents illustrations of some D4 symmetric protein octamers useful as node templates.
  • Figure 18a shows the PurE protein from Thermotoga maritima (Schwarzenbacher et. al. 2004, pdb code:lo4v) in schematic backbone representation and in surface representation (Fig 18b).
  • Figure 18c shows the sirtuin protein from Thermotoga maritima (Cosgrove et. al. 2006, pdb code:2h2i) in schematic backbone representation and in surface representation (Fig 18d).
  • Figure 18e shows the TT0030 protein from Thermus Thermophilus (Zhu et. al.
  • Nodes with Polygonal Symmetry In addition to nodes with Dn symmetry, several occurrences exist of symmetric multimeric protein complexes with tetrahedral (usually incorporating 12 protein subunits), cubeoctahedral symmetry (usually incorporating 24 protein subunits), or icosahedral symmetry (usually incorporating 2On subunits).
  • the surfaces of these multimers which usually form hollow shell structures, range from nearly spherical, to shapes that approximate truncated tetrahedra. As shown schematically in Figure 19, all of these polyhedra incorporate dyad symmetry elements.
  • Figure 19a shows a truncated tetrahedron
  • Figure 19b shows a cubeoctahedron
  • Figure 19c shows an icosahedron, together with their dyad symmetry axes. Connections made along the dyad axes of these polyhedra can be used to generate structures with features that radiate in three dimensions from a central node. Such dendritic structures may find application in new materials. Some modified polyhedra may serve as nodes in regular 3-dimensional lattices.
  • Figure 20 presents illustrations of protein multimers having the symmetry properties of regular polyhedra and utility as templates for nanostructure node proteins.
  • Figure 20a shows the ornithine carbamoyltransferase dodecameric tetrahedral protein complex protein from Pyrococcus furiosus (Massant et. al. 2003, pdb code:lpw) in schematic backbone representation.
  • Figure 20b shows the 24-subunit cubeoctahedral heat shock protein complex from Methanococcus jannaschii, (Kim et. al. 1998 pdb code:lshs) in schematic backbone representation.
  • Figure 20c shows the 60-subunit dodecahedral protein complex of the dihydrolipoyl transacetylase catalytic domain (residues 184-425) from Bacillus stearothermophilus (Izard, et. al. 1999, pdb code:lb5s) in schematic backbone representation. Additional structures with polyhedral symmetry are listed in Table IA.
  • protein multimers suitable for use as node templates can be composed of two or more protein subunits related by symmetry.
  • Node proteins are created by using site- specific mutagenesis to introduce reactive amino acids at specific sites on the template node protein surface that can be subsequently functionalized to allow the geometrically defined attachment of a linear strut through chemical linkages or non-covalent interactions between specific sites on the node and strut.
  • the envisioned nanostructures will incorporate streptavidin as a strut, or streptavidin in complex with other proteins that can preserve certain binding and geometrical features of the streptavidin tetramer as outlined above (Fig 1, Fig 12) and described elsewhere (Salemme & Weber, 2007).
  • streptavidin is a protein tetramer with D2 symmetry that incorporates 4 binding sites for the vitamin biotin.
  • Node proteins suitable for binding streptavidin are template proteins that have been modified through site-specific modification to allow covalent reaction of specific amino acid side chains to covalently attach biotin groups to the node protein.
  • amino acids can potentially be introduced as sites for specific chemical modification on the template node protein surface, including cysteine, methionine, lysine, histidine, tyrosine and arginine. Any other occurrences of an amino acid of a type that is to be introduced through site-specific modification on the node template surface must also be modified through site-specific mutagenesis by substituting a structurally similar amino acid, so that the final node protein subunit sequence incorporates reactive amino acids only at those sites that facilitate the predefined node-strut geometry.
  • the node structures are modified to incorporate cysteine residues, which can be modified with suitable reagents to incorporate covalently bound biotin groups able to bind streptavidin with defined geometry and high affinity (Fig 2). Cysteine residues occurring on the surface in the naturally occurring sequence of the node template protein are substituted with serine, alanine or another amino acid depending upon the local structural environment.
  • Figure 1 illustrates the structure of streptavidin, a D2 tetramer whose subunits are related by three mutually perpendicular two-fold rotational (dyad) axes of symmetry.
  • the biotin binding sites on streptavidin (or more specifically the coordinates of the protein-bound biotin carboxyl oxygen atoms that are the sites of bifunctional chemical reagent attachment) are separated by 20.5 Angstroms and oriented along a line at an angle of 20 degrees relative to the "y" dyad axis of the streptavidin tetramer (as defined in Fig 1) and at an angle of 72 degrees relative to the "z" dyad axis of the streptavidin tetramer.
  • the bound streptavidin In general, maximum precision and flexibility in the assembly of streptavidin-linked structures is achieved when the bound streptavidin is positioned with defined geometry relative to the node to which it is bound. For nodes with Cn symmetry, the bound streptavidin should be aligned so that either the y or z- dyad axes of the streptavidin tetramer are aligned parallel with the Cn symmetry axes of Cn symmetric nodes.
  • the y or z-dyad axes of the streptavidin tetramer are aligned with one of the D2 dyad axes, and the streptavidin x dyad axis is coincident with a second dyad axis of the D2 node (Although there are some exceptions to this rule as shown in Figure 13).
  • the y or z-dyad axes of the streptavidin tetramer is aligned with the Dn axis of the node and the streptavidin x dyad axis is coincident with a dyad axis of the Dn node.
  • the y or z-dyad axes of the streptavidin tetramer are aligned with a major symmetry axis of the node (depending on the polyhedral node symmetry), and the streptavidin x-dyad axis is coincident with a dyad axis of the polyhedral node.
  • Figures 21b and 21c show schematic views of a node with Cn rotational symmetry, where the Cn symmetry axis defines the z-axis of the structure.
  • the geometry of site-specific modifications on the node template (or more specifically the coordinates of the thiol sulfur atoms of the incorporated cysteine side chains on the node protein) must be complementary to the geometry of the biotin binding sites on streptavidin, and must align the streptavidin z-axis (Fig 21a) or y-axis (Fig 21b) with the node Cn or z-axis. This requires that the modification sites on the nodes are oriented at an angle (e.g.
  • two specific amino acid reactive residues (or site-specific modifications) of the nanostructure node multimeric protein (or node template) can be complementary to the geometry of a pair of biotin binding sites on a streptavidin or streptavidin derivative strut.
  • the Cn symmetry axis of the nanostructure node multimeric protein is substantially parallel to a dyad axis of the D2 symmetric streptavidin or streptavidin derivative strut.
  • substantially parallel can mean, for example, parallel to within less than or equal to, for example, 0.5 degree, 1 degree, 2 degrees, 5 degrees, or 10 degrees.
  • Cn Symmetric Node Specification Definition of the sites for site-specific modification on Cn symmetric node templates can be determined using computer modeling, computational methods or a combination of these methods. Generally the methods involve a constrained geometrical search for favorable interaction complexes.
  • Figure 22 schematically illustrates the variable search parameters for Cn and Dn node structures.
  • the Cn search parameters include a rotation of the Cn node about its z-axis, and a translation of streptavidin along its x-axis in the xy plane of the node (Fig 22a).
  • the method involves initially orienting the Cn template node and streptavidin so that they a) do not spatially overlap, b) are oriented with the Cn (z-axis) of the node parallel to either the y-axis or z-axis of streptavidin, and c) have similar z coordinate values for their respective centers of mass.
  • the node is incrementally rotated about the Cn axis through an angular range somewhat greater than 360/n degrees.
  • the streptavidin tetramer is translated along its dyad x- axis until van der Waals contact or near van der Waals contact is made between the atomic coordinates of the node template and atomic coordinates of streptavidin.
  • Each of the resulting streptavidin-node complexes is then examined using computer graphics (Jones et. al. 1990, Humphry et. al. 1996), geometrical, energetic computational methods (Case et. al.
  • Cn Polyhedral Node Specification The method outlined above is suitable for nodes that are incorporated into essentially planar, 2-dimensional structures oriented on surfaces. Similar constrained searches can be developed to design nodes for the assembly of 3- dimensional structures. For example, nodes can be designed that can assemble into 3- dimensional polyhedra that such as a regular a regular dodecahedron incorporating C3 symmetric nodes or a regular icosahedron incorporating C5 symmetric nodes ( Figure 11). Additional polyhedral nodes are possible as well.
  • the approach to defining sites for modification is similar to that outlined above for Cn planar nodes, except that the orientation of the approach axis between streptavidin and the Cn axis of the node complex is not 90 degrees, but is the angle ⁇ formed between the edge of the polyhedron and a vector from the center of the polyhedron to an apical node (Fig 22b).
  • the apex angle ⁇ for an icosahedron is approximately 121.92 degrees (Fig lla) and for a dodecahedron ⁇ is approximately 110.93 degrees (Fig lib).
  • Dn Node Specification Nodes based on node templates with Dn symmetry represent an extensive family with diverse structural geometry (Table IA). As noted above, structures with dyad symmetry axes such as Dn symmetric structures offer the possibility of symmetric placement of biotin linkage sites on node subunits that are complementary to the binding sites on streptavidin. The process generally produces node subunit proteins that incorporate only a single site-specific modification for the purposes of incorporating a reactive cysteine residue, so that the bound streptavidin tetramer in the complex forms a symmetric link between node subunits oriented by a dyad axis of symmetry.
  • Definition of the sites for site-specific modification on Dn symmetric node templates can be determined using a constrained computer search (Fig 21c) where a) the z-axis of the streptavidin tetramer and either the x,y, or z-dyad axes of D2 node are constrained to be parallel, and b) the approach x-axis of streptavidin (which is a dyad axis) is constrained to be coincident with a dyad axis relating subunits of the Dn-symmetric node template.
  • Final complex configurations are those where the atoms of streptavidin and the node make Van der Waals contact or near Van der Waals contact.
  • Nodes based on node templates with D2 symmetry are appropriate for many applications including formation of 2D and 3D lattices, as well as for strut extenders that connect two streptavidin tetramers in a linear array (Fig 12b,d).
  • Definition of the sites for site- specific modification on D2 symmetric node templates can be determined using a constrained computer search as outlined above, noting however, that since the D2 node has three mutually perpendicular dyad axes, and that there are 2 alternative streptavidin orientations around each dyad, that there are potentially a total of six possible complex configurations where streptavidin can be symmetrically bonded to a D2 node so that its dyad symmetry axes are coincident and/or perpendicular to the symmetry axes of the node.
  • Figure 12a illustrates the case where the streptavidin z-dyad axis is parallel to the z-dyad axis of a D2 node
  • Fig 12c illustrates the example where the streptavidin y-dyad axis is parallel to the z-dyad axis of a D2 node.
  • Locating the positions on a Dn node surface suitable for the introduction cysteine residues for biotinylation may also be performed through an alternative graphical or mathematical process. Basically this involves the superposition of "bounding boxes" (with dimensions of approximately 6.4 Angstroms by 19.5 Angstroms, Fig 21d.) that represent the projected positions of the potential biotinylation sites (e.g. sites complementary to the biotin bonding sites in each of the 2 possible streptavidin binding orientations) around each dyad axis in a structure.
  • bounding boxes with dimensions of approximately 6.4 Angstroms by 19.5 Angstroms, Fig 21d.
  • Figure 23 shows a stereoscopic view of the D2 symmetric node template pdb code: lrtw with pairs of bounding boxes embedded along each of the three dyad axes.
  • a computer program can then be used to find the shortest distances between selected amino acid side chain atoms in the exposed atom/residue list and the lines defining the bounding box that project the positions of the biotin binding sites.
  • the C ⁇ atoms can be identified by inspection using computer graphics modeling programs. The atoms so identified will generally define the amino acid residues in the template sequence that can be mutated to Cys residues, and when functionalized by biotinylation, will form sites that are symmetric to streptavidin and align the Dn axis of the node to either the y-axis or z-axis of streptavidin.
  • alternate linear couplers can be engineered that introduce twist between the streptavidin tetramers linked to the D2 node along the complex x-axis (Fig 13).
  • Identification of modifications sites on the node template involves a process that is slightly different from that described above, where the search (or alternatively, the rotational orientation of the bounding boxes around the complex x-axis) is performed with the z-axes of the streptavidin tetramer and the D2 node oriented at some predetermined angle ⁇ (Figl3a), depending on the total angular twist desired in the final linear coupler.
  • Additional nodes can be based on node templates with D3 or D4 symmetry as detailed below.
  • Definition of the sites for site-specific modification on Dn symmetric node templates can be determined using a constrained computer search process similar to that described above for Cn nodes, where the orientation of the approach axis between streptavidin and the Dn axis of the node complex is 90 degrees, but the search is additionally constrained so that the approach axis along which the streptavidin molecule advanced is coincident with a dyad axis relating subunits of the Dn- symmetric node template.
  • Polyhedral Node Specification Additional nodes, appropriate for the formation of extended 3-dimensional radial structures or 3-dimensional lattices, can be based on node templates with higher symmetry that incorporate dyad symmetry elements. Observed node symmetries include tetrahedral, cubic, cuboctahedral, and truncated icosahedral (Table IA).
  • Definition of the sites for site-specific modification on these higher symmetry node templates can be determined using a constrained computer search process similar to that described above for D2 nodes, where the orientation of the x approach axis of streptavidin is constrained to be coincident with a dyad axis relating subunits of the symmetric node template. Note that this process generally produces node subunit proteins that incorporate only a single site-specific modification per subunit, so that the streptavidin tetramers in the complex form symmetric links between node subunits oriented by a dyad axis of symmetry.
  • Table 2 provides the Protein Data Bank code (pdb code) for the node template structure, the node symmetry, the amino acid sequence of the node template (as downloaded from the Protein Data Bank), and the modifications of the sequence that are required to create a node that can be functionalized by biotinylation so that it interacts with streptavidin or other proteins with binding sites disposed with the same geometry as the streptavidin binding sites (Salemme & Weber 2007). Sequence modifications are grouped as "general” and "specific biotinylation sites”. General sequence modifications usually represent modifications to replace potentially interfering cysteine residues occurring in a template sequence with structurally similar residues.
  • pdb code Protein Data Bank code
  • the replacement amino acid may be Ala, Ser, His, Asp, or potentially some other amino acid.
  • Additional sequence modifications that "generally" alter the template protein sequence could include terminal modifications and/or the introduction of subunit linking polypeptide sequences to create single-chain structures.
  • residues designated as sites of modification in Table 2 correspond to the sequence numbering provided in the designated pdb file containing the structural coordinates of the node template.
  • biotinylation sites are sites for the introduction of Cys residues into the template sequence that will provide optimal geometry and, for Dn and tetrahedral nodes, symmetric placement of the biotinylation sites around the node dyad symmetry axes. The locations of these sites were determined by use of the computer graphical and computational methods defined above. As noted above in Figure 21 there are generally two orthogonal orientations that streptavidin can take with respect to the major symmetry axes of complexes with Cn, Dn, or higher symmetry.
  • nodes with Dn or higher symmetry offer the possibility of aligning the dyad symmetry axes of streptavidin with dyad symmetry axes of the node. These are enumerated as "H” and "V" along diad axes (x,y, or z) of a Dn or higher symmetry node (Fig 21d,e). D2 nodes have three dyad axes, so there are a total of 6 orientations by which streptavidin can be attached to a D2 node.
  • Figures 24a and 24b respectively show a schematic view and space filling view of a node based on the previously described trimeric C3 symmetric protein lthj, in covalent complex with 3 bound molecules of streptavidin.
  • biotins bound to streptavidin are shown in space filling representation in the schematic diagrams although atomic coordinates for linking atoms or amino acid side chains residues are not shown for simplicity.
  • the one shown corresponds to a node construct where a Cysl48 to Ala modification and specific biotinylation sites have been introduced at sequence positions 70 (Asp70 to Cys) and 200 (Tyr200 to Cys) in the lthj polypeptide sequence (Table 2A).
  • Table 2C also provides a node specification of for C3 trimeric planar node based on the Ij 5s protein described above.
  • Figure 25 shows a stereoscopic view of a single chain variant of the lthj trimer.
  • the sequence of the single-chain trimer incorporates three 207-residue amino acid sequences derived from the original lthj sequence that are interconnected by two seven residue linkers.
  • Table 2B gives sequence specifications for the trimer variants with both symmetric and asymmetric binding sites for streptavidin as schematically illustrated in Figure 4c,d,e.
  • Figures 24c and 24d respectively show a schematic view and space filling view of a node based on the previously described trimeric C4 symmetric protein lvcg, in covalent complex with 4 bound molecules of streptavidin.
  • the illustration shown corresponds to a node construct where Cys 14 and Cys236 modifications have been made and specific biotinylation sites have been introduced at sequence positions 44 (Ser44 to Cys) and 49 (Thr49 to Cys) in the lvcg polypeptide sequence (Table 2E).
  • FIG. 26 shows schematic and surface stereoscopic views of a C3 symmetric node, with 3 streptavidin tetramers bound at angles corresponding to an dodecahedron apex (Fig lla).
  • the dodecahedral polyhedral node is based upon the structure of a 5'-deoxy-5'-methylthioadenosine phosphorylase homologue from Sulfolobus tokodaii (Kitago et al. 2003) protein as the template node, and generated by the methods described above (pdb code: Iv4n). Specific sequence specifications are given in Table 2D. Table 2D also gives a specification for a "bucky" or truncated icosahedral apex node (See Fig 19.c), based on Iv4n as the node template.
  • FIG. 27 shows schematic and surface stereoscopic views of a C5 symmetric node, with 5 streptavidin tetramers bound at angles corresponding to an icosahedron apex (Fig lla).
  • the icosahedral polyhedral node is based upon the lvdh protein (described above Fig 1Od) as the template node, and generated by the methods described above. Specific sequence specifications are given in Table 2F.
  • Streptavidin D2 Strut Coupler As noted above (Fig 1), streptavidin itself is a tetramer with D2 symmetry and can function as a node in the context of some assemblies. Although not specifically derived from a thermophilic bacterium, streptavidin is unusual for its thermostability both in its unliganded and biotin-bound forms (Weber et. al. 1989,1992,1994). Figure 28 schematically shows streptavidin tetramers that have been modified through site- specific mutagenesis to incorporate four dyad symmetry-related biotinylation sites (e.g.
  • FIGS 28a and b respectively show schematic and surface representations where the x-axis of the central "node" streptavidin tetramer is oriented parallel to the z-axes of 2 bound streptavidin tetramers.
  • Figure 28 c and d respectively show schematic and surface representations where the z-axis of the central "node” streptavidin tetramer is oriented parallel to the z-axes of 2 bound streptavidin tetramers.
  • streptavidin "nodes” modified for binding streptavidin tetramers along streptavidin dyad are given in Table 2G.
  • This method of attaching streptavidin-linked binding or other functional protein domains provides an additional means for creating functionalized struts in nanostructures.
  • Table 2G also provides a specification for a streptavidin "node” with streptavidins bound along the "node” x-axis, so blocking access to the streptavidin "node” biotin binding sites. Such constructs may be useful when it is desirable to protect the "node" biotin binding sites during an intermediate stage of an assembly process.
  • FIGS 29a,b show stereoscopic views of a tetrameric D2 node based on the lmal node template in schematic and space filling representation respectively.
  • Table 2H gives the specifications for variations in the lmla node based on different orientations of bound streptavidin tetramers (e.g. see Fig 12,a,c) and combinations of biotinylation sites along each of the three independent node dyad axes.
  • Variations in dyad axis site substitution patterns can produce nodes suitable for the formation of orthorhombic 3D lattices (e.g. the node shown in Fig 29), 2D rectangular lattices, or linear strut extenders.
  • Figure 30 shows illustrations in schematic and space filling representation of two examples of linear struts incorporating a D2 node based on lmal and two streptavidin tetramers.
  • streptavidin tetramers are oriented with their z-axes parallel to one of the D2 node dyad axes.
  • D3 Nodes Figures 31a,b show stereoscopic views of a hexameric D3 node based on the lhyb node template in schematic and space filling representation respectively. There are 6 streptavidin tetramers bound to the node, including 3 tetramers with their y-axes oriented parallel to the D3 node symmetry axis and 3 tetramers with their z-axes oriented parallel to the D3 node symmetry axis.
  • the 2 "poles" of the D3 dyad axes are symmetrically non-equivalent, and that variations can be produced with for example, with 3 bound streptavidin tetramers bound at either pole in either of two orientations ( Figure 12a,c).
  • Table 2L gives the specifications for variations in the lhyb node based on different orientations of bound streptavidin tetramers (e.g. see Fig 12,a,c) and combinations of biotinylation sites at poles of the dyad axis.
  • Tables 2K and 2M respectively provide additional specifications for D3 symmetric nodes based on the Ib4b and 2prd node templates.
  • FIGs 32a,b show stereoscopic views of two octameric D4 node complexes based on the 2h2i node template in schematic representations. There are 4 streptavidin tetramers bound to each node, along the two symmetrically non-equivalent axes of the D4 node (Fig 15f).
  • the complex shown in Figure 31a incorporates streptavidin tetramers with their z-axes oriented parallel to the D4 node symmetry axis, while the complex shown in Figure 31b incorporates streptavidin tetramers with their y-axes oriented parallel to the D4 node symmetry axis.
  • Table 2O gives the sequence specifications for variations the 2h2i node based on different orientations of bound streptavidin tetramers (e.g. see Fig 12,a,c) and combinations of biotinylation sites along the symmetrically non-equivalent dyad axes.
  • Tables 2N and 2P respectively provide additional sequence specifications for D4 symmetric nodes based on the Io4v and 2iel node templates.
  • Tetrahedral (Cubic Lattice) Node Figures 33a,b show stereoscopic backbone and space-filling views of a dodecameric (T23) tetrahedral node based on the lpw node template in complex with 6 streptavidin complexes bound along the 3 symmetrically equivalent, mutually perpendicular dyad axes of the structure.
  • Table 2Q gives the sequence specifications for the 2 possible binding orientations for streptavidin to the node along the dyad axis.
  • Figure 34 shows schematic views of struts of different length consisting of combinations of streptavidin and nodes with D2 symmetry. Such constructs are useful in controlling the dimensions of assembled nanostructures.
  • Figure 34a shows an extended strut incorporating two streptavidin tetramers and a single D2 symmetric node (e.g see Fig 30ab).
  • Figure 34b shows an extended strut incorporating three streptavidin tetramers and two D2 symmetric nodes.
  • the central streptavidin has been modified (e.g.
  • Figure 35 schematically shows examples of radial structures as, for example, could be formed on self-assembling monolayers or anchored to discrete metal particles deposited on a silicon or other non-metallic substrate surface.
  • Figure 35a shows a C3 node which is linked through streptavidin struts to 3 single-chain C4 tetramers that have all been functionalized as described in Figure 9.
  • Figure 35b shows a C7 node which is linked through streptavidin struts to 7 single-chain C3 trimers that are variations of the functionalized trimers described in Figure 5.
  • the structures can be also be functionalized through modifications introduced into the struts (e.g. see Figs 28 and 34).
  • Two-dimensional lattices functionalized with specific binding molecules like immunoglobulin binding domains could find application in diagnostics, biological filters or other applications.
  • Figure 36 schematically shows examples of 2- dimensional lattices, as, for example, could be formed on self-assembling monolayers.
  • Figure 36a shows a hexagonal lattice incorporating C3 nodes linked through streptavidin struts.
  • Figures 36b,c show square lattices incorporating C4 nodes and struts of different lengths to control the lattice dimensions.
  • the struts in Figure 36c incorporate a D2 strut extender as outlined in Figure 30.
  • the structures can be functionalized either through modifications introduced into the nodes (e.g Figs 5 and 9) or struts (e.g Figs 28 and 34).
  • Two-dimensional lattices functionalized with specific binding molecules like immunoglobulin binding domains could find application in diagnostics, biological filters, or other applications.
  • 2-dimensional polygonal structures as, for example, could be formed on self-assembling monolayers.
  • Figure 37a shows a hexagon array incorporating single-chain C3 nodes linked through streptavidin struts.
  • Figures 37b,c show square arrays incorporating single-chain C4 nodes and struts of different lengths to control the lattice dimensions.
  • the struts in Figure 37c incorporate a D2 strut extender as outlined in Figure 30.
  • the structures can be functionalized either through modifications introduced into the nodes (e.g see Figs 5 and 9) or struts (e.g. see Figs 28 and 34).
  • Two-dimensional polygonal structures functionalized with specific binding molecules like immunoglobulin binding domains could find application in diagnostics, biological filters or other applications.
  • Radial 3 -dimensional structures can be produced by the attachment of struts incorporating streptavidin to the dyad axes of polyhedral nodes such as those shown in Figures 19 and 20.
  • Struts or terminating nodes of struts can be functionalized either through modifications introduced into the nodes (e.g see Figs 5 and 9) or struts (e.g. see Figs 28 and 34).
  • Radial structures functionalized with specific binding molecules like immunoglobulin binding domains could find application in diagnostics, biological filters or other applications.
  • 3-Dimensional Polygon Structures Three-dimensional polygonal structures with defined geometry and dimensions can be generated through the combination of struts incorporating streptavidin and nodes with the symmetry and geometry corresponding to a polygonal apex node.
  • Representative structures of regular polyhedra are shown in Figures lla,b and Figure 19c.
  • Examples of apex node structures for regular dodecahedra and icosahedra are given in Figures 26 and 27 respectively.
  • Sequence specifications for these nodes are given in Table 2D and 2F respectively.
  • Table 2D also provides a specification for a "bucky" node.
  • Three- dimensional polygonal structures can be functionalized with specific binding molecules like immunoglobulin binding domains and could find application in diagnostics, biological filters or other applications.
  • 3-dimensional polygonal structures which are generally hollow inside, can be used to encapsulate or coat organic, inorganic, or biomaterials for imaging, diagnostic, drug delivery or other applications.
  • 3-Dimensional Lattices Three-dimensional lattices can be built up from molecular nodes and struts using a number of different strategies, allowing precise control of geometrical and symmetry properties of the resulting lattice.
  • Figure 38a,b presents stereoscopic views, in schematic and space filling representation, of a 3D lattice node incorporating two variations of a D3 node derived from the node template lhyb (Fig 17cd and Table 2L).
  • the two node variations have biotinylation sites that orient bound streptavidin tetramers at 90 degrees to each other (e.g see Fig 12a,c) along their equivalent dyad axes.
  • Figure 40a schematically illustrates the 3-connected 3D lattice that can be formed incorporating such linked nodes (shown as two white dots in the schematic lattice illustration).
  • Figures 39a,b present stereoscopic views, in schematic and space filling representation, of a 3D lattice node incorporating two variations of a D4 node derived from the node template 2h2i (Fig 18cd and Table 2O).
  • FIG 40b schematically illustrates the 4- connected 3D lattice that can be formed incorporating such linked nodes (shown as two white dots in the schematic lattice illustration).
  • Figures 40a,b present stereoscopic views, in backbone and space filling representation, of a 3D lattice node derived from the dodecahedral node template lpw (Table 2Q).
  • Figure 40c schematically illustrates the 6-connected 3D cubic lattice that can be formed by linking such nodes with streptavidin or extended struts.
  • the central white dot represents the location of a node.
  • the nodes and struts of 3-dimensional lattices can be functionalized with specific binding molecules like immunoglobulin binding domains and could find application in diagnostics, biological filters or other applications.
  • specific binding molecules like immunoglobulin binding domains
  • proteinaceous nanoscale assemblies can provide a means of high- resolution patterning of silicon, glass, metal, or other substrates, to allow production of microelectronic devices, devices incorporating zero-mode waveguides (Levene et. al, 2003) or microelectromechanical systems (MEMS) using conventional semiconductor fabrication (Widman et al., 2000) and/or MEMS fabrication technology (Judy, 2001).
  • the proteinaceous nanoscale assembly can be used directly as a way of introducing a pattern on a substrate material.
  • the proteinaceous nanoscale assembly is used as a way of masking a resist to transfer the pattern of the nanoscale assembly to an underlying substrate material.
  • the approaches outlined below are applicable to both 2-dimensional and 3-dimensional assembly architectures.
  • Figure 41 schematically illustrates a method of making a nanostructure pattern on a surface.
  • Figure 41a shows, for example, a substrate that has a semiconductor material surface with a single gold atom or cluster (Haztor-di Picciotto, 2007) or, alternatively, a patch of chemically reactive molecules (e.g., Liu & Amro, 2002) located on the surface to nucleate the formation of the nanostructural assembly.
  • a substrate that has a semiconductor material surface with a single gold atom or cluster (Haztor-di Picciotto, 2007) or, alternatively, a patch of chemically reactive molecules (e.g., Liu & Amro, 2002) located on the surface to nucleate the formation of the nanostructural assembly.
  • a patch of chemically reactive molecules e.g., Liu & Amro, 2002
  • the node can be specifically immobilized on the surface (Fig 41b).
  • the immobilized node can be further reacted with nanostructural components incorporating streptavidin or streptavidin-incorporating struts to form immobilized nanostructures such as schematically illustrated in Figure 41c.
  • Figure 42 schematically presents a method of making a repetitively patterned protein nanostructure on a metallic or non-metallic substrate following the steps exemplified in Figure 41 using a simplified representation for the node and strut components.
  • a substrate Fig 42a
  • the nucleation sites can be arranged in a regular or periodic pattern, a quasiperiodic pattern (such as a Penrose tiling), or a non-periodic predetermined patter.
  • a patterned array (Fig 42c) is produced.
  • Figure 42d shows a section of the patterned surface at the section line ⁇ in Figure 42c.
  • Figure 43 presents a method of making a patterned nanostructure assembly with sub- 100 nanometer features on a substrate surface.
  • Figure 43a reiterates the patterned surface of Figure 42c and Figure 43b shows the section of Fig 43a at ⁇ l.
  • Figure 43c shows the result of using any of several methods of semiconductor fabrication (e.g., using various forms of plasma and/or chemical vapor deposition technology, Widman, et al., 2000) to coat the substrate patterned with the protein nanostructure to produce the patterned surface shown in plan in Figure 43c and in section in Figure 42d (corresponding to the section line ⁇ 2 in Figure 43c).
  • the patterned substrate can be coated with materials such as a metal (such as iron), a noble metal (such as gold, platinum, or silver), a glass (such as silicon dioxide), a ceramic, a semiconductor (such as silicon or germanium), a carbon allotrope (such as diamond or graphite), a polymer, and/or an organic polymer (such as tetrafluoroethylene).
  • a metal such as iron
  • a noble metal such as gold, platinum, or silver
  • a glass such as silicon dioxide
  • a ceramic such as silicon dioxide
  • a semiconductor such as silicon or germanium
  • a carbon allotrope such as diamond or graphite
  • a polymer such as tetrafluoroethylene
  • Figure 44 presents a method of making a patterned structure with sub- 100 nanometer features on a substrate surface using a proteinaceous nanostructure assembly as a patterned mask superimposed on a photoresist material.
  • Figure 44a,b,c shows a cross section of a protein nanostructure (Fig44c) superimposed on a layer of a resist material (Fig44b), that is in turn coated on a substrate to be patterned (Fig 44a).
  • Exposure of the assembly to, for example, irradiation of a suitable nature to modify the resist, produces the structure of Figure 44d, where the superimposed nanostructure has prevented exposure of the resist to the incident radiation.
  • Figure 44e shows the structure where the exposed resist has been dissolved away, for example using chemical means.
  • Figure 44f shows the structure where the exposed substrate surfaces have been etched producing nanoscale features that are complementary to the structural features of the proteinaceous nanoscale assembly used to pattern the resist.
  • Figure 44g shows the structure after the proteinaceous nanoscale assembly and non-reacted resist have been removed, for example by using chemical means.
  • the resulting patterned surface can be used as a template for soft lithography (Xia & Whitesides 1998, Rogers & Nuzzo 2005) or as a step in a multistep semiconductor fabrication process (Widman et al., 2000).
  • FIG. 45ab schematically shows a cubic lattice structure composed of six-connected cubic nodes (for example, see Fig 33) and streptavidin struts (Fig 45b) assembled on a solid substrate (Fig 45a).
  • Figure 45cd shows the structure (Fig. 45c) embedded in a matrix (Fig 45d) that can polymerize and/or be transformed by chemical reaction, heat, and/or radiation to form a chemically and/or thermally stable matrix material.
  • the matrix (Fig. 45d) can interpenetrate the structure (Fig. 45c).
  • the matrix (Fig 45d) can itself have the form of a cubic lattice offset from the cubic lattice of the proteinaceous nanostructure assembly.
  • the cubic lattice of the structure (Fig 45c) and the cubic lattice of the matrix (Fig 45d) can interpenetrate each other.
  • Figure 45e shows the structure after chemical, heat, and/or radiation treatment is applied to ablate the proteinaceous nanoscale structure, leaving a "negative" three-dimensional cubic channel structure in the matrix material. That is, the matrix material can occupy the space not occupied by the proteinaceous nanostructure assembly.
  • the matrix material can include a metal (such as iron), a noble metal (such as gold, platinum, or silver), a glass (such as silicon dioxide), a ceramic, a semiconductor (such as silicon or germanium), a polymer, and/or an organic polymer (such as tetrafiuoroethylene).
  • a metal such as iron
  • a noble metal such as gold, platinum, or silver
  • a glass such as silicon dioxide
  • a ceramic such as silicon dioxide
  • a semiconductor such as silicon or germanium
  • a polymer such as tetrafiuoroethylene
  • Figure 45f shows the structure of Fig 45e after further chemical treatment is applied to deposit a metallic or other second matrix material in the negative cavity originally occupied by the proteinaceous nanostructure assembly.
  • the second matrix material can include a metal (such as iron), a noble metal (such as gold, platinum, or silver), a glass (such as silicon dioxide), a ceramic, a semiconductor (such as silicon or germanium), a polymer, and/or an organic polymer (such as tetrafiuoroethylene).
  • Figure 45g shows the structure after chemical, heat, or radiation treatment is applied to remove the first matrix material, leaving a nanoscale structure composed of metal or other second matrix material that is a replica of, that is, has the same or similar form as the original proteinaceous nanostructure assembly.
  • three-dimensional nanoscale assemblies made of metal or semiconductor materials have potential utility as components in semiconductor or MEMS applications.
  • a subunit can be a tertiary polypeptide structure.
  • the amino acid residues in a subunit can be covalently linked through peptide bonds in a polypeptide sequence.
  • a subunit can be formed of one or more polypeptide chains.
  • the polypeptide subunit can, under certain conditions, e.g., certain pH conditions, aggregate with one or more other polypeptide subunits to form a multisubunit node polypeptide that is a quaternary polypeptide structure. For example, in a native streptavidin tetramer, 4 identical subunits, each formed of an identical but separate polypeptide chain, aggregate.
  • a multimeric protein having a symmetry can be formed of several essentially identical subunits that are repeated with an orientation with respect to each other to achieve the symmetry.
  • a Cn symmetric multimeric protein can be formed of n subunits placed about a common axis.
  • a C3 symmetric multimeric protein can be formed of 3 subunits placed about a common axis.
  • a Dn symmetric multimeric protein can be formed of 2n subunits, where each subunit is related to another subunit to form a pair, and each pair of subunits is placed about a common axis.
  • a D4 symmetric multimeric protein can be formed of 8 subunits, where each of 4 pairs of subunits are placed about a common axis.
  • a multimeric protein having the symmetry of a Platonic or Archimedean solid can be formed of a number of subunits equal to the number of edges in each polygonal face of the solid, summed over the polygonal faces.
  • a multimeric protein with tetrahedral symmetry can be formed of a number of subunits equal to the number of edges in a face, 3, times the number of faces, 4, that is, 12 subunits.
  • a multimeric protein with dodecahedral symmetry can be formed of a number of subunits equal to the number of edges in a pentagonal face, 5, times the number of faces, 12, to yield a total of 60 subunits.
  • Polypeptide subunits (subunits) within the quaternary polypeptide structure can be held to each other by noncovalent bonds (e.g., ionic bonds, van der Waals bonds, and/or hydrophobic bonds) and/or by covalent bonds (e.g., disulfide bridges and/or peptide bonds).
  • noncovalent bonds e.g., ionic bonds, van der Waals bonds, and/or hydrophobic bonds
  • covalent bonds e.g., disulfide bridges and/or peptide bonds
  • each subunit may be formed of one or more polypeptide chains that are not covalently bound to the polypeptide chains of any other subunit of a quaternary polypeptide structure, each subunit may be formed of a polypeptide chain that is covalently bound to a polypeptide chain of at least one other subunit (e.g., the quaternary polypeptide structure can formed of a number of polypeptide chains less than the number of subunits, for example, the quaternary polypeptide structure can be formed of a single polypeptide chain), or some subunits may be formed of a polypeptide chain not covalently bound to a polypeptide chain of another subunit whereas other subunits are formed of a polypeptide chain that is covalently bound to a polypeptide chain of at least one other subunit.
  • the quaternary polypeptide structure can formed of a number of polypeptide chains less than the number of subunits, for example, the quaternary polypeptide structure can be formed of a single poly
  • amino acid residues of a polypeptide subunit can be in a single polypeptide sequence.
  • Multimerization can refer to the process in which individual polypeptide subunits aggregate to form a multisubunit node polypeptide.
  • the structure formed by the aggregated subunits can be termed a multimer.
  • Such a multimer can be referred to as having quaternary structure.
  • Three individual polypeptide subunits, each formed of a polypeptide chain that is not covalently linked to another subunit, aggregating under the influence of non-covalent bonds to form a trimer is an example of multimerization.
  • three individual polypeptide subunits can be formed of a polypeptide sequence that is covalently linked to the polypeptide sequence of another subunit, so that the three polypeptide subunits are formed from a single polypeptide chain.
  • each individual polypeptide subunit can be folded into a separate tertiary structure without the individual polypeptide subunits being assembled into a quaternary trimer.
  • the tertiary structures of the individual polypeptide subunits can come into close proximity, for example, under the influence of non-covalent bonds, to form a quaternary trimer in which a number of amino acid residues of each polypeptide subunit are in close proximity to a number of the amino acid residues of the other polypeptide subunits.
  • a rotational symmetry axis of an object can be an axis about which a less than full rotation of the object can result in a matching superposition of the object upon itself.
  • An ordering of subunits about the rotational symmetry axis can refer to the subunits corresponding to the N-fold symmetry in a successive clockwise or counter-clockwise sequence when sighting along the rotational symmetry axis.
  • polypeptide subunits of a multisubunit node polypeptide can be related by a symmetry.
  • reference to a symmetrical relation herein is to be understood to encompass an essential symmetry relation. That is, features that are essentially related by a symmetry might not be strictly identical.
  • two of the polypeptide subunits may differ from each other in that one, two, or a short oligomeric subsequence of the polypeptide sequences from which they are formed are different. However, this minor difference in the polypeptide sequence does not affect the overall form of the subunit.
  • trimer still can be considered to have three-fold rotational symmetry.
  • a derivative of an initial molecule includes molecules resulting from the replacement of an atom, group of atoms, bond, or bonds of the initial molecule by a different atom, group of atoms, bond, or bonds and molecules resulting from the addition or deletion of an atom or a group of atoms to the initial molecule, or from the rearrangement of an atom, group of atoms, bond, or bonds of the initial molecule, for example, as in an isomer or stereoisomer.
  • 2-iminobiotin is a derivative of biotin.
  • 2-iminobiotin is the same as that of biotin, except that the oxygen double bonded to the imidazolidine is replaced with a single bonded primary amine and the single bond between the 2-carbon and the 3 -nitrogen of the imidazolidine ring is replaced by a double bond.
  • nucleobases include cytosine, guanine, adenine, thymine, uracil, 5-methylcytosine, ribothymidine, hypoxanthine, xanthine, 7- methylguanine, and 5,6-dihydrouracil.
  • nucleobase derivatives examples include isoguanine, isocytosine, 2-amino-6-(2-thienyl)purine, pyrrole-2-carbaldehyde, and acycloguanosine (Aciclovir).
  • nucleosides examples include adenosine, guanosine, 5'-methyluridine, uridine, cytidine, deoxynucleosides, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine, i nosine, xanthinosine , 7-methylguanosine, pseudouridine, dihydrouridine, 5- methylcytidine, dideoxynucleosides.
  • nucleoside derivatives examples include azidothymidine (Zidovudine), didanosine, vidarabine, cytosine arabinoside (cytarabine), emtricitabine (Emtriva), lamivudine, dideoxycytidine (zalcitabine), abacavir (Ziagen), stavudine (Zerit), idoxuridine, trifluridine (Viroptic).
  • nucleotides examples include adenosine monophosphate, adenosine diphosphate, adenosine triphospate, guanosine mono-, di-, and triphosphate, uridine mono-, di-, and triphosphate, cytidine mono-, di-, and triphosphate, thymidine mono-, di-, and triphosphate, cyclic guanosine monophosphate, cyclic adenosine monophospate
  • nucleotide derivatives include tenofovir disoproxil fumarate (Viread), adefovir dipivoxil (Preveon), and adenosine triphosphate derivatives, such as adenosine 5'-(gamma-thiotriphosphate).
  • the resultant polypeptide is a derivative of the initial polypeptide.
  • a group of atoms is added to an initial polypeptide, for example, if a linker molecule having a thiol reactive group and a biotin covalently linked to each other is reacted with a cysteine of the initial polypeptide, so that the biotin becomes bonded through a disulfide to the cysteine, the resultant polypeptide is a derivative of the initial polypeptide.
  • a streptavidin or avidin derivative can have an amino acid residue in the amino acid sequence of streptavidin or avidin replaced with a different amino acid residue.
  • streptavidin derivative strut is to be understood as including struts formed of a streptavidin derivative, struts that include streptavidin
  • streptavidin may or may not be covalently bonded to other portions of the strut
  • struts that include a streptavidin derivative wherein the streptavidin derivative may or may not be covalently bonded to other portions of the strut
  • a polypeptide extension of a polypeptide subunit can be a polypeptide sequence that is linked to an amino or carboxy terminus of a polypeptide sequence comprising the polypeptide subunit.
  • the polypeptide extension may or may not be folded into the tertiary structure of the polypeptide subunit.
  • a binding function of a polypeptide sequence can be a subsequence of amino acids to which an atom, group of atoms, or molecule, such as a portion of a protein or a metallic surface, can form a covalent or non-covalent bond.
  • a polypeptide subsequence can be a continuous set of covalently bonded amino acid residues within a polypeptide sequence.
  • the polypeptide subsequence may comprise all, less than all, or only one of the amino acid residues in the polypeptide sequence.
  • a nanostructure strut can bind covalently or non-covalently to a specific binding site of a nanostructure node multimeric protein.
  • a protein such as a multimeric protein, can include a ligand binding pocket.
  • Such a pocket can be a depression in or inward folding of the surface of the protein.
  • the ligand binding pocket can include a specific binding site.
  • a nanostructure node multimeric protein can include a ligand binding pocket.
  • a nanostructure strut can bind to the ligand binding pocket.
  • the nanostructure strut can include a region of an immunoglobulin that binds to the ligand binding pocket of the nanostructure node multimeric protein.
  • the nanostructure strut can include biotin, iminobiotin, a nucleotide, an enzyme inhibitor, an enzyme activator, an enzyme substrate, an enzyme cofactor, a coenzyme, and/or derivatives that bind to the ligand binding pocket of the nanostructure node multimeric protein.
  • a bridge molecule can serve to attach two other molecules, such as proteins.
  • a bridge molecule can include a biotin group covalently bound to an adenosine triphosphate (ATP) group.
  • the biotin group can bind to a biotin binding site, such as present on streptavidin, and the adenosine triphosphate (ATP) group can bind to an ATP binding site, such as present on the MJ0577 protein.
  • a bindable polypeptide subunit for example, of a multimeric protein, can be capable of binding, directly or through an intermediary molecule, such as a bridge molecule, to another molecule, such as a protein.
  • a bindable subunit can include a specific binding site to which a nanostructure strut, e.g., a streptavidin-containing nanostructure strut, can bind.
  • a non-bindable polypeptide subunit for example, of a multimeric protein, can be incapable of binding to another molecule, such as a protein.
  • a non-bindable subunit may lack a specific binding site to which a nanostructure strut, e.g., a streptavidin- containing nanostructure strut, can bind.
  • Synthesizing a protein can refer to synthesizing a polypeptide sequence with chemical methods, and can refer to synthesizing a polypeptide sequence with molecular biological methods, such as, for example, inserting a gene into a host organism (for example, E. col ⁇ ) to induce the host organism to express the protein.
  • a host organism for example, E. col ⁇
  • EXP14Q3193C2 were cultured in 50 mL Terrific Broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 6OO of 5.53. 0.9 mL was used to inoculate a second culture of 50 mL Terrific Broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 C C to an OD 6 oo of 0.807, induced with 0.4 mM IPTG and supplemented with 0.5 mM ZnSO 4 , then grown for 20 hours at 25 0 C to an OD 60 O of 20.97. 2.0 g of cells were collected by low speed centrifugation.
  • EXP14Q3193C2 were cultured in 50 mL Luria-Bertani broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 6O o of 5.53. 0.9 mL was used to inoculate a second culture of 50 mL Luria-Bertani Broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol.
  • the culture was grown overnight at 37 0 C to an OD OOO of 0.753, induced with 0.4 mM IPTG and supplemented with 0.5 mM ZnSO 4 , then grown for 4 hours at 25 0 C to an OD 60 O of 3.23. 0.8 g of cells were collected by low speed centrifugation.
  • EXP14Q3193C2 were cultured in 50 mL Luria-Bertani broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD ⁇ oo of 5.53. 0.9 mL was used to inoculate a second culture of 50 mL Luria-Bertani broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol.
  • the culture was grown overnight at 37 0 C to an OD 600 of 0.753, induced with 0.4 mM IPTG and supplemented with 0.5 mM ZnSO 4 , then grown for 20 hours at 25 0 C to an OD 600 of 23.64. 2.4 g of cells were collected by low speed centrifugation.
  • the nodes formed with the expression vector EXP14Q3193C2 were based on gamma-carbonic anhydrase from Methanosarcina therm ophila.
  • the C3 symmetric, 3-subunit, synthesized protein was composed of three identical polypeptide chains.
  • the synthesized protein differs from the native protein. Residues Asp70 and Tyr200 were changed to Cys. Cysl48 was changed to Ala (the amino acid residue numbering follows that assigned to the native polypeptide).
  • a His tag that can be cleaved by the Factor Xa protease was added to the
  • the assembled 3 subunit protein formed of 3 polypeptide chains, includes a total of 6 surface cysteine residues available for functionalization (for example, with a biotin group) and complexation with 3 streptavidin tetramers.
  • the gene nucleotide sequence for the synthetic sequence EXP14Q3193C2 incorporated into the EXP14Q3193C2 expression vector was: gaaggagatatacatATGC AAGAGATTACCGTTGACGAATTTAGCAATATCCGTGAAAACC
  • the corresponding amino acid sequence of the one polypeptide chain of the synthetic protein produced by the EXP14Q3193C2 expression vector was: MQEITVDEFSNIRENPVTPWNPEPSAPVIDPTAYIDPEASVIGEVTIGANVMVSPMASIRS DEGMPIFVGCRSNVQDGVVLHALETINEEGEPIEDNIVEVDGKEYA VYIGNNVSLAHQS QVHGPAAVGDDTFIGMQAFVFKSKVGNNAVLEPRSAAIGVTIPDGRYIPAGMVVTSQA EADKLPEVTDDYAYSHTNEAVVCVNVHLAEGYKETIEGRHHHHHH [SEQ ID NO 25]
  • the culture was grown overnight at 37 0 C to an OD 6O o of 0.949, induced with 0.4 raM IPTG and supplemented with 0.5 raM ZnSO 4 , then grown for 4 hours at 25 0 C to an OD 6 oo of 2.78. 0.6 g of cells were collected by low speed centrifugation.
  • EXP14Q3193C3 were cultured in 50 mL Luria-Bertani broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 6OO of 6.83. 0.73 mL was used to inoculate a second culture of 50 mL Luria-Bertani broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol.
  • the culture was grown overnight at 37 0 C to an OD 60O of 0.949, induced with 0.4 mM IPTG and supplemented with 0.5 mM ZnSO 4 , then grown for 20 hours at 25 0 C to an OD 600 of 4.49. 0.8 g of cells were collected by low speed centrifugation.
  • EXP14Q3193C3 were cultured in 50 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 60O of 6.83. 0.73 mL was used to inoculate a second culture of 50 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 6 oo of 0.796, induced with 0.4 mM IPTG and supplemented with 0.5 mM ZnSO 4 , then grown for 4 hours at 25 0 C to an OD 600 of 3.94. 0.7 g of cells were collected by low speed centrifugation.
  • EXP14Q3193C3 were cultured in 50 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 60O of 6.83. 0.73 mL was used to inoculate a second culture of 50 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 600 of 0.89, induced with 0.4 mM IPTG and supplemented with 0.5 mM ZnSO 4 , then grown for 20 hours at 25 0 C to an OD 600 of 17.52. 1.9 g of cells were collected by low speed centrifugation.
  • EXP14Q3193C3 were cultured in 50 mL Luria-Bertani broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 600 of 5.63. 0.89 mL was used to inoculate a second culture of 50 mL Luria-Bertani broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol.
  • the culture was grown overnight at 37 0 C to an OD 6 oo of 0.905, induced with 0.4 mM IPTG and supplemented with 0.5 mM ZnSO 4 , then grown for 4 hours at 25 0 C to an OD 6 oo of 2.92. 0.6 g of cells were collected by low speed centrifugation.
  • EXP14Q3193C3 were cultured in 50 mL Luria-Bertani broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 6 Oo of 5.63. 0.89 mL was used to inoculate a second culture of 50 mL Luria-Bertani broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol.
  • the culture was grown overnight at 37 °C to an OD 60O of 0.905, induced with 0.4 mM IPTG and supplemented with 0.5 mM ZnSO 4 , then grown for 20 hours at 25 0 C to an OD 6 oo of 3.62. 0.8 g of cells were collected by low speed centrifugation.
  • EXP14Q3193C3 were cultured in 50 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 6 Oo of 5.63. 0.89 mL was used to inoculate a second culture of 50 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 6 oo of 0.796, induced with 0.4 mM IPTG and supplemented with 0.5 mM ZnSO 4 , then grown for 4 hours at 25 0 C to an OD 600 of 3.87. 1.3 g of cells were collected by low speed centrifugation.
  • EXP14Q3193C3 were cultured in 50 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 6O o of 5.63. 0.89 mL was used to inoculate a second culture of 50 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 6 oo of 0.796, induced with 0.4 mM IPTG and supplemented with 0.5 mM ZnSO 4 , then grown for 20 hours at 25 0 C to an OD 60 O of 18.22. 1.9 g of cells were collected by low speed centrifugation.
  • EXP14Q3193C3 were cultured in 375 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 6 O 0 of 4.276. The culture was used to inoculate a second culture of 16 L Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/niL chloramphenicol.
  • the culture was grown overnight at 37 0 C with 30% dissolved oxygen and 400-550 rpm to an OD 6 oo of 1.053, induced with 0.4 mM IPTG and supplemented with 0.5 mM ZnSO 4 , then grown for 19.75 hours at 25 0 C to an OD OOO of 7.34. 182.5 g of cells were collected by low speed centrifugation. [00433]
  • the 3-fold node protein was isolated from the collected E. coli cells with expression vector EXP14Q3193C3 as follows. 10 grams of E.
  • coli cells with EXP14Q3193C3 were suspended in 20 mL 50 mM KPO 4 buffer pH 6.8, 30 mg lysozyme, 1 mg DNase I, and one pellet EDTA-free protease inhibitors (Roche). The suspension was held at 4 0 C and stirred for 1 hour, then sonicated in 3 sets of 30 1 -second pulses. The suspension was centrifuged at 12500 x g for 20 min. The soluble portion was subjected to column chromatography on Q- Sepharose equilibrated with 50 mM KPO 4 buffer pH 6.8, 0.001 mM ZnSO 4 .
  • Node protein was eluted by a linear gradient between 50 mM KPO 4 buffer pH 6.8, 0.001 mM ZnSO 4 and 50 mM KPO 4 buffer pH 6.8, 0.001 mM ZnSO 4 , 1 M NaCl.
  • Node protein fractions were identified by PAGE SDS analyses, then pooled and loaded onto a Phenyl-Sepharose chromatography column equilibrated with 50 mM KPO 4 buffer pH 6.8, 0.001 mM ZnSO 4 , 1 M NaCl.
  • Node protein was eluted from the column by a linear gradient between 50 mM KPO 4 buffer pH 6.8, 0.001 mM ZnSO 4 , 1 M NaCl and 50 mM KPO 4 buffer pH 6.8, 0.001 mM ZnSO 4 .
  • Node protein fractions identified by PAGE SDS analyses were combined and dialyzed against 2 changes of 25 mM NaPO 4 buffer pH 8.0 with each change corresponding to at least 1Ox node protein volume. Dialyzed node protein was mixed with 3 mL Ni agarose resin equilibrated with 25 mM NaPO 4 buffer pH 8.0, then reacted for 18 hours with rocking at 4 °C. The resin was washed with twice with 15 mL 25 mM NaPO 4 buffer pH 8.0, then the node protein was eluted with 25 mM NaPO 4 buffer pH 8.0, 250 mM imidazole.
  • a second, alternative isolation procedure was carried out in a similar manner, except that the Ni agarose resin was used before the Q-sepharose and phenyl-Sepharose chromatographic steps.
  • a third, alternative isolation procedure was carried out in a similar manner, except that the E. coli cells were disrupted by addition of nonionic detergent (B-PER ThermoScientific) instead of by addition of lysozyme followed by stirring and sonication.
  • B-PER ThermoScientific nonionic detergent
  • the nodes formed with the expression vector EXP14Q3193C3 were based on gamma-carbonic anhydrase from Methanosarcina thermophila.
  • the C3 symmetric, 3-subunit, synthesized protein was composed of a single polypeptide chain. That is, whereas the native protein has a quaternary structure formed from 3 polypeptide chains, in the protein produced from the expression vector EXP14Q3193C3, the 3 polypeptide chains are fused together into a single polypeptide chain that folds into a structure having 3 subunits.
  • the 3 polypeptide chains were fused together with two identical linkers, each having the sequence GGSGGG (Gly-Gly-
  • Ser-Gly-Gly-Gly The linker extended from the natural C-terminus (residue 212) of a subsequence corresponding to a polypeptide chain in the native protein and forming a subunit to residue 6 of the subsequence in a polypeptide chain forming the adjacent subunit (the amino acid residue numbering follows that assigned to the native polypeptide).
  • Residues Asp 70 and Tyr200 were changed to Cys
  • Cysl48 was changed to Ala.
  • the assembled 3 subunit protein formed of a single polypeptide chain, includes a total of 6 surface cysteine residues available for functionalization (for example, with a biotin group) and complexation with 3 streptavidin tetramers.
  • EXP14Q3193C3 expression vector was: ggggacaagtttgtacaaaaagcaggcaccgaagg «gfltotoc ⁇ tATGGATGAATTTAGCAATATCCGCGA
  • EXP14Q3193C4 were cultured in 50 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 6 Oo of 6.04. 0.83 mL was used to inoculate a second culture of 50 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C to an OD 6 Oo of 0.963, induced with 0.4 mM IPTG and supplemented with 0.5 mM ZnSO 4 , then grown for 20 hours at 25 0 C to an OD 60 O of 22.8. 2.1 g of cells were collected by low speed centrifugation.
  • the nodes formed with the expression vector EXP14Q3193C4 were based on gamma-carbonic anhydrase from Methanosarcina thermophila.
  • the C3 symmetric, 3-subunit, synthesized protein was composed of a single polypeptide chain. That is, whereas the native protein has a quaternary structure formed from 3 polypeptide chains, in the protein produced from the expression vector EXP14Q3193C4, the 3 polypeptide chains are fused together into a single polypeptide chain that folds into a structure having 3 subunits.
  • the 3 polypeptide chains were fused together with two identical linkers, each having the sequence GGSGGG (Gly-Gly- Ser-Gly-Gly-Gly).
  • the linker extended from the natural C-terminus (residue 212) of a subsequence corresponding to a polypeptide chain in the native protein and forming a subunit to residue 6 of the subsequence in a polypeptide chain forming the adjacent subunit (the amino acid residue numbering follows that assigned to the native polypeptide). Following the N-terminus, within the first two polypeptide subsequences corresponding to a polypeptide chain in the native protein, the following substitutions were made: Residues Asp70 and Tyr200 were changed to
  • Cys; and Cysl48 was changed to Ala.
  • Cys 148 was changed to Ala, but residues Asp70 and Tyr200 were left unchanged.
  • a His tag that can be cleaved by the Factor Xa protease was added to the third polypeptide subsequence, that is, the subsequence before the C-terminus.
  • two of the subunits of the assembled 3 subunit protein, formed of a single polypeptide chain included a total of 4 surface cysteine residues available for f ⁇ inctionalization (for example, with a biotin group) and complexation with 2 streptavidin tetramers. That is, two of the subunits can complex with a streptavidin each, but the third subunit cannot complex with a streptavidin.
  • EXP14Q3193C4 expression vector was: cgatgcgtccggcgtagaggatcgagatctcgatcccgcgaaattaatacgactcactatagggagaccacaacggtttccctctagatca caagtttgtacaaaaagcaggcaccgaagg ⁇ g ⁇ totocotATGGATGAATTTAGC AATATTCGCGAAAAC
  • EXP14Q3193C3 (Example IB), and EXP14Q3193C4 (Example 1C), could be (and were) expressed in E. coli.
  • the proteins were stable to proteolysis by E. coli proteases as evidenced by the presence of bands of the appropriate molecular weight that appeared in Western blots using anti-His tag antibodies. This strongly suggested that the proteins were properly folded. It was found that the protein expression by vector EXP14Q3193C3 (Example IB) was higher than that for EXP14Q3193C4 (Example 1C).
  • the isolated band resulting from the EXP14Q3193C3 variant was sequenced by mass spectrometry and confirmed the identity of the protein.
  • EXP14Q3164 were cultured in 9 mL Terrific Broth supplemented with 0.1 mg/mL ampicillin and 0.2 mg/mL riboflavin. The culture was grown overnight at 37 0 C with rotation at 225 rpm. The culture was used to inoculate a second culture of 1.0 L Terrific Broth supplemented with 0.1 mg/mL ampicillin and 0.2 mg/mL riboflavin. The culture was grown to an OD OOO of 1.39 when 0.4 mM IPTG was added. The culture continued to grow for 20.25 additional hours at 25.0 0 C to an OD 60 O of 0.70. 4.6 g of cells were collected by low speed centrifugation. [00447] In a third batch, E. coli cells BL21 StarTM (DE3) pLysS with expression vector
  • EXP14Q3164 were cultured in 8.6 mL Terrific Broth supplemented with 0.1 mg/mL ampicillin, 0.034 mg/mL chloramphenicol and 0.2 mg/mL riboflavin. The culture was grown overnight at 37 0 C with rotation at 225 rpm. The culture was used to inoculate a second culture of 1 L Terrific Broth supplemented with 0.1 mg/mL ampicillin, 0.034 mg/mL chloramphenicol and 0.2 mg/mL riboflavin. The culture was grown to an OD ⁇ oo of 0.868 when 0.4 mM IPTG was added. The culture continued to grow for 3.0 additional hours at 25.8 0 C to an OD 600 of 4.23. 8.6 g of cells were collected by low speed centrifugation.
  • EXP14Q3164 were cultured in 50 mL Luria-Bertani broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C with rotation at 225 rpm. The culture with an OD 600 of 0.761 was induced with 0.4 mM IPTG and supplemented with 0.2 mg/mL riboflavin, then grown for 4 hours at 37.0 0 C to an OD 6 oo of 4.46. 0.5 g of cells were collected by low speed centrifugation.
  • E. coli cells BL21 StarTM (DE3) with expression vector
  • EXP14Q3164 were cultured in 50 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C with rotation at 225 rpm. The culture with an OD 600 of 0.797 was induced with 0.4 mM IPTG and supplemented with 0.2 mg/mL riboflavin, then grown for 4 hours at 37.0 0 C to an OD 6 Oo of 14.06. 1.1 g of cells were collected by low speed centrifugation.
  • E. coli cells BL21 StarTM (DE3) with expression vector
  • EXP14Q3164 were cultured in 50 mL Luria-Bertani broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C with rotation at 225 rpm. The culture with an OD 60O of 0.774 was induced with 0.4 mM IPTG and supplemented with 0.2 mg/mL riboflavin, then grown for 4 hours at 37.0 0 C to an OD 6 oo of 8.46. 0.7 g of cells were collected by low speed centrifugation.
  • EXP14Q3164 were cultured in 50 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C with rotation at 225 rpm. The culture with an OD 600 of 0.797 was induced with 0.4 mM IPTG and supplemented with 0.2 mg/mL riboflavin, then grown for 4 hours at 37.0 °C to an OD 6 oo of 14.06. 1.1 g of cells were collected by low speed centrifugation.
  • E. coli cells BL21 StarTM (DE3) with expression vector
  • EXP14Q3164 were cultured in 50 mL Luria-Bertani broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C with rotation at 225 rpm. The culture with an OD 600 of 0.825 was induced with 0.4 mM IPTG and supplemented with 0.2 mg/mL riboflavin, then grown for 4 hours at 25.0 0 C to an ODO 00 of 4.17. 0.6 g of cells were collected by low speed centrifugation.
  • EXP14Q3164 were cultured in 50 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C with rotation at 225 rpm. The culture with an OD O00 of 0.75 was induced with 0.4 mM IPTG and supplemented with 0.2 mg/mL riboflavin, then grown for 4 hours at 25.0 °C to an OD 60O of 5.36. 0.9 g of cells were collected by low speed centrifugation. [00454] In a tenth batch, E. coli cells BL21 StarTM (DE3) pLysS with expression vector
  • EXP14Q3164 were cultured in 50 rnL Luria-Bertani broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C with rotation at 225 rpm. The culture with an OD 6 oo of 0.694 was induced with 0.4 niM IPTG and supplemented with 0.2 mg/mL riboflavin, then grown for 4 hours at 25.0 °C to an OD 6 oo of 2.66. 0.6 g of cells were collected by low speed centrifugation.
  • E. coli cells BL21 StarTM (DE3) pLysS with expression vector EXP14Q3164 were cultured in 50 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C with rotation at 225 rpm. The culture with an OD 60O of 0.795 was induced with 0.4 mM IPTG and supplemented with 0.2 mg/mL riboflavin, then grown for 4 hours at 25.0 0 C to an OD 6O o of 4.81. 0.8 g of cells were collected by low speed centrifugation.
  • EXP14Q3164 were cultured in 345 mL Terrific broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown overnight at 37 0 C with rotation at 400-800 rpm. The culture was used to inoculate a second culture of 16 L Terrific Broth supplemented with 0.1 mg/mL ampicillin and 0.034 mg/mL chloramphenicol. The culture was grown to an OD 60 O of 0.885 when 0.4 mM IPTG and 200 microg/mL riboflavin were added. The culture continued to grow for 4.0 additional hours at 37 0 C to an OD 6 oo of 12.154. 323.3 g of cells were collected by low speed centrifugation.
  • the 4-fold node protein was isolated from the collected E. coli cells with expression vector EXP14Q3164 through a first isolation procedure as follows. 4.5 grams of E. coli cells BL21 StarTM (DE3) pLysS with expression vector EXP14Q3164 were suspended in 40 mL nonionic detergent (B-PER ThermoScientific) and allowed to react for 15 min. The cell suspension was clarified by centrifugation at 12500 x g for 15 min. The supernatant was heated to 60 0 C and held at that temperature for 15 min.
  • B-PER ThermoScientific nonionic detergent
  • Insoluble proteins were removed by centrifugation at 12500 x g for 15 min and the clear yellow supernatant was dialyzed against two changes of a solution of 50 mM NaPO 4 buffer at pH 8.0 and 0.5 M NaCl, where each change was at least 1OX the supernatant volume. After dialysis, the supernatant was incubated with 1 mL Ni agarose resin equilibrated with a solution of 50 mM NaPO 4 buffer at pH 8.0 and 0.5 M NaCl while held at 4 0 C and gently rocked for 18 hr.
  • a second, alternative isolation procedure was carried out in a similar manner as the first isolation procedure, except that E. coli cells BL21 StarTM (DE3) pLysS with expression vector EXP14Q3164 were suspended in 40 mL of solution with 50 mM NaPO 4 buffer at pH 8.0, 0.5 M NaCl, 40 mg lysozyme, 1 mg DNase I, and protease inhibitors (one pellet EDTA-free protease inhibitors (Roche) or 0.1 mL HALTTM protease cocktail (Pierce)), incubated for 1 hr with stirring at 4 ° C, and then sonicated for three increments of 30 pulses of 1 -second duration.
  • E. coli cells BL21 StarTM (DE3) pLysS with expression vector EXP14Q3164 were suspended in 40 mL of solution with 50 mM NaPO 4 buffer at pH 8.0, 0.5 M NaCl, 40 mg lysozyme, 1 mg DNase I,
  • a third, alternative isolation procedure was carried out in a similar manner as the first isolation procedure, except that the 4-fold node protein was further purified by dialysis against PBS pH 7.4, and then chromatographed on a size exclusion column of Superose-12 equilibrated with PBS pH 7.4 operated at a flow rate of either 0.1 mL/min or 0.4 mL/min.
  • a fourth, alternative isolation procedure was carried out in a similar manner as the second, alternative isolation procedure, except that the 4-fold node protein was further purified by dialysis against PBS pH 7.4, and then chromatographed on a size exclusion column of Superose-12 equilibrated with PBS pH 7.4 operated at a flow rate of either 0.1 mL/min or 0.4 mL/min.
  • the gene nucleotide sequence for the sequence EXP14Q3164 incorporated into the EXP14Q3164 expression vector was as follows. For the synthetic gene sequence shown, the open reading frame is in upper case with the initiating Methionine and Stop codons in bold.
  • EXP14Q3164 expression vector was as follows. The amino acid sequence is provided using the Standard one letter representation for each amino acid.
  • EXP14Q3164 expression vector was confirmed by mass spectrometry.
  • Biotin-containing reagents were covalently linked to cysteine residues on the 4- fold node using the following procedure.
  • the node was equilibrated in neutral or acidic buffers such as phosphate buffer saline (PBS) pH 7.4 or 20 mM sodium phosphate buffer pH 6.8 for reaction with biotinylation reagents N-d-biotinamido-N'-(3-maleimidopropionamido)-4,7,10- trioxatridecane- 1,13 -diamine (MAL-dPEGTM3-biotin, Quanta BioDesign, Powell OH) and N-d- biotinamido-N'-(3-maleimidopropionamido)-3,6,9,12,15,18,21,24,27,30,33- undecaoxapentatriacontane- 1,35 -diamine (MAL-dPEGTMl l- biotin, Quanta Bio
  • Node was then concentrated to a volume of about 0.5 mL and a concentration of least 1 mg/mL using centrifugal protein concentrator (PierceNet) and protein concentration determined by using an A 4 60 extinction coefficient of 11,300 M "1 cm "1 .
  • PierceNet centrifugal protein concentrator
  • a 4 60 extinction coefficient of 11,300 M "1 cm "1 For example, 0.2 mL of node solution at a concentration of 21 mg/mL and 0.13 mL of a node solution at 28 mg/mL were used.
  • Solutions of biotin-containing reagents were prepared by adding solid reagent to buffer or solvent.
  • the buffer was 20 niM sodium phosphate buffer pH 6.8 or PBS pH 7.4 and for the sulfur-reactive biotinylation reagent biotin-HPDP, the dissolving solution was dimethyl sulfoxide (DMSO).
  • DMSO dimethyl sulfoxide
  • 2.7 mg MAL-dPEGTM3 -biotin was dissolved in 0.05 niL PBS pH 7.4, 3.3 mg MAL-dPEGTMl l- biotin was dissolved in 0.05 mL PBS pH 7.4, and 2.8 mg biotin-HPDP was dissolved in 0.5 mL DMSO.
  • Biotinylation reagents were added to the node solutions very soon after dissolution of the solid reagent. The final molar concentration of biotinylation reagent in the reaction was in excess of the node concentration. In separate reactions, the molar ratios of MAL-dPEGTM3-biotin:node were 2.5:1 and 3.6:1, and the molar ratios of MAL-dPEGTMl l- biotin:node were 2.0: 1 and 2.8:1. The reaction was allowed to progress for at least 2 hours. Unreacted reagent was then removed by centrifugation through a size exclusion resin (Zeba Desalting Column, PierceNet).
  • a size exclusion resin Zeba Desalting Column, PierceNet
  • NODE:SAV complexes were formed in solution by mixing the streptavidin and derivatized (biotinylated) 4-fold node (NODE) solutions, generally by the addition of more concentrated streptavidin to the biotinylated NODE.
  • Streptavidin solutions were prepared by dissolving lyophilized Streptomyces avidinii streptavidin (ProZyme, San Leandro, CA) in 50 mM sodium phosphate buffer pH 6.8, 0.25 M NaCl to achieve a final concentration of 1 or lO mg/mL.
  • Streptavidin solutions were also prepared by dissolving lyophilized Streptomyces avidinii streptavidin (ProZyme, San Leandro, CA) in PBS pH 7.4 at a concentration of 15 mg/niL. 0.50 mL of streptavidin solution was chromatographed on a Superosel2 column equilibrated with PBS pH 7.4 and operated at flow rates from 0.2 to 0.4 mL/min. Eluted streptavidin fractions were combined and concentrated using centrifugal concentrators (iCON concentrators, Pierce). Streptavidin concentration was determined using an A 28O extinction coefficient of 41326 M '1 cm "1 .
  • NODE:SAV complexes were formed in solution by adding 0.002 mL aliquots of streptavidin solution at a concentration of 10 mg/mL in 50 mM sodium phosphate buffer pH 6.8, 0.25 M NaCl to a reaction volume of 100 ⁇ L derivatized node at a concentration of 30 mg/mL in 20 mM sodium phosphate buffer pH 6.8 until an equimolar stoichiometry of NODE to SAV was achieved. The reaction equilibrated at room temperature for three days.
  • NODE:SAV complexes were analyzed using 4-12% TRIS- Glycine PAGE gels under denaturing conditions where solutions to be analyzed were heated in the presence of dithiothreitol (DTT) and sodium dodecyl sulfate (SDS).
  • DTT dithiothreitol
  • SDS sodium dodecyl sulfate
  • NODE:SAV complexes were also formed by immobilizing the derivatized node on a surface, then reacting SAV with the immobilized NODE. Following the manufacturer's instructions, 0.30 rnL of Ni-NTA agarose resin (Invitrogen) was equilibrated with PBS pH 7.4. After 0.9 mg node derivatized with MAL-dPEGTM3-biotin was added to the resin and allowed to equilibrate for 2 hrs, 0.7 mg SAV in PBS pH 7.4 was added. The resin with derivatized node and SAV was equilibrated by mixing for 12 hours on an orbital rotator.
  • Ni-NTA agarose resin Invitrogen
  • NODE:SAV complex was eluted from the resin by washing with 50 mM phosphate buffer pH 8, 0.5 M NaCl, 0.25 M imidazole.
  • NODE: SAV complexes were analyzed using 4-12% TRIS-Glycine PAGE gels under denaturing conditions where solutions to be analyzed were heated in the presence of DTT and SDS.
  • NODE:SAV complexes were also formed by immobilizing the derivatized node on a resin, adding streptavidin, eluting the NODE:SAV complex, then adding additional streptavidin to the eluted NODE:SAV complex.
  • 0.30 mL of Ni-NTA agarose resin (Invitrogen) was equilibrated with PBS pH 7.4.
  • 0.25 mg SAV in PBS pH 7.4 was added to the mixture.
  • the resin with derivatized node and SAV was equilibrated by mixing for 12 hours on an orbital rotator.
  • the NODE: SAV complex was eluted from the resin by washing with 50 mM phosphate buffer pH 8, 0.5 M NaCl, 0.25 M imidazole.
  • To the NODE:SAV complex in solution 0.3 mg SAV was added and the mixture allowed to equilibrate for 12 hrs.
  • NODE:SAV complexes were analyzed using 4-12% TRIS-Glycine PAGE gels under denaturing conditions where solutions to be analyzed were heated in the presence of DTT and SDS.
  • 0.30 mL of Ni-NTA agarose resin Invitrogen was equilibrated with PBS pH 7.4.
  • Electrophoretic analysis of NODE SAV complexes was carried out.
  • the panels presented in Fig. 48 show PAGE analyses using 8-16% PreciseTM gels (Pierce) and BupHTM
  • Tris-HEPES-SDS running buffer (Pierce). Lanes are numbered on the top. Novex Sharp
  • the streptavidin tetramer subunits which were obtained by fermentation from Streptomyces avidinii, had a range of molecular weights.
  • the average molecular weight of the streptavidin tetramer was understood to be approximately 52 kDa.
  • the 4-fold node is IPP isomerase from Thermus thermophilus.
  • the native molecular weight is 35.9 kDa per chain, that is, 143.6 kDa per tetramer. With the added tags, the molecular weight of the construct used is higher.
  • lanes 1, 2, 3, and 4 and lanes 7, 9, 11, 13, and 15 complexes formed by first immobilizing the node, reacting it with streptavidin, eluting the complex from a solid support, and then reacting that complex with excess streptavidin in solution were analyzed.
  • the bands shown in lane 10 were understood to correspond to protein entities as follows.
  • the lower molecular weight band of 15 kDa or less was understood to correspond to streptavidin monomer arising from the unliganded tetramer that denatures under the conditions (compare lane 28, which analyzed the unliganded SAV tetramer) and to smaller fragments from IPP isomerase (compare lane 27, which analyzed IPP isomerase prior to biotinylation).
  • the two bands about 20 kDa were understood to correspond to degraded IPP isomerase. These 2OkDa MW bands were present in Lane 27, which analyzed IPP isomerase prior to biotinylation.
  • IPP isomerase appears as a doublet of molecular weight just less than 40 kDa. Both subbands of the doublet were sequenced by mass spectrometry (MS). Both subbands gave the same sequence; the coverage of both samples was about 68%, and both sequences confirmed that the bands were IPP isomerase. Because IPP isomerase was isolated by affinity chromatography using the N-terminal His tag, it was understood that the cleavage site was near the C-terminus.
  • Lane 10 The lane 10 band at about 40 kDa was understood to correspond to the IPP isomerase monomer. Lane 27 represented the control analysis, which showed IPP isomerase prior to biotinylation.
  • the lane 10 band at about 50 kDa was understood to correspond to the streptavidin:biotin tetramer.
  • Lanes 6 and 29 represent the control analyses.
  • the liganded tetramer was stable under these conditions (the unliganded streptavidin was unstable, compare lanes 28 and 29 which differed only in that biotin was added for 15 minutes to the sample analyzed in lane 29).
  • the lane 10 band at about 70 kDa was understood to correspond to the streptavidin tetramer (52 kDa) in complex with one chain (36 kDa) of the IPP isomerase 4-fold node. This band was entirely absent from the control analyses of streptavidin (lane 28), streptavidin: biotin complex (lanes 6 and 29), and IPP isomerase (lane 27).
  • the conclusion that the 70 kDa band corresponded to the NODE: SAV complex was reached upon consideration of the control analyses of lanes 6, 27, 28, and 29 and of analyses of a number of NODE: SAV complexes prepared by different methods and with different biotinylation reagents.
  • the lane 10 band at about 110 kDa may correspond to the streptavidin tetramer bound to two node chains.
  • the streptavidin would be bound to two node chains by acting as a link between the two node chains.
  • Fitzpatrick PA Steinmetz ACU, Ringe D, Klibanov AM "Enzyme Crystal Structure in a Neat Organic Solvent” Proc Nat Acad Sci USA (1993)90:8653.
  • Liu GY, Amro NA Partitioning protein molecules on surfaces: A nanoengineering approach to supramolecular chemistry" Proc Nat Acad Sci (2002)99:5165-5170.

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Abstract

L'invention porte sur des protéines synthétisées par génie génétique qui sont utilisées dans l'assemblage d'ensembles nanostructures bidimensionnels et tridimensionnels, sur la base d'une conception systématique et sur la production de structures de nœud de protéine qui peuvent être interconnectées, par exemple, par de la streptavidine ou des entretoises incorporant de la streptavidine pour produire des structures avec des dimensions et une géométrie définies. Des ensembles nanostructures ayant une utilité en tant que dispositifs fonctionnels ou en tant que réserves pour la formation de motifs de substrats ont des architectures comprenant des polygones, des polyèdres, des réseaux bidimensionnels et des réseaux tridimensionnels.
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US8993714B2 (en) 2007-10-26 2015-03-31 Imiplex Llc Streptavidin macromolecular adaptor and complexes thereof
US9102526B2 (en) 2008-08-12 2015-08-11 Imiplex Llc Node polypeptides for nanostructure assembly
EP2385982A2 (fr) * 2009-01-09 2011-11-16 Codexis, Inc. Polypeptides d'anhydrase carbonique et leurs utilisations
EP2385982A4 (fr) * 2009-01-09 2013-05-29 Codexis Inc Polypeptides d'anhydrase carbonique et leurs utilisations
US9285363B2 (en) 2009-05-11 2016-03-15 Imiplex Llc Method of protein nanostructure fabrication
US20130230562A1 (en) * 2010-06-23 2013-09-05 Hyung-Jun Ahn Fusion protein comprising small heat shock protein, cage protein formed thereby, and novel use thereof
US8916685B2 (en) * 2010-06-23 2014-12-23 Korea Institute Of Science And Technology Fusion protein comprising small heat shock protein, cage protein formed thereby, and novel use thereof
WO2014124301A1 (fr) * 2013-02-07 2014-08-14 University Of Washington Through Its Center For Commercialization Nanostructures protéiques à auto-assemblage
US10248758B2 (en) 2013-02-07 2019-04-02 University Of Washington Through Its Center For Commercialization Self-assembling protein nanostructures
US10253302B2 (en) 2013-08-21 2019-04-09 Crysalin Ltd. Method for producing ordered protein lattice
US9630994B2 (en) 2014-11-03 2017-04-25 University Of Washington Polypeptides for use in self-assembling protein nanostructures
US11485759B2 (en) 2014-11-03 2022-11-01 University Of Washington Polypeptides for use in self-assembling protein nanostructures
US10351603B2 (en) 2014-11-03 2019-07-16 University Of Washington Polypeptides for use in self-assembling protein nanostructures
WO2018047856A1 (fr) * 2016-09-06 2018-03-15 学校法人慶應義塾 Protéine fusionnée, corps structural, agent de piégeage, méthode de piégeage, adn et vecteur
JPWO2018047856A1 (ja) * 2016-09-06 2019-07-11 学校法人慶應義塾 融合タンパク質、構造体、捕集剤、捕集する方法、ｄｎａ、及びベクター
US11732011B2 (en) 2017-04-04 2023-08-22 University Of Washington Self-assembling protein nanostructures displaying paramyxovirus and/or pneumovirus F proteins and their use
US11192926B2 (en) 2017-04-04 2021-12-07 University Of Washington Self-assembling protein nanostructures displaying paramyxovirus and/or pneumovirus F proteins and their use
CN108444957A (zh) * 2018-02-05 2018-08-24 南京医科大学 一种基于dna-银纳米簇与聚吡咯纳米粒的转录因子检测方法
CN108444957B (zh) * 2018-02-05 2020-07-17 南京医科大学 一种基于dna-银纳米簇与聚吡咯纳米粒的转录因子检测方法
US11771755B2 (en) 2018-02-28 2023-10-03 University Of Washington Self-asssembling nanostructure vaccines

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