ROULD, Mark A. (1970 Timber Lakes Drive, Yardley, Pennsylvania, 19067, US)
WEBER, Patricia C. (1970 Timber Lakes Drive, Yardley, Pennsylvania, 19067, US)
ROULD, Mark A. (1970 Timber Lakes Drive, Yardley, Pennsylvania, 19067, US)
WEBER, Patricia C. (1970 Timber Lakes Drive, Yardley, Pennsylvania, 19067, US)
| WE CLAIM: Claim 1. A nanostructure node, comprising a multimeric protein having at least one polypeptide chain and a known three- dimensional structure with Cn, Dn, or higher symmetry, wherein the multimeric protein has stable tertiary structure at a temperature of 70 0C or greater and a modified amino acid sequence comprising a specific binding site positioned with predefined stoichiometry and orientation for attachment of a nanostructure strut, wherein the specific binding site comprises two reactive amino acid residues, each covalently attached to a moiety independently selected from the group consisting of a biotin group, a biotin derivative group, iminobiotin, an adenosine triphosphate (ATP) group, an adenosine triphosphate (ATP) derivative group, a nucleobase group, a nucleobase derivative group, a nucleoside group, a nucleoside derivative group, a nucleotide group, and a nucleotide derivative group. Claim 2. The nanostructure node of Claim 1, wherein the nanostructure strut comprises two moiety binding sites separated from each other by a first distance and wherein the two reactive amino acid residues are separated from each other by a second distance approximately equal to the first distance. Claim 3. The nanostructure node of Claim 1, wherein the moiety covalently attached to the reactive amino acid residue is selected from the group consisting of a biotin group and a biotin derivative group and binds to streptavidin or a streptavidin derivative strut. Claim 4. The nanostructure node of Claim 3, wherein the moieties are separated from each other by a distance of from about 15 Angstroms to about 25 Angstroms. Claim 5. The nanostructure node of Claim 3, wherein the multimeric protein is selected from the group consisting of proteins having an amino acid sequence with 80 percent or greater sequence identity to that of a pdb code protein lfsz, Ige8, lisq, Ij2v, lkht, Iki9, lkwg, Ills, ImW, In2m, Inl3, Io5j, lqrf, lthj, lufy, luku, Iv4n, Iv8d, lvke, lwvq, lwzn, 1x25, 2b33, 2cz4, 2dcl, 2dhr, 2dt4, 2hik, 2nwl, lbkb, Inc7, lvrd, 2cuO, 2fk5, 2flf, ItOt, lvdh, Iw8s, 2b99, 2bbh, 2bbj, 2hnl, 2hn2, 2iub, Ii8f, lljo, 2alb, 2al8, 2di4, 2dr3, 2ekd, 2ewh, Ih64, Ii4k, H81, ljbm, ljri, Im5q, lmgq, IaOe, lbxb, Ido6, ldof, lgtd, lhyg, lilg, Iik6, lixr, Ij Iy, Ij2w, Ijg8, ljvb, lknv, Ilk5, llvw, Im8k, lmal, Into, lnvg, Io2a, Io54, Ir37, Iris, lrtw, Iu9y, ludd, luir, lusy, luxt, Iv6t, Iv8o, Iv8p, Ivc2, lvco, lvdk, lvjp, Ivk8, Ivl2, lvlv, Ivr6, Iw3i, Iwb7, Iwb8, lwlu, Iws9, lwyt, IxOl, lxle, 1x10, lxtt, Iy56, Iz54, 2afb, 2b5d, 2bri, 2cbl, 2cd9, 2cdc, 2cx9, 2czc, 2dly, 2d8a, 2d29, 2df5, 2dfa, 2drh, 2dsl, 2ela, 2e9f, 2eba, 2ebj, 2eo5, 2ep5, 2glO, 2gm7, 2h6e, 2hae, 2hmf, 2iss, 2j4k, 21db, 2p3n, 2ph3, 2yym, 3pfk, laup, lbgv, lbvu, If9a, lfxk, lgtm, lhyb, ljeO, ljku, lodi, lodk, Ipg5, Iqw9, It57, luan, lude, luiy, lvla, lvls, Iv91, Ivl9, lwkl, Iwz8, lwzn, IxOu, 2a8y, 2afb, 2anu, 2bja, 2bjk, 2cqz, 2cz8, 2dcn, 2ddz, 2dev, 2dqb, 2dxf, 2dya, 2eez, 2g3m, 2il4, 2ide, 2j4j, 2j9d, 2prd, 2q8n, ljpu, Ijq5, Im4y, Io4v, lsaz, lumg, lvcf, Ix9j, 2ax3, 2cwx, 2d69, 2h2i, 2iel, lgeh, Iw8s, Iwm9, lwuq, lwur, lwxO, lznn, 2djw, Im4y, Im5q, Ith7, lpvv, lvlg, lxfo, IyOr, IyOy, lyoy, 2clb, 2glf, 2vl8, 2vl9, lshs, lvlg, or Ib5s. Claim 6. The nanostructure node of Claim 1, wherein at least one polypeptide chain of the multimeric protein is bonded to a bifunctional reagent with additional binding or a functionality selected from the group consisting of catalytic, chemomechanical, electromechanical, optomechanical, and optoelectronic functionality. Claim 7. The nanostructure node of Claim 1, wherein at least one polypeptide chain of the multimeric protein comprises an amino acid sequence coding for protein binding or a functionality selected from the group consisting of catalytic, chemomechanical, electromechanical, optomechanical, and optoelectronic functionality. Claim 8. The nanostructure node of Claim 3, wherein the multimeric protein comprises a single polypeptide chain. Claim 9. The nanostructure node of Claim 3, wherein the multimeric protein has Cn symmetry and a Cn symmetry axis, wherein the specific binding site comprises two specific amino acid reactive residues, wherein the streptavidin or streptavidin derivative strut has 3 dyad axes and two pair of biotin binding sites, the first pair related to the second pair by a dyad axis, and the first binding site of a pair related to the second binding site of the pair by a dyad axis, wherein the two specific amino acid reactive residues are complementary to the geometry of a pair of biotin binding sites on the streptavidin or streptavidin derivative strut, so that when the two specific amino acid reactive residues are aligned with a pair of biotin binding sites, the Cn symmetry axis of the nanostructure node multimeric protein is substantially parallel to a dyad axis of the streptavidin or streptavidin derivative strut. Claim 10. The nanostructure node of Claim 9, wherein the multimeric protein has an amino acid sequence given in Table 2B or has an amino acid sequence with greater than 80 percent sequence identity with a sequence provided in Table 2B. Claim 11. The nanostructure node of Claim 9, having a C3 -symmetric planar structure, wherein the multimeric protein has an amino acid sequence with greater that 80 percent identity to the amino acid sequence of the pdb coderlthj or of the pdb code:lj5s protein trimer. Claim 12. The nanostructure node of Claim 9, having a C4-symmetric planar structure, wherein the multimeric protein has an amino acid sequence with greater than 80 percent identity to the amino acid sequence of the pdb code:lvcg or of the pdb code:2cuO protein tetramer. Claim 13. The nanostructure node of Claim 9, selected from the group consisting of a C5-symmetric planar node wherein the protein has an amino acid sequence with greater than 80 percent identity to that of the pdb code:lvdh protein pentamer, a Co-symmetric planar node wherein the protein has an amino acid sequence with greater than 80 percent identity to that of the pdb code:2ekd protein hexamer, and a Cl- symmetric planar node wherein the protein has greater than 80 percent identity to that of the pdb code:li81 protein heptamer. Claim 14. The nanostructure node of Claim 3, selected from the group consisting of a C3-symmetric apex node, wherein the protein has an amino acid sequence with at least 80 percent identity to the amino acid sequence of the pdb code:lv4n protein trimer, and a C5-symmetric apex node wherein the protein has an amino acid sequence with at least 80 percent identity to the amino acid sequence of the pdb coderlvdh protein pentamer and wherein the streptavidin or streptavidin derivative strut binds to the multimeric protein with a geometry selected from the group consisting of dodecahedral apex geometry, "buckyball" geometry, truncated icosahedral apex geometry, and icosahedral apex geometry. Claim 15. The nanostructure node of Claim 3, having D2 symmetry, wherein the multimeric protein has an amino acid sequence with at least 80 percent identity to an amino acid sequence selected from the group consisting of the pdb code:lmal protein tetramer, the pdb code: Into protein tetramer, the pdb code:lrtw protein tetramer, and the pdb code:lstp protein tetramer and wherein the streptavidin or streptavidin derivative strut binds to the multimeric protein with a geometry selected from the group consisting of linear, 2-dimensional rectangular, and 3- dimensional orthorhombic lattice geometry. Claim 16. The nanostructure node of Claim 3, having D3 symmetry, wherein the multimeric protein has an amino acid sequence with at least 80 percent identity to the amino acid sequence selected from the group consisting of the pdb code:lb4b protein hexamer, the pdb code:lhyb protein hexamer, and the pdb code:2prd protein hexamer and wherein the streptavidin or streptavidin derivative strut binds to the multimeric protein with hexagonal geometry. Claim 17. The nanostructure node of Claim 3, having D4 symmetry, wherein the multimeric protein has an amino acid sequence with at least 80 percent identity to an amino acid sequence selected from the group consisting of the pdb code:lo4v protein octamer, the pdb code:2h2i protein octamer, and the pdb code:2iel protein octamer and wherein streptavidin or streptavidin derivative strut binds to the multimeric protein with rectangular geometry. Claim 18. The nanostructure node of Claim 3, having tetrahedral T23 symmetry, wherein the multimeric protein has an amino acid sequence with at least 80 percent identity to the amino acid sequence of the pdb coderlpw protein dodecamer and wherein the streptavidin or streptavidin derivative strut binds to the multimeric protein with cubic lattice geometry. Claim 19. A nanostructure node chain: streptavidin complex, comprising: a polypeptide chain of a nanostructure node; and a streptavidin or streptavidin derivative tetramer having biotin binding sites, wherein the nanostructure node comprises a multimeric protein having a known three- dimensional structure, with Cn, Dn, or higher symmetry, wherein the multimeric protein has stable tertiary structure at a temperature of 70 0C or greater and a modified amino acid sequence, wherein the polypeptide chain comprises two cysteine residues, each functionalized with a biotin or biotin derivative group, and wherein the biotin or biotin derivative groups of the two cysteine residues are bound to two biotin binding sites of the streptavidin or streptavidin derivative tetramer. Claim 20. The nanostructure node chain:streptavidin complex of Claim 19, wherein the multimeric protein comprises an IPP isomerase or an IPP isomerase derivative. Claim 21. The nanostructure node chain: streptavidin complex of Claim 19, further comprising a second polypeptide chain of the nanostructure node, wherein the second polypeptide chain comprises two cysteine residues, each functionalized with a biotin or biotin derivative group, and wherein the biotin or biotin derivative groups of the two cysteine residues of the second polypeptide chain are bound to two biotin binding sites of the streptavidin or streptavidin derivative tetramer. Claim 22. An extended nanostructure strut, comprising: the nanostructure node of Claim 3, and further comprising a first streptavidin strut and a second streptavidin strut, wherein the nanostructure node has at least two specific binding sites and wherein the first streptavidin strut is bound to a first specific binding site and the second streptavidin strut is bound to the second specific binding site. Claim 23. A nanostructural assembly, comprising: a nanostructure node according to Claim 1 ; and a nanostructure strut bound to the specific binding site. Claim 24. The nanostructural assembly of Claim 23, wherein the nanostructure strut comprises streptavidin and/or a streptavidin derivative. Claim 25. The nanostructural assembly of Claim 1, wherein at least one polypeptide chain of the multimeric protein is bonded to a bifunctional reagent with additional binding or a functionality selected from the group consisting of catalytic, chemomechanical, electromechanical, optomechanical, and optoelectronic functionality, and/or at least one polypeptide chain of the multimeric protein comprises an amino acid sequence coding for protein binding or a functionality selected from the group consisting of catalytic, chemomechanical, electromechanical, optomechanical, and optoelectronic functionality. Claim 26. The nanostructural assembly of Claim 23, wherein the nanostructural assembly has the form of a radial planar array. Claim 27. The nanostructural assembly of Claim 23, wherein the nanostructural assembly has the form of a planar polygon and the nanostructure node has a symmetry selected from the group consisting of Cn symmetry and Dn symmetry. Claim 28. The nanostructural assembly of Claim 23, wherein the nanostructural assembly comprises a lattice having the form of a regular plane tiling selected from the group consisting of a triangular tiling (with the nanostructure node having C6 symmetry or D6 symmetry), a square tiling (with the nanostructure node having C4 symmetry or D4 symmetry), and a hexagonal tiling (with the nanostructure node having C3 symmetry or D3 symmetry). Claim 29. The nanostructural assembly of Claim 23, wherein the nanostructural assembly comprises a lattice having the form of a square tiling (with the nanostructure node having D2 symmetry). Claim 30. The nanostructural assembly of Claim 23, wherein the nanostructural assembly has the form of a 3-dimensional radial array, and wherein the nanostructure node has a symmetry selected from the group consisting of Dn, tetrahedral, cubeoctahedral, icosahedral, and dodecahedral symmetry. Claim 31. The nanostructural assembly with the form of a 3-dimensional radial array of Claim 30, wherein the nanostructural assembly has the form of a 3-dimensional radial array with six arms directed along three mutually perpendicular axes and wherein the nanostructure node has tetrahedral (T23) symmetry. Claim 32. A nanostructural assembly with the form of a 3-dimensional radial array of Claim 30, wherein the nanostructural assembly has the form of a 3-dimensional radial array with 18 arms directed toward the apices of a cuboctahedron and wherein the nanostructure node has cuboctahedral symmetry. Claim 33. A nanostructural assembly with the form of a 3-dimensional radial array of Claim 30, wherein the nanostructural assembly has the form of a 3-dimensional radial array with 30 arms directed along the dyad axes of a dodecahedron and wherein the nanostructure node has dodecahedral symmetry. Claim 34. The nanostructural assembly of Claim 23, having a form selected from the group consisting of a regular 3-dimensional polyhedron, an Archimedean solid, and a Catalan solid. Claim 35. The nanostructural assembly of Claim 23, having the form of a three-connected, hexagonal-pattern, three-dimensional lattice. Claim 36. The nanostructural assembly of Claim 23, having the form of a four-connected, cubic-pattern, three-dimensional lattice. Claim 37. The nanostructural assembly of Claim 23, having the form of a six-connected, cubic, three-dimensional lattice. Claim 38. The nanostructure node of Claim 1, wherein a polypeptide chain comprises an amino acid sequence having a designated amino and/or carboxy terminus and further comprising a polypeptide extension of from 5 to 1000 amino acid residues linked with a peptide bond to the designated amino and/or carboxy terminus. Claim 39. The nanostructure node of Claim 38, wherein the polypeptide extension comprises a binding function for a protein, metallic, or other surface. Claim 40. The nanostructure node of Claim 38, wherein the polypeptide extension comprises an amino acid sequence that is a substrate for an enzyme. Claim 41. The nanostructure node of Claim 38, wherein the polypeptide extension comprises a 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. Claim 42. The nanostructure node of Claim 38, wherein the polypeptide extension comprises an antibody binding polypeptide subsequence and further comprising an antibody bound to the antibody binding polypeptide subsequence. Claim 43. The nanostructure node multimeric protein of Claim 1, wherein the multimeric protein comprises three subunits, wherein at least one and at most two subunits comprise a specific binding site. Claim 44. The nanostructure node of Claim 1, wherein the multimeric protein comprises four subunits, wherein at least one and at most three subunits comprise a specific binding site. Claim 45. The nanostructural assembly of Claim 23, wherein the streptavidin is immobilized on a surface. Claim 46. The nanostructural assembly of Claim 23, further comprising: a protein comprising an adenosine triphosphate (ATP) binding site; and a bridge molecule having a biotin group covalently bound to an adenosine triphosphate (ATP) group; wherein the bridge molecule is bound to the biotin binding sites on the streptavidin and the ATP binding site. Claim 47. A nanostructural assembly comprising: a nanostructure node according to Claim 3; and a protein comprising an adenosine triphosphate (ATP) binding site, wherein the moiety covalently attached to each specific amino acid reactive residue is selected from the group consisting of an adenosine triphosphate (ATP) group or an adenosine triphosphate (ATP) derivative group; wherein the ATP or ATP derivative group is bound to the ATP binding site. Claim 48. A device, comprising: a substrate having a surface; a nucleation site on the substrate surface; and the nanostructure node of Claim 1 coupled to the nucleation site. Claim 49. The device of Claim 48, wherein the substrate is selected from the group consisting of a metal, a noble metal, a glass, a ceramic, a semiconductor, a polymer, an organic polymer, an organic material, and combinations and wherein the nucleation site is selected from the group consisting of a metal atom, a noble metal atom, a metal cluster, a noble metal cluster, a chemically reactive molecule, a patch of chemically reactive molecules, and combinations. Claim 50. The device of Claim 48, wherein the substrate is selected from the group consisting of iron, gold, platinum, silver, silicon dioxide, silicon, germanium, a carbon allotrope, diamond, graphite, polytetrafluoroethylene, and combinations and wherein the nucleation site is selected from the group consisting of an iron atom, a gold atom, a platinum atom, a silver atom, a copper atom, and combinations. Claim 51. The device of Claim 48, wherein a polypeptide chain of the nanostructure node comprises an amino acid sequence having a designated amino and/or carboxy terminus and further comprising a polypeptide extension of from 5 to 1000 amino acid residues linked with a peptide bond to the designated amino and/or carboxy terminus, wherein the polypeptide extension comprises a binding function coupled to the nucleation site. Claim 52. The device of Claim 48, further comprising a nanostructure strut attached to the specific binding site. Claim 53. A device, comprising: a substrate having a surface with a node-occupied area and a node-unoccupied area; the nanostructure node of claim 1 on the node-occupied area of the surface; and a coating that covers the nanostructure node and covers the surface node-unoccupied area of the surface. Claim 54. The device of Claim 53, wherein the coating is selected from the group consisting of a metal, a noble metal, a glass, a ceramic, a semiconductor, a carbon allotrope, a polymer, an organic polymer, an organic material, and combinations. Claim 55. A device, comprising: a substrate having a surface with a node-occupied area and a node-unoccupied area; the surface coated with a resist layer; and the nanostructure node of claim 1 on the resist layer. Claim 56. A device, comprising the nanostructural assembly of Claim 23; and a first matrix, wherein the first matrix interpenetrates the nanostructural assembly. Claim 57. The device of Claim 56, wherein the nanostructural assembly has the form of a cubic lattice and wherein the first matrix has the form of a cubic lattice. Claim 58. The device of Claim 56, wherein the first matrix comprises a metal, a noble metal, a glass, a ceramic, a semiconductor, a polymer, an organic polymer, and an organic material. Claim 59. A device, comprising a second matrix material having the same or similar form as a nanostructural assembly. Claim 60. The device of Claim 59, wherein the second matrix comprises a metal, a noble metal, a glass, a ceramic, a semiconductor, a polymer, an organic polymer, and an organic material. Claim 61. A method of using a multimeric protein as a nanostructure node, comprising dissolving a nanostructure strut in an aqueous solution, dissolving the nanostructure node of Claim 1 in the aqueous solution or immobilizing the nanostructure node on a surface and exposing the immobilized nanostructure node to the aqueous solution, and allowing the nanostructure strut to bind to the nanostructure node at the specific binding site. Claim 62. The method of Claim 61, wherein the nanostructure strut comprises streptavidin, wherein the specific binding site comprises at least two reactive amino acid residues, wherein each reactive amino acid residue has a covalently attached iminobiotin group, wherein allowing the nanostructure strut to bind to the nanostructure node comprises increasing the pH of the aqueous solution to at least about 7 to induce the iminobiotin group to bind to the streptavidin of the nanostructure strut. Claim 63. The method of Claim 61, wherein the specific binding site comprises at least two reactive amino acid residues, wherein each reactive amino acid residue has a covalently attached photo-activated nucleotide, wherein the nanostructure strut comprises an adenosine triphosphate binding (ATP) binding site, and wherein allowing the nanostructure strut to bind to the nanostructure node comprises irradiating the aqueous solution with light to induce the photo-activated nucleotide to bind to the ATP binding site. Claim 64. A method of making a nanostructure node comprising synthesizing a multimeric protein with a modified amino acid sequence, so that the specific binding site comprises at least two reactive amino acid residues, and the multimeric protein has Cn subunit symmetry, wherein each reactive amino acid residue is capable of covalently attaching a biotin group, so that at least one streptavidin strut can be attached to the multimeric protein with predefined stoichiometry and orientation. Claim 65. A method comprising: using a computer to generate a mathematical and/or graphic representation of the 3- dimensional molecular structure of a template multimeric protein and a streptavidin tetramer; replacing each surface cysteine residue of the template multimeric protein with an alternative amino acid in the representation; iterating through several spatial configurations of the streptavidin tetramer relative to the template multimeric protein in the representation, with the streptavidin tetramer in approximate Van der Waals contact with the template multimeric protein; for each spatial configuration, assigning cysteine to replace two amino acid side chains on the surface of the template multimeric protein that are geometrically complementary to positions in the streptavidin tetramer that correspond to the terminal chemical groups on biotin (e.g., the biotin valeric acid carbon atom) when bound to the streptavidin tetramer to generate a nanostructure node multimeric protein representation; assigning a measure of imperfection to each spatial configuration; storing each spatial configuration and associated multimeric protein; and outputting an optimal multimeric protein design for a nanostructure node. Claim 66. The method according to Claim 65, wherein the measure of imperfection is selected from the group consisting of a root- mean-square (rms) error between the coordinates of the projected positions of valeric acid carbon atoms of a biotin group bound to the streptavidin tetramer and of the sulfur atoms of the nearest cysteine on the surface of the nanostructure node multimeric protein, and the potential energy of electrostatic interaction between the nanostructure node multimeric protein and the streptavidin tetramer and wherein the multimeric protein having the least measure of imperfection is selected as the optimal multimeric protein design. Claim 67. A method according to Claim 65 of operating on a template sequence of a template multimeric protein with Cn subunit symmetry having a surface to define the amino acid sequence of a nanostructure node multimeric protein that can form planar nanoassemblies incorporating Cn planar nodes and streptavidin or streptavidin-incorporating struts attached with predefined stoichiometry and orientation, comprising: a. using a computer to generate a mathematical and/or graphic representation of the 3- dimensional molecular structure of the Cn symmetric template multimeric protein and a streptavidin tetramer; b. using a graphical and/or mathematical method to identify surface cysteine residues on the surface of the template multimeric protein; c. assigning an alternative amino acid(s) (e.g., Ala, Serine, Asp, etc.) to replace the identified surface cysteine residues in the template sequence; d. using the mathematical and/or graphic representation to initially position the 3- dimensional coordinates of the template multimeric protein and streptavidin tetramer so that o the Cn symmetry (or z) axis of the template multimeric protein is parallel to the streptavidin tetramer z-dyad axis or y-dyad axis, o the centers of mass of the template multimeric protein and streptavidin coordinates have the same or nearly the same z coordinate, and o the molecules do not physically intersect; e. using the mathematical and/or graphic representation to incrementally translate the 3- dimensional coordinates of the streptavidin tetramer along one of its dyad axes that is normal to and intersects the Cn axis of the template multimeric protein, until the template multimeric protein and streptavidin tetramer approximately reach Van der Waals contact; f. using a computational and/or graphics method to identify as specific amino acid reactive sites two amino acid residues on the surface of the template multimeric protein that are geometrically complementary to positions in the streptavidin tetramer that correspond to the terminal chemical groups on biotin (e.g., the biotin valeric acid carbon atom) when bound to the streptavidin tetramer; g. assigning a cysteine to replace each of two amino acid residues identified as specific amino acid reactive sites, wherein the assigned cysteine has an associated biotin group, to generate a nanostructure node multimeric protein; h. using a computational and/or computer graphics method, creating a model of the complex formed between the nanostructure node multimeric protein, having the biotin groups associated with the assigned cysteines bound to the streptavidin tetramer, evaluating the overall quality of a potential linkage between the nanostructure node multimeric protein and the streptavidin tetramer, and assigning a measure of binding imperfection; i. using a computational and/or computer graphics method to evaluate the overall quality of the complementarity of fit between the surface of the nanostructure node multimeric protein and the surface of the streptavidin tetramer and assigning a measure of complementarity of fit; j. storing the 3-dimensional coordinates of the nanostructure node multimeric protein : streptavidin complex along with measures of binding imperfection and complementarity of fit in a database; k. beginning with the initial orientation of d., incrementing a rotation of the template multimeric protein about the Cn axis; L iterating steps d. to k. over an angular increment of at least 360/n degrees, where n defines the foldedness of the multimeric protein symmetry axis; and m. outputting an optimal multimeric protein design for a nanostructure node. Claim 68. The method of Claim 67, wherein the measure of binding imperfection comprises the root-mean-square (rms) error between the coordinates of the projected positions of valeric acid carbon atoms of the biotin group as bound to the streptavidin tetramer and of the sulfur atoms of the assigned cysteine with which the biotin group is associated, wherein the measure of complementarity of fit is selected from the group consisting of steric and electrostatic complementarity of amino acid residues at the interface, maintenance of preferred amino acid side chain rotomer conformations, potential energy as estimated using a computational method such as molecular mechanics, quantum mechanics, or potential energy calculations, experimental methods of measuring complex stability, including affinity measuremenst, calorimetry, or other experimental methods, and combinations, wherein the optimal multimeric protein design is selected based on a criterion consisting of small binding imperfection, high complementarity of fit, approp riate coordinates of the multimeric protein : streptavidin complex, and combinations. Claim 69. The method of Claim 67 wherein the angular increment is in a range of from about 0.001 degrees to about 5 degrees. Claim 70. The method of Claim 67, further comprising producing a protein according to the optimal multimeric protein design. Claim 71. The method of Claim 70, further comprising modifying at least one polypeptide chain of the optimal multimeric protein produced through reaction with a bifunctional reagent to incorporate additional binding or functionality selected from the group consisting of catalytic, chemomechanical, electromechanical, optomechanical, and optoelectronic functionality into the at least one polypeptide chain. Claim 72. The method of Claim 70 further comprising modifying at least one polypeptide chain of the optimal multimeric protein produced through covalent incorporation of an amino acid sequence coding for protein binding or functionality selected from the group consisting of catalytic, chemomechanical, electromechanical, optomechanical, and optoelectronic functionality. Claim 73. The method of Claim 70, further comprising introducing at least one linking polypeptide sequence (e.g., through a genetic method) to covalently interconnect at least two subunits of the template multimeric protein, so that the optimal nanostructure node multimeric protein comprises a number of polypeptide chains reduced from the template number of polypeptide chains, wherein the linking polypeptide sequence is determined through application of a graphic, mathematical, or empirical method. Claim 74. The method of Claim 70, wherein the optimal multimeric protein is produced by expression in an E. coli bacterium or another heterologous protein expression system. Claim 75. The method of Claim 67, further comprising modifying the template sequence of the template multimeric protein to improve the complementarity of fit quality between the multimeric protein and the streptavidin tetramer. Claim 76. The method of Claim 75, wherein modifying the template sequence of the template multimeric protein is selected from the group consisting of adding at least one amino acid, removing at least one amino acid, and replacing at least one amino acid at the surface of the template multimeric protein. Claim 77. A method according to Claim 65 of operating on a template sequence of a template multimeric protein with Cn subunit symmetry having a surface and a template number of polypeptide chains to define the amino acid sequence of a nanostructure node multimeric protein that can form polyhedral nanoassemblies incorporating Cn polyhedral nodes and streptavidin or streptavidin-incorporating struts attached with predefined stoichiometry and orientation, comprising: a. in a computer, generating a mathematical and/or graphic representation of the 3- dimensional molecular structure of the Cn symmetric template multimeric protein and a streptavidin tetramer; b. using a graphical and/or mathematical method to identify surface cysteine residues on the surface of the template multimeric protein; c. assigning an alternative amino acid(s) (e.g., Ala, Serine, Asp, etc.) to replace the identified surface cysteine residues in the template sequence; d. using the mathematical and/or computer representation to initially position the 3- dimensional coordinates of the template multimeric protein and streptavidin tetramer so that o the Cn symmetry (or z) axis of the template multimeric protein and streptavidin tetramer z-dyad axis or y-dyad axis are oriented at an angle corresponding to a polyhedral node apex angle, o the centers of mass of the template multimeric protein and streptavidin coordinates are variably displaced along their z coordinates to facilitate the generation of polyhedron apex node geometry, and o the molecules do not physically intersect; e. using the mathematical and/or graphic representation to incrementally translate the 3- dimensional coordinates of the streptavidin tetramer along one of its dyad axes that intersects the Cn axis of the template multimeric protein, until the template multimeric protein and the streptavidin tetramer approximately reach Van der Waals contact; f. using a computational and/or graphics method to identify as specific amino acid reactive sites two amino acid residues on the surface of the template multimeric protein that are geometrically complementary to positions in the streptavidin tetramer that correspond to the terminal chemical groups on biotin (e.g., the biotin valeric acid carbon atom) when bound to the streptavidin tetramer; g. assigning a cysteine to replace each of two amino acid residues identified as specific amino acid reactive sites, wherein the assigned cysteine has an associated biotin group, to generate a nanostructure node multimeric protein; h. using a computational and/or computer graphics method, creating a model of the complex formed between the nanostructure node multimeric protein and the streptavidin tetramer, having the biotin groups associated with the assigned cysteines bound to the streptavidin tetramer, evaluating the overall quality of a potential linkage between the node multimeric protein and the streptavidin tetramer, and assigning a measure of binding imperfection; i. using a computational and/or computer graphics method to evaluate the overall quality of the complementarity of fit between the surface of the nanostructure node multimeric protein and the surface of the streptavidin tetramer and assigning a measure of complementarity of fit; j. storing the 3-dimensional coordinates of the nanostructure node multimeric protein : streptavidin complex along with measures of binding imperfection and complementarity of fit in a database; k. beginning with each member of a set of initial orientations of d. where the centers of mass of the template multimeric protein and streptavidin coordinates are variably displaced along their z coordinates by increments of from about 0.001 to about 5 angstroms, up to a total of 50 Angstroms, and incrementing a rotation of the template multimeric protein about the Cn axis by increments of from about 0.001 to about 5 degrees; 1. iterating steps d. to k. over an angular increment of at least 360/n degrees, where n defines the foldedness of the template multimeric protein symmetry axis; and ra. selecting an optimal nanostructure node multimeric protein for production. Claim 78. The method of Claim 77, wherein the measure of binding imperfection comprises the root-mean-square (rms) error between the coordinates of the projected positions of valeric acid carbon atoms of the biotin group as bound to the streptavidin tetramer and of the sulfur atoms of the assigned cysteine with which the biotin group is associated, wherein the measure of complementarity of fit is selected from the group consisting of steric and electrostatic complementarity of amino acid residues at the interface, maintenance of preferred amino acid side chain rotomer conformations, potential energy as estimated using a computational method such as molecular mechanics, quantum mechanics, or potential energy calculations, experimental methods of measuring complex stability, including affinity measuremenst, calorimetry, or other experimental methods, and combinations, wherein the optimal nanostructure node multimeric protein is selected for production based on a criterion consisting of small binding imperfection, high complementarity of fit, appropriate coordinates of the nanostructure node multimeric protein : streptavidin complex, and combinations. Claim 79. A method of operating on a template sequence of a Dn or higher symmetry template multimeric protein having a surface and a template number of polypeptide chains to define the amino acid sequence of a nanostructure node multimeric protein that can form nanoassemblies incorporating nodes of Dn or higher symmetry and streptavidin or streptavidin- incorporating struts attached with predefined stoichiometry and orientation, comprising: a. in a computer, generating a mathematical and/or graphic representation of the 3- dimensional molecular structure of the Dn or higher symmetry template multimeric protein; b. using a mathematical and/or graphical method, identifying, and listing amino acid residues with backbone or side chain atoms exposed at the protein surface; c. assigning an alternative amino acid(s) (e.g., Ala, Serine, Asp, etc.) to replace cysteine residues in the list of amino acid residues with backbone or side chain atoms exposed at the protein surface; c. generating a list of 3 -dimensional coordinates of candidate amino acid carbons comprising Cβ amino acid atoms for all non-Gly and backbone Ca carbon atoms for all GIy amino acid residues with backbone or side chain atoms exposed at the protein surface; d. generating a mathematical and/or graphic representation of a "bounding box" of which edge lines correspond to projected positions complementary to the biotin binding sites of streptavidin, wherein the bounding box has dimensions of about 6.4 Angstroms by about 19.5 Angstroms by a lengthwise dimension; e. using a graphics and/or mathematical method to identify the location of the dyad axes in the template multimeric protein; f. using a mathematical and/or graphic method to superimpose representations of the "bounding box" on the representation of the 3-dimensional molecular structure of the template multimeric protein, wherein a pair of bounding boxes are positioned with lengthwise dimension parallel to a dyad axis of the template multimeric protein and symmetrically about each dyad axis, wherein the longest dimension of each bounding box is greater than the largest dimension of the template multimeric protein along the dyad axis about which the bounding box is positioned, wherein for a pair of bounding boxes positioned about a dyad axis, a first member of the pair of bounding boxes is positioned with its 19.5 Angstrom dimension parallel to a Dn or higher symmetry axis of the template multimeric protein, and a second member of the pair of bounding boxes is positioned with its 19.5 Angstrom dimension perpendicular to the Dn or higher symmetry axis of the template multimeric protein; g. using a mathematical and/or computer graphic method, identifying candidate amino acid carbons within 5 Angstroms of an edge line along the lengthwise dimension of each bounding box; h. identifying amino acid residues that include a candidate amino acid carbon as specific amino acid reactive sites; i. assigning a cysteine to replace an amino acid residue identified as specific amino acid reactive site, wherein the assigned cysteine has an associated biotin group, to generate a representation of the 3-dimensional molecular structure of a nanostructure node multimeric protein; j. using a computational and/or graphics method, creating a model of the complex formed between the nanostructure node multimeric protein and the streptavidin tetramer, having the biotin groups associated with the assigned cysteines bound to the streptavidin tetramer, evaluating the overall quality of a potential linkage between the node multimeric protein and the streptavidin tetramer, and assigning a measure of binding imperfection; k. using a computational and/or graphics method to evaluate the overall quality of the complementarity of fit between the surface of the nanostructure node multimeric protein and the surface of the streptavidin tetramer and assigning a measure of complementarity of fit; 1. storing the 3 -dimensional coordinates of the nanostructure node multimeric protein : streptavidin complex along with measures of binding imperfection and complementarity of fit quality in a database; m. iterating steps i. through 1. over the amino acid residues identified as specific amino acid reactive sites; and n. selecting an optimal nanostructure node multimeric protein for production. Claim 80. The method of Claim 79, wherein the measure of binding imperfection comprises the root-mean-square (rms) error between the coordinates of the projected positions of valeric acid carbon atoms of the biotin group as bound to the streptavidin tetramer and of the sulfur atoms of the assigned cysteine with which the biotin group is associated, wherein the measure of complementarity of fit comprises the potential energy of electrostatic interaction, wherein the optimal nanostructure node multimeric protein is selected for production based on a criterion consisting of small binding imperfection, high complementarity of fit, appropriate coordinates of the nanostructure node multimeric protein : streptavidin complex, and combinations. Claim 81. A method of producing a nanostructure node multimeric protein, comprising: culturing a cell host in a culture medium to express the optimal multimeric protein of Claim 67, Claim 77, or Claim 79; and heating the culture medium above a temperature at which the cell host lyses and below the denaturation temperature of the nanostructure node multimeric protein to produce a lysate comprising the nanostructure node multimeric protein, and isolating the thermostable nanostructure node multimeric protein in substantially pure form from the lysate. Claim 82. The method of Claim 81, further comprising using recombinant DNA technology, site-specific modification techniques, and/or gene fusion techniques to modify a nucleotide sequence of a thermophilic organism for directing the expression of the nanostructure node multimeric protein. Claim 83. A method of using a nanostructural assembly to mask a resist material for the fabrication of devices with sub- 100 nanometer features, comprising providing the nanostructural assembly of Claim 23 and a resist materialm and placing the nanostructural assembly on the surface of the resist material. Claim 84. The method of Claim 83, where the device is selected from the group consisting of a nanolithography stamp, a semiconductor device, a waveguide, a zero-mode waveguide, a microelectromechanical system (MEMS), a nanofluidics system, a filter, a two-dimensional planar filter, and combinations. Claim 85. A method of using a nanostructural assembly as a patterning material for the fabrication of devices with sub- 100 nanometer features comprising providing the nanostructural assembly of Claim 23, preparing a substrate surface to introduce at least one specific protein attachment site, binding the nanostructure node to the surface at the at least one specific protein attachment site through chemical linkages, and coating the nanostructural assembly with a material. Claim 86. The method of Claim 85, wherein the coating material is selected from the group consisting of a metal, a noble metal, a glass, a ceramic, a semiconductor, a polymer, an organic polymer, and combinations and wherein the substrate is selected from the group consisting of a metal, a noble metal, a glass, a self-assembling monolayer, a plastic, a polymer, an organic polymer, an organic material, a ceramic, a semiconductor, and combinations. Claim 87. A method of using a nanostructural for patterning a resist material for the fabrication of devices with sub- 100 nanometer features comprising providing the nanostructural assembly of Claim 23, coating a substrate surface with a continuous resist layer whose chemical properties are altered by irradiation with a suitable type of radiation; preparing the resist layer surface to introduce at least one specific protein attachment site; placing the nanostructure node on the resist layer surface at a predetermined location; exposing the surface with the nanostructure node to radiation, wherein the surface of the resist lying underneath the nanostructure node is protected from the radiation; removing the irradiated resist material through a chemical action; etching the surface with the nanostructure node and underlying resist to form a pattern that is complementary to the structure of the nanostructure node; removing, using chemical or other means, the nanostructure node and underlying resist to leave a pattern on the substrate surface that is complementary to the structure of the nanostructure node. Claim 88. The method of Claim 87, further comprising binding the nanostructure node to the resist layer surface at specific attachment sites through a chemical linkage. Claim 89. The method of Claim 87, wherein the substrate is selected from the group consisting of a metal, a noble metal, a glass, a self-assembling monolayer, a plastic, a polymer, an organic polymer, an organic material, a ceramic, a semiconductor, and combinations. Claim 90. A method of using a nanostructural assembly as a patterning material for the fabrication of devices with sub- 100 nanometer channels comprising providing the nanostructural assembly of Claim 23, embedding the nanostructural assembly with a matrix material; treating the nanostructural assembly with radiation, light, heat, and/or chemicals to remove or ablate the nanostructural assembly, leaving the matrix material with internal channels presenting a negative impression of the original nanostructural assembly. Claim 91. The method of Claim 90, wherein the matrix material is selected from the group consisting of metal, a noble metal, a glass, a self-assembling monolayer, a plastic, a polymer, an organic polymer, a ceramic, an organic material, a semiconductor, and combinations. Claim 92. The method of Claim 90, further comprising: coating or chemically treating the negative impression created in the matrix material to deposit a second matrix material in the negative space originally occupied by the nanostructural assembly; treating the assembly with radiation, light, heat, and/or chemicals to remove or ablate the matrix material, leaving a three-dimensional nanostructure assembly comprising the second matrix material with features of the original nanostructural assembly. Claim 93. The method of Claim 92, wherein the second matrix material is selected from the group consisting of a metal, a noble metal, a glass, a self-assembling monolayer, a plastic, a polymer, an organic polymer, an organic material, a ceramic, a semiconductor, and combinations. Claim 94. A nanostructure node, comprising: a modified amino acid sequence that has a stable tertiary structure at a temperature of 70 0C or greater, a known 3-dimensional structure that essentially has Cn, Dn, or higher symmetry, and a specific binding site for the attachment of a nanostructure strut with predefined stoichiometry and orientation, wherein the specific binding site comprises at least two specific reactive amino acid residues. |
This invention was made with government support under Grant Number 1R43GM077743-01A1 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION
[0001] Miniaturization is required for the improvement of existing technologies and the enablement of new ones. For example, increases in the speed and processing power of computing machinery are dependent on further miniaturization. Silicon semiconductor devices, are presently fabricated by a "top down" sequential patterning technology using photolithography, far-ultraviolet lithography, or, more recently, electron beam lithography. Although progress with this technology has been made to produce ever smaller devices, it is generally recognized that the reliable production of structures with consistent sub- 10 nanometer features probably lies beyond the capabilities of top-down silicon fabrication technology. [0002] Self-assembling nanosystems might create complex and higher density novel device architectures. Such devices could potentially have applications as biosensors, actuators, biomaterials, or nanoelectronic devices for a wide variety of applications in fields as diverse as medicine and material science.
[0003] "Bottom up" techniques of self-assembly are common to biological systems
(Padilla et al. 2001; Whitesides et al. 2002; Liu & Amro 2002; Lee et al. 2002; Ringler & Schulz 2003). Several companies are developing nanotechnology based on carbon or silicon-based nanostructures, functionalized carbon nanotubes, or buckyballs. An alternative approach to the development of self-assembled nanostructures makes use of biomolecules like nucleic acids and proteins. Several 2-dimensional and 3-dimensional nanostructures formed of DNA have been generated. (Rothemund 2006; Seeman, 2005ab; Shih 2004).
[0004] Whole viruses have been used as substrates for nanostructures, as described in
Blum et al. (2004), Blum et al. 2005, Chatterji et al. (2004), Chatterji et al. 2005, and Falkner et al. (2005). Cambrios uses virus structures for material sciences applications (www.cambrios.com).
[0005] There have been reports of 1 -dimensional (e.g. Medalsy et. al., 2008), 2- dimensional (e.g. Sleytr et. al. 2007), and 3-dimensional protein arrays (e.g. protein crystals) have been reported. Padilla et. al (2001) and Yeates et. al (2004) discuss engineered fusion proteins, produced by using recombinant DNA technology to link the genes coding for subunits of protein multimers of different symmetry, and describe the spontaneous assembly in solution of both tetrahedral complexes and a linear helical filament using the fused protein domain approach.
[0006] An alternative approach to the formation of 2-dimensional self-assembling lattices of biomolecules involves diffusional organization on self-assembled monolayers (SAMs)
(Liu et. al 1996, Liu & Amro 2002, Lee et al. 2002, Sleytr et al. 2007).
[0007] The protein-based assemblies cited above primarily result from the spontaneous association of molecules and so only allow limited control over nanostructure assembly.
[0008] In 2003, Ringler & Schulz described the formation of a structure that incorporated a modified form of the tetrameric aldolase RhoA from E. coli and streptavidin.
They reported the assembly of a 2-dimensional lattice formed of the RhoA tetramers and streptavidin through interaction of the proteins with a self-assembled monolayer.
[0009] There are severe limitations in the prior work. For example, the assembly process of Padilla and Ringler & Schulz resulted in the formation of many non-uniform or defective structures with poor quality of the structural assemblies. Because assembly occurred spontaneously, there was no control over the steps of assembly, resulting in partial structures and aggregated complexes. Also, the proteins used in the prior studies were not conformationally stable.
SUMMARY OF THE INVENTION
[0010] Several methods according to the invention include the following:
[0011] A method of using a template multimeric protein with Cn, Dn, or higher symmetry, that incorporates specific attachment sites for nanostructure struts with predefined stoichiometry and orientation, and is derived from a thermophilic microorganism, as a nanostructure node.
[0012] A method of using a list of sequences of multimeric proteins with Cn, Dn or higher symmetries derived from template multimeric proteins (having a template number of polypeptide chains) of thermostable organisms with utility as node templates for the generation of nanostructure nodes including nanostructure node multimeric proteins incorporating specific binding sites for the symmetric attachment of nanostructure struts with defined stoichiometry and orientation.
[0013] A method of using a set of sequences with greater than 80 per cent sequence identity with a list of multimeric proteins with Cn, Dn or higher symmetries derived from thermostable organisms with utility as node templates for the generation of nanostructure nodes incorporating specific binding sites for the symmetric attachment of nanostructure struts with defined stoichiometry and orientation.
[0014] A method of using a list of sequences of multimeric proteins with Cn symmetry derived from template multimeric proteins (having a template number of polypeptide chains) of thermostable organisms with utility as node templates for the generation of nanostructure nodes including nanostructure node multimeric proteins incorporating specific binding sites for the symmetric attachment of nanostructure struts with defined stoichiometry and orientation. [0015] A method of using a set of sequences with greater than 80 per cent sequence identity with a list of multimeric proteins with Cn symmetry derived from thermostable organisms with utility as node templates for the generation of nanostructure nodes incorporating specific binding sites for the symmetric attachment of nanostructure struts with defined stoichiometry and orientation.
[0016] A method of using a protein node incorporating multiple subunit polypeptide chains related by Cn symmetry, with each subunit incorporating two specific amino acid reactive sites (specific amino acid reactive residues) permitting the covalent attachment of biotin groups, subsequently allowing interconnection with streptavidin tetramers (or streptavidin derivative tetramers or avidin tetramers, or avidin derivative tetramers) with defined stoichiometry and orientation.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] A method of making an optimal nanostructure node that is produced through expression in an E, coli bacterium or another heterologous protein expression system. [0022] 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.
[0023] A method of making a planar protein node based on a template node sequence from a thermophilic organism that incorporates multiple subunit polypeptide chains related by C3, C4, C5, C6, and C7 symmetry, and that has been modified according to a computer graphical or mathematical method to define and incorporate two reactive amino acid groups permitting the covalent attachment of biotin groups, subsequently allowing Cn-symmetric interconnection between the node and n streptavidin tetramers in a planar orientation. [0024] 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. [0025] 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. [0026] 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.
[0027] 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.
[0028] 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.
[0029] A method of making optimal nanostructure nodes using computer graphics, mathematical methods, or experimental methods for defining amino acid sequences of nanostructure nodes with improved interface interactions between a Dn or higher symmetry node and streptavidin.
[0030] A method of making 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.
[0031] A method of making a protein node where at least one subunit polypeptide chains have been modified through covalent incorporation of a polypeptide chain sequence coding for protein binding or other functionality.
[0032] A method of making 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.
[0033] Several embodiments of the invention include the following:
[0034] In an embodiment, 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. [0035] In an embodiment, 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.
[0036] In an embodiment, 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.
[0037] In an embodiment, 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.
[0038] In an embodiment, a protein node with subunit polypeptide chains related by Cn,
Dn or higher symmetry, 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.
[0039] In an embodiment, 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.
[0040] In an embodiment, a protein node with subunit polypeptide chains related by Cn,
Dn or higher symmetry, where all of subunits have been covalently interconnected to form a protein multimer composed of a single polypeptide chain, and modified to incorporate specific binding sites for chemical modification leading to the covalent attachment of biotin groups.
[0041] In an embodiment, 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.
[0042] In an embodiment, 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.
[0043] In an embodiment, 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.
[0044] In an embodiment, a planar protein node based on the template protein pdb code: lthj, 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 (or streptavidin derivative tetramers, or avidin tetramers, or avidin derivative tetramers, or combinations) in a planar orientation.
[0045] In an embodiment, 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.
[0046] In an embodiment, 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.
[0047] In an embodiment, 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.
[0048] In an embodiment, 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.
[0049] In an embodiment, 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.
[0050] In an embodiment a planar protein node based on the template node sequence pdb code: Ii81 that incorporates seven subunit polypeptide chains related by C7 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 C7 symmetric interconnection with 7 streptavidin tetramers in a planar orientation.
[0051] In an embodiment, 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.
[0052] In an embodiment, 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. [0053] In an embodiment, 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.
[0054] In an embodiment, a protein node based on the tetrameric D2-symmetric node template pdb coderlnial, where positions on subunits related by D2 symmetry have been modified to incorporate specific cysteine residues allowing covalent attachment of biotin groups and subsequent interconnection with streptavidin tetramers with defined stoichiometry and orientation. According to whether cysteine modifications are introduced along one, two, or all three of the independent dyad axes of the tetramer, streptavidin linked structures with linear, 2- dimensional rectangular, or 3 -dimensional orthorhombic lattice geometry may be formed. [0055] In an embodiment, a protein node based on the tetrameric D2-symmetric node template pdb code:lnto. According to whether cysteine modifications are introduced along one, two, or all three of the independent dyad axes of the tetramer, streptavidin linked structures with linear, 2-dimensional rectangular, or 3 -dimensional orthorhombic lattice geometry may be formed.
[0056] In an embodiment, a protein node based on the tetrameric D2-symmetric node template pdb code:lrtw. According to whether cysteine modifications are introduced along one, two, or all three of the independent dyad axes of the tetramer, streptavidin linked structures with linear, 2-dimensional rectangular, or 3 -dimensional orthorhombic lattice geometry may be formed.
[0057] In an embodiment, 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.
[0058] In an embodiment, 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.
[0059] In an embodiment, 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.
[0060] In an embodiment, 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.
[0061] In an embodiment, 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.
[0062] In an embodiment, 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.
[0063] In an embodiment, 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.
[0064] In an embodiment, 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.
[0065] In an embodiment, 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
[0066] In an embodiment, 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.
[0067] In an embodiment, 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.
[0068] In an embodiment, 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. [0069] In an embodiment, 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.
[0070] In an embodiment, 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.
[0071] In an embodiment, 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.
[0072] In an embodiment, a nanostructure with 2-dimensional polygonal geometry incorporating Cn-symmetric nodes and streptavidin or streptavidin-incorporating struts. [0073] In an embodiment, a nanostructure with 2-dimensional polygonal geometry incorporating single-chain Cn-symmetric nodes and streptavidin or streptavidin-incorporating struts.
[0074] In an embodiment, a nanostructure with 2-dimensional hexagonal polygonal geometry incorporating single-chain C3-symmetric nodes and streptavidin or streptavidin- incorporating struts.
[0075] In an embodiment, a nanostructure with 2-dimensional hexagonal polygonal geometry incorporating single-chain C3 -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 streptavidin or streptavidin-incorporating struts. [0076] In an embodiment, a nanostructure with 2-dimensional hexagonal polygonal geometry incorporating single-chain C3-symmetric nodes based on the node templates pdb code:lthj.
[0077] In an embodiment, a nanostructure with 2-dimensional square polygonal geometry incorporating single-chain C4-symmetric nodes and streptavidin or streptavidin- incorporating struts.
[0078] In an embodiment, a nanostructure with 2-dimensional square polygonal geometry incorporating single-chain C4-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 streptavidin or streptavidin-incorporating struts. [0079] In an embodiment, a nanostructure with 2-dimensional square polygonal geometry incorporating single-chain C4-symmetric nodes based on the node template pdb code: 1 vcg.
[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.
[0082] In an embodiment, a 2-dimensional hexagonal lattice incorporating C3- symmetric nodes and streptavidin or streptavidin-incorporating struts.
[0083] In an embodiment, a 2-dimensional hexagonal lattice incorporating C3- symmetric nodes based on node templates corresponding to the pdb code:lthj protein trimer or the pdb code:lj5s protein trimer and streptavidin or streptavidin-incorporating struts. [0084] In an embodiment, a 2-dimensional square lattice incorporating C4-symmetric nodes and streptavidin or streptavidin-incorporating struts.
[0085] In an embodiment, 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.
[0086] In an embodiment, a 2-dimensional square lattice incorporating C4-symmetric nodes based on the node template sequence pdb code:lvcg and streptavidin or streptavidin- incorporating struts.
[0087] In an embodiment, 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.
[0088] In an embodiment, 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 [0089] In an embodiment, a 3-dimensional radial nanostructure incorporating a node with tetrahedral (T23) symmetry based on a dodecameric node and streptavidin or streptavidin- incorporating struts.
[0090] In an embodiment, a 3-dimensional radial nanostructure incorporating a node template with tetrahedral (T23) symmetry 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.
[0091] In an embodiment, a 3-dimensional radial nanostructure incorporating a node with tetrahedral (T23) symmetry based on the dodecameric node template pdb code:lpw and streptavidin or streptavidin-incorporating struts. [0092] In an embodiment, a 3 -dimensional radial nanostructure incorporating a node with cubeoctahedral symmetry based on the 24-subunit node template derived from a thermophilic organism and streptavidin or streptavidin-incorporating struts.
[0093] In an embodiment, 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
X-ray crystallography, and streptavidin or streptavidin-incorporating struts.
[0094] In an embodiment, 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.
[0095] In an embodiment, 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.
[0096] In an embodiment, 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 .
[0097] In an embodiment, a 3-dimensional polyhedron formed of streptavidin or streptavidin-incorporating struts, and nodes with Cn symmetry template derived from a list of known three-dimensional protein structures with corresponding symmetry as determined from
X-ray crystallography, and incorporating binding interactions corresponding to the apex geometry of a polyhedron .
[0098] In an embodiment, 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.
[0099] In an embodiment, 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.
[00100] In an embodiment, 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. [00101] In an embodiment, 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.
[00102] In an embodiment, 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.
[00103] In an embodiment, 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.
[00104] In an embodiment, a 3-dimensional, three-connected hexagonal-pattern lattice formed of streptavidin or streptavidin-incorporating struts, and nodes with D3 symmetry, alternatively modified to allow binding to streptavidin in two orientations. [00105] In an embodiment, a 3-dimensional, three-connected hexagonal-pattern lattice formed of streptavidin or streptavidin-incorporating struts, and different nodes with D3 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.
[00106] In an embodiment, 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.
[00107] In an embodiment, 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.
[00108] In an embodiment, a nanostructure comprising a 3-dimensional, four-connected, cubic pattern lattice formed of nodes with D4 symmetry and streptavidin or streptavidin- incorporating struts. [00109] In an embodiment, 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.
[00110] In an embodiment, 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.
[00111] In an embodiment, a 3-dimensional, six-connected cubic lattice formed of streptavidin or streptavidin-incorporating struts.
[00112] In an embodiment, a 3-dimensional, six-connected cubic lattice formed of streptavidin or streptavidin-incorporating struts, and nodes with tetrahedral symmetry.
[00113] In an embodiment, 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.
[00114] In an embodiment, 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.
[00115] In an embodiment, a nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits and specific binding functionality.
[00116] In an embodiment, a nanostructure node incorporating 3, 5, or 6 subunits.
[00117] In an embodiment, a nanostructure node incorporating 3, 5, or 6 subunits, where the subunits are related by rotational symmetry.
[00118] In an embodiment, a nanostructure node multimeric protein incorporating multiple polypeptide subunits related by tetrahedral, octahedral, or icosahedral symmetry.
[00119] In an embodiment, 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.
[00120] In an embodiment, 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.
[00121] In an embodiment, 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.
[00122] In an embodiment, 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.
[00123] In an embodiment, 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.
[00124] In an embodiment, 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.
[00125] In an embodiment, 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.
[00126] In an embodiment, 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.
[00127] In an embodiment, a thermostable nanostructure node protein based on a template sequence derived from a thermostable microorganism and comprising multiple polypeptide chain subunits and specific binding functionality.
[00128] In an embodiment, 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.
[00129] In an embodiment, 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. [00130] In an embodiment, 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).
[00131] In an embodiment, 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
Thermus thermophilus.
[00132] In an embodiment, 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
20 Angstroms.
[00133] In an embodiment, 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.
[00134] In an embodiment, 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.
[00135] In an embodiment, 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.
[00136] In an embodiment, 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.
[00137] In an embodiment, 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. [00138] In an embodiment, 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.
[00139] In an embodiment, a nanostructure assembly incorporating a multimeric nanostructure node protein together with a specifically bound nanostructure strut. [00140] In an embodiment, 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. [00141] In an embodiment, 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.
[00142] In an embodiment, 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.
[00144] 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 90 degree rotation about the C4 axis, the nanostructure node functions as a right angle "L" linker between two nanostructure struts.
[00145] In an embodiment, 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.
[00146] In an embodiment, a protein superstructure, comprising a multisubunit nanostructure node with specifically bound strut components.
[00147] In an embodiment, 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.
[00148] In an embodiment, 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.
[00149] In an embodiment, 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.
[00150] In an embodiment, 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.
[00151] In an embodiment, 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.
[00152] In an embodiment, a kit, comprising a nanostructure multisubunit node and a monostructure strut. [00153] In an embodiment, a kit, comprising a nanostructure multisubunit node and a monostructure strut comprised of streptavidin.
[00154] Several methods according to the invention include the following:
[00155] A method of making a 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.
[00156] A method of making a 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.
[00157] A method of making a 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.
[00158] A method of making a 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.
[00159] A method of making a nanostructure node multimeric protein by combining subunits, some of which have a linker binding site and others of which do not have linker binding sites.
[00160] 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.
[00161] 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.
[00162] 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.
[00163] 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.
[00164] 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
[00165] 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
[00166] A method of making a protein nanostructure that includes a nanostructure node multimeric protein binding to a nanostructure strut.
[00167] 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.
[00168] A method of making a protein nanostructure that includes a nanostructure node multimeric protein and nanostructure struts comprising streptavidin.
[00169] A method of making a protein nanostructure that includes a nanostructure node multimeric protein incorporating covalently bound iminobiotin groups and nanostructure struts comprising streptavidin.
[00170] 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.
[00171] A method of using a proteinaceous nanostructure assembly as a pattern or resist masking material for the fabrication of devices with sub- 100 nanometer features.
[00172] A method of using a 2-dimensional proteinaceous nanostructure assembly as a pattern for the fabrication of devices with sub-100 nanometer features.
[00173] A method of using a 2-dimensional proteinaceous nanostructure assembly as a mask for a resist material for the fabrication of devices with sub-100 nanometer features. [00174] A method of using a 3-dimensional proteinaceous nanostructure assembly as a negative patterning material 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.
[00176] A method of using a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a soft lithography stamp for nanolithography.
[00177] A method of using a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a semiconductor device.
[00178] A method of using a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a zero-mode waveguide.
[00179] A method of using a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a microelectromechanical system (MEMS) device.
[00180] A method of using a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a nanofluidics device.
[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.
[00183] A method of making devices with sub- 100 nanometer features using a 2- dimensional proteinaceous nanostructure assembly as a patterning material on substrates composed of metal, glass, a self-assembling monolayer, plastic, ceramic, or a semiconductor material.
[00184] A method of making devices with sub- 100 nanometer features using a 2- dimensional proteinaceous nanostructure assembly as a pattern that is assembled from engineered nodes derived from a list of thermostable multimers with known structure and optionally, streptavidin or streptavidin-incorporating struts.
[00185] 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.
[00186] 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 and binding a node protein or the node protein assembly to the resist layer surface at specific attachment sites through a chemical linkage. [00187] 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 for patterning a substrate composed of metal, glass, a self-assembling monolayer, plastic, ceramic, or a semiconductor material.
[00188] A method of making devices with sub-100 nanometer features using a 2- dimensional proteinaceous nanostructure assembly as a pattern for a resist material where the proteinaceous pattern is assembled from engineered nodes derived from a list of thermostable multimers with known structure and optionally, streptavidin or streptavidin-incorporating struts. [00189] 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.
[00190] 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.
[00191] 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).
[00192] 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).
[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.
[00195] A method of making devices with sub-100 nanometer, 3-dimensional features, wherein the node protein or the node protein assembly is bound to the resist layer surface at specific attachment sites through a chemical linkage.
[00196] A method of making devices with sub-100 nanometer, 3-dimensional features, 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).
[00197] 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.
[00198] 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, wherein the replica image is composed of metal, glass, plastic, ceramic, or a semiconductor material.
[00199] 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.
[00200] A method of making a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a soft lithography stamp for nanolithography.
[00201] A method of making a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a semiconductor device.
[00202] A method of making a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a zero-mode waveguide.
[00203] A method of making a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a microelectromechanical system (MEMS) device. [00204] A method of making a proteinaceous nanostructure assembly as a pattern or resist material for the fabrication of a nanofluidics device.
[00205] Several embodiments of the invention include the following:
[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. [00207] 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.
[00208] 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. [00209] 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.
[00210] 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. [00211] 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. [00212] 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, and 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. [00213] 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. BRIEF DESCRIPTION OF THE DRAWINGS
[00214] Table IA lists template protein structures useful for the construction of nanostructure nodes having various symmetries.
[00215] 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.
[00216] Table 2 lists specifications and amino acid sequences for node embodiments with various symmetries.
[00217] 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.
[00218] Figure 2 shows the reaction of protein cysteine sulfhydryl groups with biotinylation reagents.
[00219] 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.
[00220] Figure 4 presents schematic illustrations of single-chain nodes based on protein multimers with three-fold rotational (C3) symmetry.
[00221] 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.
[00222] Figure 6 shows the reaction of protein cysteine sulfhydryl groups with bifunctional crosslinking reagents.
[00223] 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.
[00224] Figure 8 presents schematic illustrations of single-chain nodes based on a protein multimers with four-fold rotational (C4) symmetry.
[00225] 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.
[00226] 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.
[00227] Figure 11 presents schematic illustrations of 3-dimensional polyhedra incorporating nodes with C3 and C5 symmetry.
[00228] Figure 12 presents schematic illustrations of D2 symmetric nodes used as strut extenders.
[00229] Figure 13 presents illustrations of a D2 node used as a strut extender that introduces an axial rotation along the strut axis.
[00230] Figure 14 presents illustrations of D2 symmetric protein multimers from thermostable microorganisms useful as node templates.
[00231] Figure 15 presents schematic illustrations of a hexameric node with D3 symmetry and an octameric node with D4 symmetry.
[00232] Figure 16 presents schematic illustrations of a doubly-modified hexameric node with D3 symmetry and an doubly-modified octameric node with D4 symmetry.
[00233] Figure 17 presents illustrations of hexameric protein multimers with D3 symmetry from thermostable microorganisms useful as node templates.
[00234] Figure 18 presents illustrations of octameric protein multimers with D4 symmetry from thermostable microorganisms useful as node templates.
[00235] Figure 19 presents illustrations of regular polyhedra with dyad axes of symmetry.
[00236] 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.
[00237] Figure 21 requirements for complementary binding geometry between streptavidin and node surfaces.
[00238] 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.
[00239] 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.
[00240] 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.
[00241] Figure 25 presents a stereoscopic representation of an engineered single-chain
C3 symmetric node.
[00242] 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.
[00243] 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.
[00244] Figure 28 presents schematic illustrations of D2 symmetric nodes engineered from streptavidin.
[00245] 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.
[00246] 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.
[00247] 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.
[00248] 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.
[00249] 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.
[00250] Figure 34 presents schematic illustrations of complexes of streptavidin with linear strut connectors having D2 symmetry to produce struts of various lengths.
[00251] 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. [00252] Figure 36 presents schematic illustrations of streptavidin-linked two-dimensional lattices formed using nodes with three-fold (C3) and four-fold (C4) rotational symmetry.
[00253] 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.
[00254] Figure 38 presents a molecular illustration of two hexameric D3 nodes interconnected by streptavidin enabling formation of a three-connected three-dimensional lattice.
[00255] Figure 39 presents a molecular illustration of two octameric D4 nodes interconnected by streptavidin enabling formation of a four-connected three-dimensional lattice.
[00256] Figure 40 presents schematic illustrations of various three-dimensional lattices with different node connectivity.
[00257] Figure 41 presents a method of making a proteinaceous nanostructure pattern on a substrate surface.
[00258] Figure 42 presents a method of making a repetitively patterned proteinaceous nanostructure on a substrate surface.
[00259] Figure 43 presents a method of making a coated patterned nanostructure on a substrate surface.
[00260] Figure 44 presents a method of making a patterned structure using a proteinaceous nanostructure as a mask for a photoresist material.
[00261] 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.
[00262] Figures 46A-46C present diagrams of the expression vectors EXP14Q3193C2,
EXP14Q3193C3, and EXP14Q3193C4.
[00263] Figure 47 presents a diagram of the expression vector EXP14Q3164.
[00264] Figure 48 presents images of electrophoretic analysis of NODE:SAV complexes.
DETAILED DESCRIPTION
[00265] Embodiments of the invention are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent parts can be employed and other methods developed without parting from the spirit and scope of the invention. All references cited herein are incorporated by reference as if each had been individually incorporated. [00266] In this document, an amino acid may be indicated by its standard one-letter abbreviation, as understood by one of skill in the art. For example, a polypeptide sequence may be represented by a string of letters.
[00267] In this document, 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. For example, 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. For example, 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". For example, a second polypeptide having every eleventh residue of a first polypeptide deleted would have "80 percent or greater sequence identity" to the first polypeptide, because each string of ten amino acids of the second polypeptide would be identical to a string of ten amino acids of the first polypeptide - such a second polypeptide would have 10/11 = 91% sequence identity to the first polypeptide. For example, a second polypeptide having an additional residue inserted after every ten amino acids of a first polypeptide would have "80 percent or greater sequence identity" to the first polypeptide - such a second polypeptide would have 10/11 = 91% sequence identity to the first polypeptide. For example, 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. According to the invention, 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. Generally, proteins that are homologous or have sufficient sequence identity are those without changes that would detract from adequate structural integrity, reactivity, and symmetry.
[00268] The 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. As such, the 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. For example, 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. However, as is standard in the art, residue numbers indicated in Table 2 (for example, in the context of suggested modifications to the template sequence) 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.
Overview of components and approach
[00269] An objective of the work leading to the present invention, of which several embodiments are presented in this text, is the development of biomolecular components allowing for the systematic and precise fabrication of complex nanodevices with two and 3- dimensional architectures. Proteins, typically having (subunit) dimensions in the range of 3 to 20 nm (or the equivalent, 30 to 200 Angstrom units), and other organic molecules serve as the biomolecular components, and allow for unprecedented miniaturization of devices. By providing proteins with two or more points of controllable attachment, a limited set of a small number of biomolecular components allows for construction of an unlimited number of structures, over the design of which a user has full control. Thus, the biomolecular components will advance research and development into nanodevice applications. The control over assembly and reproducible precision of structures formed by these biomolecular components allows for the fabrication of nanodevices of unprecedented complexity, extent, and diversity. [00270] 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. [00271] In an embodiment, 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.
[00272] In an embodiment, 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.
"Parts box" philosophy
[00273] 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. [00274] Structures organized in three dimensions can also be usefully classified as finite or infinite. Common examples of 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.
[00275] Assembly of biomolecular components such as struts and nodes can proceed in stages that provide the user with the efficiency and parallel nature characteristic of "bottom-up" self-assembly and the control and ability to form asymmetric and complex structures characteristic of "top-down" manufacturing. Because a limited number of biomolecular components can be combined to produce any one of an unlimited number of structures, attention can be focused on developing a small number of these biomolecular components that serve as a "parts box". Because only a limited number of biomolecular components and associated assembly techniques need be designed, produced, and tested, economies of scale can be achieved, so that inexpensive development and production of nanodevices can be realized. That is, the 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. Use of proteins
[00276] 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.
[00277] 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.
[00278] In nature, 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. Because 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.
[00279] 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.
[00280] The techniques for modifying proteins by the techniques of molecular biology and synthetic organic chemistry are well established. For example, 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. Reliable production of large numbers of proteins is a well- established biotechnical procedure. Thus proteins are excellent candidates for a "parts box" with which the philosophies of interchangeable parts and mass production can be applied at the nanoscale.
Applications
[00281] The diverse and sophisticated functionality of naturally occurring proteins allows them to perform a wide range of processing and signal transduction functions in nature, including catalysis, chemomechanical, electromechanical, optomechanical, and optoelectronic transduction for sensing and actuation purposes. This anticipates a diverse range of man-made devices that can be produced with a "parts box" of proteins as biomolecular components. [00282] Structures, for example, node : strut nanostructure assemblies, can be assembled from the struts and nodes described herein.
[00283] A "parts box" of proteins may initially be applied to make devices that are analogous to or in some way emulate natural systems. For example, 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.
[00284] Other applications may not have direct natural analogs, but are intended to interact with natural biological systems. For example, the strut-node technology presented herein 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. [00285] Applications of a "parts box" of proteins as biomolecular components are not limited to devices analogous to or for interacting with natural biological systems. For example, structures can be assembled that emulate the architecture and functions of silicon-based microprocessor architecture and computer memory or possess novel material properties. Many materials science and computer applications depend upon the miniaturization of structural features in two or three dimensions to allow the separation and storage of charge, control of electrical conductance or optical properties, or the addressable storage of data in electrical or magnetic form. As such, the technology described is applicable to the development of new electronic devices including improved batteries, capacitors, computer memory, microprocessors, nonlinear optical devices and materials. Additional applications include ultrafilters that provide protection from pathogens like viruses, or have utility in liquid separations or the desalination of salt water.
[00286] 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.
[00287] Alternatively, 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). Thus, 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.
Biomolecular Components
Protein Stability, Selection, and Engineering
[00288] The 3-dimensional atomic structures of over 25,000 proteins are known (see, http://www.rcsb.org, accessed Oct. 2, 2007), providing an extensive set from which biomolecular components having desired structural and functional characteristics can be selected for a "parts box" (see, http://scop.mrc-lmb.cam.ac.uk/scop/, accessed Oct. 2, 2007). Moreover, the tools of recombinant DNA technology enable the synthesis of virtually any polypeptide sequence or functional domain fusion, providing the basis for rapidly designing and optimizing novel assemblies from engineered biological macromolecules.
[00289] In this document, unless otherwise specified, 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. For example, in the application of a gene fusion technique, 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). For example, 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. For example, in the application of a circular or cyclic permutation technique, 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. Gene fusion and circular or cyclic permutation techniques can be combined to create new polypeptide sequences from polypeptide sequences. For example, the 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.
[00290] Although not widely recognized, numerous studies show that the structural and functional properties of proteins that normally function in aqueous solution are preserved intact when the protein is dehydrated to the level of a few water molecules per protein molecule (Rupley & Careri 1991 ; Zaks & Klibanov 1988; Fitzpatrick et. al. 1993; Castro & Knubovets 2003; Gupta & Roy 2004). Many examples exist of structural proteins, for example spider silk, that form essentially solid-state structural materials and have thermal stabilities in excess of 100 0 C. In addition, many proteins that form unusually stable complexes (Weber et al. 1992), or that carry out the biological functions of 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. [00291] 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. A subclass of extremeophiles are thermophilic organisms (again, usually microorganisms such as bacteria or algae), which live at substantially higher temperatures (typically above 60 deg C) than the vast majority of plants and animals populating the terrestrial ecosystem (usually termed 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. is completely dependent on the preservation of its properly folded, 3-dimensional structure. The vast majority of proteins of mesophilic organisms become thermally denatured when subjected to temperatures above about 50 deg C. In contrast, and although they are generally composed of exactly the same chemical components (amino-acids) as mesophilic proteins, all of the proteins in 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. Although 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. Typically, these applications have included industrial processes that use enzymes to carry out chemical reactions. There have been no reports of using thermostable proteins for nanotechnology applications. The use of engineered thermostable proteins for nanotechnology applications has many advantages.
[00292] One advantage is the ease of production of thermostable proteins for nanotechnology applications. 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. 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.
[00293] A second advantage is the ease of production of engineered and chemically modified variants of thermostable proteins for nanotechnology applications. In many cases 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. However, owing to the very high initial stability of the native forms of thermostable proteins, such 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.
[00294] Useful modifications of the native 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.
[00295] Introduction of modifications in the sequence of proteins using recombinant
DNA technology is broadly used in biomedical research and is the basis of many pharmaceutical products. However, with the exception of Salemme & Weber (2007), no reports exist for using protein engineering for structural nanotechnology applications using thermostable proteins.
Structural modifications of 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:
[00296] a. The introduction of one or more individual substitutions of one amino acid for another at defined positions in the native sequence (commonly termed a site-specific modification). Examples of the utility of such modifications include the substitution of an amino acid like cysteine with a chemically reactive side chain for a non-reactive amino acid like alanine to provide a specific chemical linkage site on the surface of a protein.
[00297] b. The addition or deletion of one or more contiguously-bonded amino acids (a polypeptide extension) from either the amino or carboxy terminus of the native protein polypeptide chain. Examples of the utility of such modifications include the addition or removal of sequences or protein domains that may confer additional binding or catalytic functionality to the native protein or that may be structurally disordered.
[00298] c. The insertion or deletion of one or more amino acids into the sequence of the native or modified protein sequence. Examples of the utility of such modifications include the insertion of sequences or protein domains that may confer additional binding or catalytic functionality to the native protein.
[00299] d. The reconnection of the protein polypeptide chain of the native or native-like sequence, so as to allow the preservation of essentially the same 3 -dimensional folded structure of the native protein, but folded from a sequence where the positions of the amino and carboxy termini have been altered or permuted. Examples of the utility of such modifications include the covalent connection of multiple polypeptide chains that normally form an associated complex into a single contiguous polypeptide sequence.
[00300] e. The interconnection of multiple copies or types of protein sequences that naturally form multimeric structures in nature composed of multiple polypeptide chains, into a structure made up of a smaller number of continuous polypeptide chains. [00301] In actual application, any or all of the types of the modifications of the native protein sequence described in a. through e. above can be used in combination to produce a modified protein sequence.
[00302] 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. Examples of the utility of such chemical modifications include the formation of a covalent connection between the polypeptide structure and chemical groups with specific protein binding activity. For example, 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. Consequently, 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.
[00303] A third advantage afforded to the use of thermostable proteins is the 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.). Similarly, the production of nanodevices that are assembled on self-assembling monolayers or semiconductor substrates like silicon wafers will often involve solution conditions and/or the use of reactive or photo-chemistries where the improved stability of thermostable proteins relative to mesophilic proteins will result in better yields of the desired products and more reliable devices. [00304] A fourth advantage afforded to the use of 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. In particular, 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. Thus, 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.
[00305] 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
[00307] 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.
[00308] Throughout the following descriptions we use standard scientific nomenclature to discuss the symmetry properties of node templates and nodes (Vainstein 1994). For a complete description of point group symmetry and symmetry operation nomenclature see: http://www.phys.ncl.ac.uk/staff/nipg/symmetry/index.html and <http://csi.chemie.tu- darmstadt.de/ak/immel/script/redirect.cgi?filename=http://cs i.chemie.tu- darmstadt.de/ak/immel/tutorials/symmetry/index.html>
[00309] 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. Also shown in 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. In Figure If, a schematic view down the x-axis dyad of the streptavidin tetramer, the 2 facing biotin binding sites (shown schematically as open circles) 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.
[00310] Weber et al. (1989) determined the X-ray structure of streptavidin and described the origins of its ability to bind the vitamin biotin. Although the biotin: streptavidin interaction is non-covalent, the biotin dissociation constant is about 10 "14 M, so that the biotin:streptavidin bond is essentially irreversible. The strength of the biotin:streptavidin bond has led to the broad application of streptavidin in research and diagnostics applications where interaction specificity is required in a complex biological milieu.
[00311] In streptavidin, 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. 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). Thus, when serving as a strut, a streptavidin tetramer can form be linked to other biomolecular components, such as nodes, at two sites through biotin molecules.
[00312] 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.
[00313] In addition to streptavidin and its homologues, many other stable protein tetramers with D2 symmetry, such as those derived from thermostable microorganisms, could function as struts either in their native state or through suitable modification of their amino acid sequence, ligand binding functionality, or chemical modification state. Examples of alternative thermostable strut templates with D2 symmetry are given in Table 1.
Nodes
[00314] A node can connect two or more struts with predefined orientation of each strut with respect to the other connected struts.
[00315] For example, a node can be a symmetric protein multimer. For example, 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. Alternatively, 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. [00316] In general, such 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.
[00317] As outlined in Table 1, 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. In addition, 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. [00318] For example, 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. Thus, 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.
[00319] 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. At low pH (~pH4) the imino group becomes charged, causing imino-biotin displacement from the streptavidin biotin binding site. Iminobiotin linking is useful for the formation of reversible interactions between streptavidin struts and node proteins. Figure 2e shows the imino-biotin reaction product. Figure 2f shows the above reaction sequences schematically as used in schematic illustrations elsewhere in this application. Although 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.
General Descriptions of Node Geometry
[00320] 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, while 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.
[00321] Single chain constructs of a node protein can be formed. For example, 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. For example, 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. In addition to the controlling strut-binding geometry, other features of the individual multimer subunits may be individually varied to introduce asymmetry into the node. For example, if 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. Conversely, 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.
[00322] Some variations of the structure of C3 multimeric nodes are illustrated in Fig. 4
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. 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 the same subunits. Figure 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.
[00323] 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. Specifically, 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). Upon irradiation with UV light, 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. Although 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.
[00324] The aforementioned 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.
[00325] Many proteins and enzymes naturally incorporate 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.
[00326] Since 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. For many classes of enzymes, 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. For example, immunoglobulins, lectin, or other specific binding molecules could be linked to nanostructures constructed of struts and nodes using this means, as outlined below. Figure 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.
[00327] In addition to the use of chemical crosslinking agents as a way to couple proteins to the underlying strut-node structure, it is possible to engineer either nodes or strut components where the 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. When these "fused" genes incorporating the binding domain sequences are expressed, the result will be a single continuous polypeptide chain incorporating the encoded linked protein domain. 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. Examples of such fused domain binding sequences (for example, a polypeptide extension) 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. Starting from upper right, and going counter-clockwise, 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, and 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.
[00328] Figure 7a shows a schematic view of a four-fold (C4) symmetric multimer, while
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. Each chain of the tetramer incorporates 486 amino acid residues, and a non-covalently bound xanthosine-5'-monophosphate substrate analog. Additional C4 symmetric node templates are presented in Table IA. [00329] Figures 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. 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 the same subunits. Figure 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. As such, 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. Thus, 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.
[00330] Figure 9 shows variations of C4 symmetric nodes that have been functionalized and have various geometrical properties. As noted above for C3 nodes, 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.
[00331] As in the case of C3 symmetric nodes, 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. Starting from the top, and going counter-clockwise, 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. As outlined above, 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. As outlined below, the ability to precisely position two different kinds of proteins or antibodies in nanostructures with close apposition could have many applications in functional or diagnostic applications. 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.
[00332] The following descriptions of 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. Similarly, 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. Thus, 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.
[00333] In addition to nodes with C2, C3, and C4 symmetry, natural protein multimers from thermophilic organisms occur with higher Cn rotational symmetry. Figure 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.
[00334] Non-planar Cn Nodes: In addition to Cn nodes with radial planar symmetry
(e.g. with biotinylation sites introduced to orient bound streptavidin tetramers in a plane normal to the Cn axis of the multimeric node), Cn multimers with suitable geometrical features can be site-specifically modified to orient streptavidin tetramers at an angle α to the Cn multimer axis. As shown in Figure 11, such nodes have utility in the assembly of closed polyhedra for specific values of n and α consistent with polyhedron formation (Pugh 1976, Williams 1979, Pearce, 1979). For example, for the generation of an icosahedron, n=5, and the approximate apex angle OC= 121.72 degrees (Fig lla). For the generation of a dodecahedron, n=3, and the approximate apex angle α=z 110.73 degrees (Fig lib). Similar considerations apply to the generation of other regular and irregular polyhedra, such as buckyballs (truncated icosahedron) and buckytubes (Weber 1999) where n=3 and the approximate apex angle α=z 104.15 degrees.
[00335] 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).
[00336] The simplest D n symmetry is D2, a symmetric tetramer where the multimer subunits are related by 3 mutually perpendicular dyad axes. As noted in Fig 1, the streptavidin molecule is itself a tetramer with D2 symmetry. Although 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). Some are relatively flat and rectangular in shape, while others approximate tetrahedral geometry. As outlined in Figure 12 and the specific embodiments described below, 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). In Figures 12a and 12c, the 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). As illustrated in Figures 12 a and c, there are generally two possible orientations for pairs of biotinylation sites that are symmetrically disposed about the D2 (or any other multimer) dyad axis; one where the orientation of the bounding box defining the biotinylation sites is horizontal and which aligns the z-axis dyad of the node with the z-axis dyad of streptavidin (Fig 12a), and the second where the orientation of the bounding box defining the biotinylation sites is vertical and which aligns the z-axis dyad of the node with the y-axis dyad of streptavidin (Figs Ig and 12c). 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. As illustrated, 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. As noted above for Cn symmetric nodes, 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. [00337] In addition to forming struts that maintain terminal biotin binding site geometry it is possible to construct extended struts where the terminal streptavidin binding sites are oriented at angles other than 180 degrees relative to each other around the common complex x-axis. Figure 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 β 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. Figure 13b schematically illustrates a streptavidin:node:streptavidin complex where β=90 degrees, so that the relative orientations of the free biotin binding sites on the complex are rotated by 90 degrees along the corresponding streptavidin x-axes (Fig 1). 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.
[00338] 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. 2005, pdb code:lrtw) in schematic backbone representation and in surface representation (Fig 14f). Also shown in Figure 14 a through f are "bounding boxes" that are aligned along the dyad symmetry axes of the respective D2 symmetric tetramers. The intersections between the molecular surface and the diagonal edges of the box define sites that are symmetric and complementary to the biotin binding in streptavidin, as described in more detail below. Additional structures with D2 symmetry are listed in Table 1.
[00339] Figure 15 presents schematic illustrations of a hexameric node with D3 symmetry and an octameric node with D4 symmetry. As noted above, 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). As schematically illustrated in Figure 15a,b,c,d, this will generally involve introduction of a single site-specific modification in each polypeptide chain of the multimer to introduce a suitable biotinylation site. Note that for multimers with D3 and higher symmetry, there may be several different dyad-related symmetrical sites on the individual multimer subunits that are potentially complementary to the streptavidin dyad-symmetric binding sites. For example, the subunits of the D3 node shown in Fig 15e are shown with biotinylation modification sites on their "faces", but alternative symmetric sites occur that "bridge" between subunits. The situation is similar for the D4 node shown in Figure 15f, except that the 4-fold symmetry creates an additional, symmetrically non- equivalent, set of dyad axes (labeled x' and y' in Fig 15f) in the structure. If the 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.
[00340] 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. 1994, pdb code:2prd) in schematic backbone representation and in surface representation (Fig 17f). Also shown in Figure 17 a through f are "bounding boxes" that are aligned along the dyad symmetry axes of the respective D3 symmetric hexamers. The intersections between the molecular surface and the diagonal edges of the box define sites that are symmetric and complementary to the biotin binding in streptavidin, as described in more detail below. Additional structures with D3 symmetry are listed in Table 1.
[00341] 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. 2006, pdb code:2iel) in schematic backbone representation and in surface representation (Fig 18f). Also shown in Figure 18 a through f are "bounding boxes" that are aligned along the dyad symmetry axes of the respective D4 symmetric octamers. The intersections between the molecular surface and the diagonal edges of the box define sites that are symmetric and complementary to the biotin binding in streptavidin, as described in more detail below. Additional structures with D4 symmetry are listed in Table IA. [00342] In addition to multimers with D3 and D4 symmetry, multimers with higher Dn symmetry are also found in thermophilic organisms (Table IA). These protein multimers have utility as node templates in applications where nanostructures with certain geometrical properties and higher node connectivity is desired than is possible using nodes with D3 and D4 symmetry.
[00343] 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. For example, Figure 19a shows a truncated tetrahedron, Figure 19b shows a cubeoctahedron, and 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.
[00344] 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.
Method Of Determining Sites of Site-Specific Modifications On Proteins Suitable For Production Of Multimeric Node Proteins With Geometrically Defined Attachment Points For Binding Streptavidin
[00345] In general, 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. In the current application, 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). As shown in Figure 1, 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.
[00346] Many 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.
[00347] In the present embodiments, 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.
[00348] 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. As illustrated, 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. 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. For nodes with D2 symmetry, 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). For nodes with Dn symmetry, 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. For polyhedral nodes with dyad axes, 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.
[00349] For nanostructures that are assembled through a combination of components linked chemically, it is necessary to ensure precise control of the linking geometry between the structural components. Without intending to be limited by theory, it is likely that imperfections in the interaction geometry of the structures described by Ringler & Schulz (2003) produced cumulative twist that ultimately limited the size of the stuctures that could self-assemble. [00350] Figure 21a reiterates the geometry of the biotin binding sites on streptavidin.
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. In order to assemble extended structures that do not twist when interconnected by streptavidin, 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. -72 degrees) relative to the Cn rotational (z) axis of the node protein complex so that the modification sites are complementary to the biotin binding sites on streptavidin. There can be some variation (for example, 20.5 plus or minus 5 Angstroms) in the separation of the node modification sites, since this variation can be accommodated by adjusting the length of the chemical linking reagent that couples the biotin to the cysteine sulfhydryl groups. However, significant (>5 degrees) variations in the angle from - 72 degrees will accumulate to cause extended structures to twist and potentially introduce strain into extended structures. [00351] Stated in other words, 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. When the two specific amino acid reactive residues are aligned with the pair of biotin binding sites, for example, so that biotin or biotin derivative groups covalently bound to each of the specific amino acid reactive residues can bind to each biotin binding site of a pair on the 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. [00352] The above criteria represent general requirements for the assembly of any planar structure incorporating streptavidin struts and nodes with Cn rotational symmetry. It is notable that in the prototype 2-dimensional lattice structure assembled by Ringler and Schulz (2003), the two cysteine residues (Asn 133 to Cy s, and Lys 261 to Cys) introduced through site-specific modification on their C4 node protein template, L-rhamnulose-1 -phosphate aldolase from E. coli, are oriented at 52 degrees relative to the C4 axis of the tetramer, giving each bound streptavidin a slight "propeller" twist relative to the central node. It is consequently evident that extended structures must have been quite strained, and that this was an important contributing factor to their inability to build very extensive 2-dimensional lattices.
[00353] 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. For each angular increment about the Cn axis, 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. 2005), or a combination of these methods to determine the quality of overall fit and feasibility and locations of site-specific modifications on the node template that can provide chemical attachment points for biotin, including the use of coupling reagents with different linker lengths. Parameters describing the complex structure (e.g 3-dimensional model coordinates, computed energy, pictures, etc.) may be then entered into a table. The process outlined above is repeated for relatively small incremental changes in rotation around the template node Cn symmetry axis (for example, about 0.1 to 2.0 degrees in rotation), so that interactions of the Cn node surface and streptavidin are extensively sampled, evaluated and compared. Complexes with the best features are then selected for manufacture using recombinant DNA technology.
[00354] 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 α formed between the edge of the polyhedron and a vector from the center of the polyhedron to an apical node (Fig 22b). As outlined above, the apex angle α for an icosahedron is approximately 121.92 degrees (Fig lla) and for a dodecahedron α is approximately 110.93 degrees (Fig lib). Similar considerations apply to the generation of other regular and irregular polyhedra, such as buckyballs (truncated icosahedron) and buckytubes (Weber 2199) where n=3 and the approximate apex angle α=104.15 degrees.
[00355] 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.
[00356] As outlined in Figures 21d and 21e, there are generally two symmetric orientations that are best suited for the formation of extended structures composed of Dn symmetric nodes. These alternative orientations correspond to streptavidin alignments where either the streptavidin z-axis (Fig 21d, "H" or horizontal) or y-axis (Fig 21e "V" or vertical) is oriented parallel to the node Dn or z-axis. 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.
[00357] 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. As examples, Figure 12a illustrates the case where the streptavidin z-dyad axis is parallel to the z-dyad axis of a D2 node, while Fig 12c illustrates the example where the streptavidin y-dyad axis is parallel to the z-dyad axis of a D2 node.
[00358] 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. For example, 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. By examination of the 3-dimensional atomic coordinates of the node template protein using a computer method (Lee and Richards, 1971) it is possible to compile a list of the coordinates of the atoms and/or amino acid residue side chains that lie on the surface of a protein. Specific side chain atoms can be selected for reference in a list; e.g. Ca backbone carbon atoms for GIy residues, and Cβ side chain atoms for all other amino acid residues. 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. Alternatively, the Cβ 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.
[00359] Several of the multimeric nodes shown in this application are shown with embedded bounding boxes (in projection) along node dyad axes.
[00360] For D2 nodes with appropriate geometrical features, 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 β (Figl3a), depending on the total angular twist desired in the final linear coupler. Figure 13ab shows a special case where a D2 node with nearly tetrahedral symmetry is modified to produce a D2 linear coupler that orients the terminal streptavidin molecules with β=90 degrees.
[00361] Additional nodes, appropriate for the formation of extended 3-dimensional lattices, 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. Note that this process generally produces node subunit proteins that incorporate only a single site-specific modification, so that the streptavidin tetramer in the complex forms a symmetric link between node subunits oriented by a dyad axis of symmetry.
[00362] 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.
[00363] For any given modeled complex it may be possible, using computational and modeling methods (Jones 1990, Case et. al. 1995), to further improve the complex through the introduction of site-specific modifications in streptavidin or the template node to improve electrostatic complementarity, van der Waals interactions or other features that will improve the stability or functionality of the complex.
Examples of Specific Node Embodiments
[00364] The sequence and symmetry specifications of the several embodiments described below are detailed in Table 2. 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. Depending on the structural environment and role of the cysteine side chain in the template protein, 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. Note that many proteins expressed in E.coli are modified by addition of an N-terminal methionine residue, which is by often counted as residue "zero" of the polypeptide chain for structural purposes and so designated in Protein Data Bank (pdb) coordinate files. In any case, 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. [00365] Specific 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. For Cn nodes, these are enumerated as "H" and "V", where the y-axis or z-axis of streptavidin is parallel to the node Cn axis, respectively (Fig 21b,c). Since streptavidin tetramer makes an asymmetric interaction with Cn node, there are potentially a large number of possible complementary interactions that are feasible for a Cn-node streptavidin-strut interaction. Table 1 generally shows one "H" interaction that analysis suggests provides the best steric and charge complimentarity between streptavidin and the node surface. [00366] As noted above, 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. Although it is not physically possible to bind streptavidin in both "H" and "V" orientations simultaneously, there nevertheless arise a large number of combinations for node streptavidin complexes that are possible as outlined in Table 2 G,H,I, J. D3 nodes are special, since interactions made at one end of the dyad axis are different from the other (Fig 15e, Fig 17), so that there are a total of 4 possible streptavidin node interaction geometries, producing a total of 8 strut-node interaction patterns (Table 2 KLM). For D4 symmetric nodes (Fig 15f, Fig 18) there are two independent dyad axes, giving a total 8 different streptavidin substitution patterns (Table 2 NOP). For tetrahedral nodes, all the dyad axes are symmetrically equivalent, so that there are only 2 possible node:streptavidin orientations possible (Table 2Q).
[00367] Three-Fold (CS) Symmetric Planar 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. (In this an succeeding figures of such complexes, the 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.) Although there are several potential sites of interaction between the surface of lthj and streptavidin that can be generated using the methods described above, 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).
[00368] Table 2C also provides a node specification of for C3 trimeric planar node based on the Ij 5s protein described above.
[00369] Single Chain Variants of Three-Fold (C3) Symmetric Planar Node: 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.
[00370] Four-Fold (C4) Symmetric Planar Node: 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. Although there are several potential sites of interaction between the surface of lvcg and streptavidin that can be generated using the methods described above, 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).
[00371] Three-Fold (C3) Symmetric Polyhedral Node: Figure 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.
[00372] Five-Fold (C5) Symmetric Polyhedral Node: Figure 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.
[00373] 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. surface cysteine residues), allowing in situ functionalization with biotin to allow the attachment of additional streptavidin tetramers. Figures 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. The specifications for the 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.
[00374] D2 Nodes: Figures 29a,b show stereoscopic views of a tetrameric D2 node based on the lmal node template in schematic and space filling representation respectively. There are 6 streptavidin tetramers bound to the node, two along each symmetrically independent dyad axis. 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. [00375] 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. In Figures 30a,b streptavidin tetramers are oriented with their z-axes parallel to one of the D2 node dyad axes. In Figures 30c,d streptavidin tetramers are oriented with their y-axes parallel to the same D2 node dyad axis. Since there are a total of three independent dyad axes, there are a total of six alternative linear strut complexes that can be formed with a D2 node and two streptavidin tetramers to form linear struts. The specifications for these nodes are included in Table 2H. Table 21 and 2J respectively provide additional specifications for D2 symmetric nodes based on the Into and lrtw node templates.
[00376] 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. Note that the 2 "poles" of the D3 dyad axes (Fig 15e) 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. [00377] D4 Nodes: Figures 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.
[00378] 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.
Examples of One-Dimensional, Two-Dimensional and Three Dimensional Assemblies Constructed with Streptavidin Struts and Nodes of Different Symmetry.
[00379] The following describes representative nanoassemblies that can be constructed using the node and strut components described above. Many more possibilities exist than are shown, although the structures outlined fall into several basic classifications.
[00380] One Dimensional Structures: 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. see Fig 28) by the introduction of cysteine residues along one orthogonal dyad axis to allow the biotinylation of the strut after it is incorporated in a nanostructure (Fig 34c). [00381] 2-Dimensional Radial Structures: 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.
[00382] 2-Dimensional Lattices: 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.
[00383] 2-Dimensional Polygon Structures: Figure 37 schematically shows examples of
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. [00384] 3-Dimensional Radial Structures: 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.
[00385] 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. Given the great variety of known carbon-based "buckyball" geometries (Weber 1999), it is probable that a corresponding variety of protein-based nanostructures can be generated. 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. In addition, 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.
[00386] 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. Consequently, when a streptavidin tetramer bridges two such nodes, they are rotated 90 degrees relative to each other. 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). [00387] 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). 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. Consequently, when a streptavidin tetramer bridges two such nodes, they are rotated 90 degrees relative to each other. Figure 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).
[00388] 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. In Figure 40c, the central white dot represents the location of a node.
[00389] 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. In addition, there are many applications where the ability to immobilize magnetic centers, charge, chromophoric groups, or other inorganic, organic, or biological groups at high density and with controlled geometry can lead to useful applications such as batteries, capacitors, non-linear optical materials, data storage, and other devices.
Examples of Nanostructural Assemblies for Nanoscale Patterning and Resist Masks
[00390] In addition to applications where the protein components of nanoscale assemblies play a functional role, 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. Alternatively, 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.
[00391] 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. Upon addition (e.g. by contacting the surface with a solution containing the nanostructure node trimer) of a trimeric node construct functionalized with bound biotin groups and modified at one terminus with a reactive moiety (binding function) that enables coupling to the nucleation site on the substrate, 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. [00392] 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. Here a substrate (Fig 42a) is patterned with an array of nucleation sites. 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. Following the steps of addition of the node proteins to the surface (Fig 42b) and addition of the streptavidin-incorporating struts, a patterned array (Fig 42c) is produced. Figure 42d shows a section of the patterned surface at the section line ε in Figure 42c.
[00393] 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 εl. 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 ε2 in Figure 43c). For example, 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). The resulting patterned surface (Fig 43c) 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).
[00394] 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 (Fig 44g) 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).
[00395] Additional example of finite or periodic 2-dimensional proteinaceous nanostructural assemblies that can serve as patterning templates on surfaces are described above and schematically illustrated in Figures 35, 36, and 37.
[00396] 3 -dimensional, as well as 2-dimensional, proteinaceous nanostructure assemblies can be used as nanoscale patterning elements. The structures can be coated as outlined in the process of Figure 43 or, alternatively, serve as a 3-dimensional resist to form a negative of the proteinaceous nanostructural assembly. For example, Figure 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). For example, 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 (first 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). For example, such "negative" structures incorporating nanoscale channels have potential utility as components in nanofluidics systems. 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. For example, three-dimensional nanoscale assemblies made of metal or semiconductor materials have potential utility as components in semiconductor or MEMS applications.
[00397] Additional examples of finite or periodic 3 -dimensional proteinaceous nanostructural assemblies are described above and some are schematically illustrated in Figure 40. Such 3-dimensional structures with nano-dimensional features can have utility as optical or physical waveguides or filters, nanofluidic devices, or in other semiconductor or MEMS applications.
Terms and Definitions
[00398] 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. For example, a Cn symmetric multimeric protein can be formed of n subunits placed about a common axis. For example, a C3 symmetric multimeric protein can be formed of 3 subunits placed about a common axis. For example, 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. For example, a D4 symmetric multimeric protein can be formed of 8 subunits, where each of 4 pairs of subunits are placed about a common axis. For example, 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. For example, 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. For example, 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. [00399] 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). Thus, 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.
[00400] For example, the amino acid residues of a polypeptide subunit can be in a single polypeptide sequence.
[00401] 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. Alternatively, 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. Even though the polypeptide subunits are covalently linked through the 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. When these polypeptide subunits undergo multimerization, 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.
[00402] 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.
[00403] Features, such as polypeptide subunits of a multisubunit node polypeptide, can be related by a symmetry. Unless otherwise stated, 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. For example, 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. For example, if one subunit of a trimer has one amino acid in the polypeptide sequence from which it is formed that is different than the corresponding amino acid in the polypeptide sequences of the other two subunits, but the folding of all the subunits is similar, the trimer still can be considered to have three-fold rotational symmetry.
[00404] 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. For example, 2-iminobiotin is a derivative of biotin. The structure of 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. Examples of nucleobases include cytosine, guanine, adenine, thymine, uracil, 5-methylcytosine, ribothymidine, hypoxanthine, xanthine, 7- methylguanine, and 5,6-dihydrouracil. Examples of nucleobase derivatives include isoguanine, isocytosine, 2-amino-6-(2-thienyl)purine, pyrrole-2-carbaldehyde, and acycloguanosine (Aciclovir). Examples of nucleosides include adenosine, guanosine, 5'-methyluridine, uridine, cytidine, deoxynucleosides, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine, i nosine, xanthinosine , 7-methylguanosine, pseudouridine, dihydrouridine, 5- methylcytidine, dideoxynucleosides. Examples of nucleoside derivatives include azidothymidine (Zidovudine), didanosine, vidarabine, cytosine arabinoside (cytarabine), emtricitabine (Emtriva), lamivudine, dideoxycytidine (zalcitabine), abacavir (Ziagen), stavudine (Zerit), idoxuridine, trifluridine (Viroptic). Examples of nucleotides 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 Examples of nucleotide derivatives include tenofovir disoproxil fumarate (Viread), adefovir dipivoxil (Preveon), and adenosine triphosphate derivatives, such as adenosine 5'-(gamma-thiotriphosphate). Similarly, if a residue of an initial polypeptide is replaced with a different residue, the resultant polypeptide is a derivative of the initial polypeptide. If 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. For example, 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. For example, a derivative of avidin is deglycosylated avidin (NeutrAvidin). An analog of a molecule is included within the term derivative. [00405] In the context of a streptavidin strut, the term streptavidin derivative strut, is to be understood as including struts formed of a streptavidin derivative, struts that include streptavidin
(wherein the streptavidin may or may not be covalently bonded to other portions of the strut), and struts that include a streptavidin derivative (wherein the streptavidin derivative may or may not be covalently bonded to other portions of the strut).
[00406] When a chemical or biochemical group is mentioned, derivatives and analogs of that chemical or biochemical group are also implied. For example, if biotin is recited, 2- iminobiotin is also implied.
[00407] 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.
[00408] A binding function of a polypeptide sequence (such as a polypeptide extension) 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.
[00409] 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.
[00410] A nanostructure strut can bind covalently or non-covalently to a specific binding site of a nanostructure node multimeric protein.
[00411] 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. For example, a nanostructure node multimeric protein can include a ligand binding pocket. A nanostructure strut can bind to the ligand binding pocket. For example, the nanostructure strut can include a region of an immunoglobulin that binds to the ligand binding pocket of the nanostructure node multimeric protein. For example, 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.
[00412] A bridge molecule can serve to attach two other molecules, such as proteins. For example, 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.
[00413] 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. For example, a bindable subunit can include a specific binding site to which a nanostructure strut, e.g., a streptavidin-containing nanostructure strut, can bind.
[00414] A non-bindable polypeptide subunit, for example, of a multimeric protein, can be incapable of binding to another molecule, such as a protein. For example, a non-bindable subunit may lack a specific binding site to which a nanostructure strut, e.g., a streptavidin- containing nanostructure strut, can bind.
[00415] 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. colϊ) to induce the host organism to express the protein.
EXAMPLES
Example 1: Expression of 3-Fold Symmetric Node
[00416] Synthetic genes and expression vectors for a 3 -fold symmetric (C3) protein to be used as a node were constructed. For the synthetic gene and expression vector sequences shown, the vector sequence is in lower case with the promoter underlined and the ribosome binding site in italics, and the open reading frame is in upper case with the initiating Methionine and Stop codons in bold. Amino acid sequences are provided using the standard one letter representation for each amino acid.
Example IA: EXP14Q3193C2
[00417] E. coli cells BL21 Star™ (DE3) pLysS with expression vector EXP14Q3193C2
(represented in Fig. 46A) 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 ODβoo 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 0 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 4 hours at 25 0 C to an OD 60 O of 2.69. 0.6 g of cells were collected by low speed centrifugation.
[00418] In a second batch, E. coli cells BL21 Star™ (DE3) pLysS with expression vector
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.
[00419] In a third batch E. coli cells BL21 Star™ (DE3) pLysS with expression vector
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.
[00420] In a fourth batch E. coli cells BL21 Star™ (DE3) pLysS with expression vector
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 ό 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.
[00421] 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
C-terminus. Thus, 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.
[00422] The gene nucleotide sequence for the synthetic sequence EXP14Q3193C2 incorporated into the EXP14Q3193C2 expression vector was: gaaggagatatacatATGC AAGAGATTACCGTTGACGAATTTAGCAATATCCGTGAAAACC
CGGTTACCCCGTGGAACCCGGAACCGAGCGCCCCCGGTTATTGACCCGACCGCCTA
TATTGACCCGGAAGCAAGCGTGATTGGTGAAGTTACGATTGGCGCAAATGTTATGG
TTAGCCCGATGGCGAGCATTCGCAGCGATGAAGGTATGCCGATTTTTGTGGGTTGTC
GTAGCAATGTTCAAGATGGTGTTGTCCTGCACGCACTGGAAACGATTAATGAAGAA
GGTGAACCGATTGAAGATAATATTGTTGAAGTTGATGGCAAAGAATACGCAGTTTA
TATTGGTAATAATGTTAGCCTGGCCCATCAGAGCCAAGTCCACGGTCCGGCCGCAG
GCGATGATACGTTTATTGGCATGCAAGCGTTCGTTTTTAAAAGCAAAGTGGGTAATA
ATGCAGTTCTGGAACCGCGTAGCGCAGCGATTGGTGTCACGATCCCGGATGGTCGC
TATATCCCGGCCGGTATGGTCGTTACCAGCCAAGCAGAAGCAGACAAACTGCCGGA
AGTCACCGATGATTACGCCTATAGCCATACCAATGAAGCCGTTGTTTGTGTGAATGT
TCATCTGGCGGAAGGTTACAAAGAAACGATTGAAGGCCGTCATCACCACCACCCAC
CACTAAgacccagctttcttgtacaaagtggtcccc. [SEQ ID NO 22]
[00423] The corresponding amino acid sequence of the one polypeptide chain of the synthetic protein produced by the EXP14Q3193C2 expression vector was: MQEITVDEFSNIRENPVTPWNPEPSAPVIDPTAYIDPEASVIGEVTIGANVMVSPMASIR S DEGMPIFVGCRSNVQDGVVLHALETINEEGEPIEDNIVEVDGKEYA VYIGNNVSLAHQS QVHGPAAVGDDTFIGMQAFVFKSKVGNNAVLEPRSAAIGVTIPDGRYIPAGMVVTSQA EADKLPEVTDDYAYSHTNEAVVCVNVHLAEGYKETIEGRHHHHHH [SEQ ID NO 25]
Example IB - EXP14Q3193C3
[00424] E. coli cells BL21 Star™ (DE3) with expression vector EXP14Q3193C3
(represented in Fig. 46B) 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 °C to an ODβoo 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 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.
[00425] In a second batch, E. coli cells BL21 Star™ (DE3) with expression vector
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.
[00426] In a third batch E. coli cells BL21 Star™ (DE3) with expression vector
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.
[00427] In a fourth batch E. coli cells BL21 Star™ (DE3) with expression vector
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.
[00428] In a fifth batch E. coli cells BL21 Star™ (DE3) pLysS with expression vector
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.
[00429] In a sixth batch E. coli cells BL21 Star™ (DE3) pLysS with expression vector
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.
[00430] In a seventh batch E. coli cells BL21 Star™ (DE3) pLysS with expression vector
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.
[00431] In an eighth batch E. coli cells BL21 Star™ (DE3) pLysS with expression vector
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.
[00432] In a ninth batch E. coli cells BL21 Star™ (DE3) pLysS with expression vector
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.
[00434] 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. [00435] 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). Within each polypeptide subsequence corresponding to a polypeptide chain in the native protein, the following substitutions were made: Residues Asp 70 and Tyr200 were changed to Cys; and Cysl48 was changed to Ala. A His tag that can be cleaved by the Factor Xa protease was added to the C- terminus of the single polypeptide chain. Thus, 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.
[00436] The gene nucleotide sequence of sequence EXP14Q3193C3 incorporated into the
EXP14Q3193C3 expression vector was: ggggacaagtttgtacaaaaaagcaggcaccgaagg«gfltotocαtATGGATGAATT TAGCAATATCCGCGA
AAATCCGGTGACCCCGTGGAATCCGGAACCGAGCGCCCCCGGTTATTGATCCGACG
GCATACATCGACCCGGAAGCCAGCGTGATTGGTGAAGTTACCATCGGCGCCAATGT
TATGGTCAGCCCGATGGCGAGCATCCGCAGCGATGAAGGCATGCCGATCTTTGTGG
GCTGTCGTAGCAATGTGCAGGATGGCGTTGTTCTGCACGCGCTGGAAACCATTAAT
GAAGAAGGCGAACCGATTGAAGACAATATTGTTGAAGTGGACGGTAAGGAATATG
CAGTGTACATCGGTAACAACGTCAGCCTGGCCCATCAGAGCCAAGTCCATGGTCCG
GCCGCCGTGGGCGATGATACCATTGGCATGCAAGCGTTCGTGTTTAAAAGCAAAGT
TGGCAATAATGCAGTTCTGGAACCGCGCAGCGCGGCGATCGGCGTGACCATTCCGG
ATGGTCGTTACATCCCGGCCGGCATGGTGGTCACCAGCCAAGCGGAGGCCGATAAA
CTGCCGGAAGTCACCGATGACTATGCCTATAGCCACACCAATGAGGCCGTCGTGTG
CGTGAACGTTCATCTGGCCGAAGGTTATAAAGAAACGGGTGGTAGCGGCGGCGGCG
ATGAATTTAGCAATATCCGCGAAAATCCGGTGACCCCGTGGAATCCGGAGCCGAGC
GCACCGGTTARRGATCCGACCGCATATATTGATCCGGAGGCCAGCGTTATCGGCGA
AGTTACGATCGCGAATGTTATGGTGAGCCCGATGGCGAGCATTCGCAGCGATGAGG
GTATGCCGATTTTTGTGGGCTGCCGTAGCAATGTGCAAGATGGTGTGGTCCTGCACG CACTGGAGACGATTAACGAGGAAGGTGAACCGATCGAGGACAACATTGTCGAAGT
GGACGGTAAGGAGTATGCGGTGTATATCGGCAACAACGTTAGCCTGGCCCACCAGA
GCCAGGTGCACGGCCCGGCAGCAGTGGGCGATGACACGTTTATTGGCATGCAGGCG
TTCGTTTTCAAAAGCAAAGTTGGCAATAACGCAGTTCTGGAACCGCGTAGCGCAGC
GATTGGCGTTACCATCCCGGATGGCCGTTATATCCCGGCCGGTATGGTCGTTACGCA
GGCGGAAGCAGATAAACTGCCGGAAGTTACCGATGACTATGCCTATAGCCATACCA
ATGAGGCAGTTGTTTGTGTCAATGTCCATCTGGCGGAAGGCTACAAAGAAACGGGT
GGTAGCGGTGGCGGTGATGAATTCAGCAACATCCGTGAAAACCCGGTGACCCCGTG
GAACCCGGAACCGAGCGCGCCGGTCATTGATCCGACCGCATATATCGATCCGGAGG
CAAGCGTCATTGGCGAAGTTACGATTGGCGCCAACGTGATGGTCAGCCCGATGGCC
AGCATCCGCAGCGATGAAGGCATGCCGATTTTTGTTGGTTGCCGTAGCAACGTTCAG
GATGGCGTGGTCCTGCACGCACTGGAAACCATTAACGAAGAAGAGCCGATTGAAGA
TAACATCGTTGAGGTCGACGGTAAAGAATATGCCGTGTATATCGGCAACAACGTTA
GCCTGGCCCATCAAAGCCAAGTTCATGGTCCGGCCGCGGTTGGTGATGACACGTTC
ATTGGCATGCAGGCGTTTGTGTTTAAGAGCAAAGTGGGTAATAATGCCGTTCTGGA
GCCGCGCAGCGCCGCAATCGGCGTCACCATCCCGGACGGTCGCTACATTCCGGCAG
GCATGGTCGTGACCAGCCAAGCCGAAGCGGACAAACTGCCGGAAGTCACCGATGAT
TAGCATACAGCCACACCAACGAGGCGGTCGTGTGTGTTAATGTGCATCTGGCGGAA
GGTTATAAAGAAACGATTGAAGGCCGTCATCACCACCATCATTGAacccagctttct tgtacaa agtggtgatgatccggctgctaacaaagcccgaaaggaagctga [SEQ ID NO: 23]
[00437] The corresponding amino acid sequence produced by the EXP14Q3193C3 expression vector was:
MDEFSNIRENPVTPWNPEPSAPVIDPTAYIDPEASVIGEVTIGANVMVSPMASIRSD EGM
PIFVGCRSNVQDGVVLHALETINEEGEPIEDNIVEVDGKEYAVYIGNNVSLAHQSQV HG
PAAVGDDFTIGMQAFVFKSKVGNNAVLEPRSAAIGVTIPDGRYIPAGMVVTSQAEAD K
LPEVTDDYAYSHTNEAVVCVNVHLAEGYKETGGSGGGDEFSNIRENPVTPWNPEPSA P
VIDPTAYIDPEASVIGEVTIGANVMVSPMASIRSDEGMPIFVGCRSNVQDGVVLHAL ETI
NEEGEPIEDNIVEVDGKEYAVYIGNNVSLAHQSQVHGPAAVGDDTFIGMQAFVFKSK V
GNNAVLEPRSAAIGVTIPDGRYIPAGMVVTSQAEADKLPEVTDDYAYSHTNEAVVCV N
VHLAEGYKETGGSGGGDEFSNIRENPVTPWNPEPSAPVIDPTAYIDPEASVIGEVTI GAN
VMVSPMASIRSDEGMPIFVGCRSNVQDGVVLHALETINEEGEPIEDNIVEVDGKEYA VYI GNNVSLAHQSQVHGPAAVGDDTFIGMQAFVFKSKVGNNAVLEPRSAAIGVTIPDGRYIP
AGMVVTSQAEADKLPEVTDDYAYSHTNEAVVCVNVHLAEGYKETIEGRHHHHHH
[SEQ ID NO 26]
Example 1C - EXP14Q3193C4
[00438] E. coli cells BL21 Star™ (DE3) with expression vector EXP14Q3193C4
(represented in Fig. 46C) 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 ODόoo 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 6O o of 0.963, 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 7.57. 0.7 g of cells were collected by low speed centrifugation.
[00439] In a second batch E. coli cells BL21 Star™ (DE3) with expression vector
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.
[00440] 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. In the third polypeptide subsequence, that is, the subsequence before the C-terminus, 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
C-terminus of the single polypeptide chain. Thus, 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 fϊinctionalization (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.
[00441] The gene nucleotide sequence of sequence EXP14Q3193C4 incorporated into the
EXP14Q3193C4 expression vector was: cgatgcgtccggcgtagaggatcgagatctcgatcccgcgaaattaatacgactcactat agggagaccacaacggtttccctctagatca caagtttgtacaaaaaagcaggcaccgaaggαgαtotocotATGGATGAATTTAGC AATATTCGCGAAAAC
CCGGTTACCCCGTGGAACCCGGAACCGAGCGCGCCGGTTATCGACCCGACGGCCTA
CATTGATCCGGAGGCAAGCGTGATTGGTGAAGTGACGATTGGTGCAAATGTCATGG
TGAGCCCGATGGCGAGCATTCGTAGCGATGAAGGTATGCCGATTTTCGTTGGTTGTC
GTAGCAATGTTCAAGATGGTGTTGTTCTGCACGCCCTGGAAACCATTAATGAAGAA
GGTGAGCCGATTGAAGACAACATCGTTGAAGTTGATGGTAAAGAATACGCGGTTTA
TATCGGCAACAACGTCAGCCTGGCACATCAGAGCCAAGTTCATGGTCCGGCAGCAG
TGGGCGATGATACGATTGGTATGCAAGCATTCGTTTTTAAAAGCAAAGTTGGTAAT
AATGCAGTTCTGGAACCGCGCAGCGCAGCAATTGGTGTTACCATTCCGGATGGTCG
TTATATCCCGGCCGGTATGGTGGTGACGAGCCAGGCGGAAGCAGATAAACTGCCGG
AAGTGACGGATGATTATGCCTATAGCCATACCAATGAAGCAGTCGTGTGTGTTAAC
GTGCACCTGGCCGAAGGTTACAAAGAAACGGGCGGTGGTAGCGGTGGCGGCGATG
AATTTAGCAATACCGTGAAAACCCGGTTACCCGTGGAATCCGGAACCGAGCGCACC
GGTTATTGATCCGACGGCATATATCGACCCGGAGGCAAGCGTGATTGGCGAAGTTA
CGGGCGCAAATGTGATGGTTAGCCCGATGGCCAGCATTCGTAGCGATGAAGGCATG
CCGATTTTTGTGGCTGCCGCAGCAATGTTCAAGATGGTGTTGTCCTGCACGCACTGG
AGACCATCAATGAAGAAGGTGAACCGATTGAAGATAACATCGTCGAAGTTGACGGC
AAAGAATATGCGGTGTATATTGGCAATAATGTCAGCCTGGCACATCAAAGCCAAGT
TCACGGTCCGGCAGCAGTGGGCGATGATACCTTTATTGGCATGCAAGCGTTTGTTTT
CAAAAGCAAAGTCGGCAATAATGCAGTTCTGGAACCGCGCGCAGCGCAGCGATTGG CGTCACGATCCCGGATGGTCGTTATATTCCGGCCGGCATGGTGGTGAGCCAGGCAG
AAGCAGATAAACTGCCGGAAGTGACCGATGACTATGCCTATAGCCATACGAACGAA
GCCGTTGTTTGCGTGAACGTGCACCTGGCAGAAGGCTACAAAGAAACCGGTGGTGG
CAGCGGCGGCGGTGATGAATTCAGCAATATTCGCGAAAATCCGGTCACCCCGTGGA
ATCCGGAACCGAGCGCCCCGGTCATTGACCCGACGGCATATATTGATCCGGAAGCA
AGCGTTATTGGTGAAGTTACGATTGGTGCAAACGTGATGGTGAGCCCGATGGCGAG
CATTCGCAGCGATGAGGGCATGCCGATTTTTGTGGGCGATCGCAGCAATGTTCAAG
ATGGTGTTGTCCTGCACGCCCTGGAAACCATCAATGAAGGCGAACCGATTGAAGAC
AATATTGTGGAAGTCGATGGTAAAGAATACGCAGTCTATATTGGTAATAATGTTAG
CCTGGCACATCAGAGCCAAGTCCACGGTCCGGCCGCAGTGGGTGATGACAGTTTAT
TGGTATGCAAGCATTTGTGTTTAAAAGCAAAGTCGGTAACAATGCAGTTCTGGAAC
CGCGCAGCGCAGCAATCGGCGTTACGATCCCGGATGGCCGTTATATCCCGGCGGGT
ATGGTGGTTACGAGCCAAGCAGAAGCGGATAAACTGCCGGAAGTTACGGATGATTA
TGCCTATAGCCATACGAACGAAGCGGTTGTCTACGTTAACGTGCATCTGGCGGAGG
GTTACAAAGAAACGATTGAGGGTCATCATCACCATCATCATTGAaacccagctttc
[SEQ ID NO 24]
[00442] The corresponding amino acid sequence produced by the EXP14Q3193C4 expression vector was:
MDEFSNIRENPVTPWNPEPSAPVIDPTAYIDPEASVIGEVTIGANVMVSPMASIRSD EGM
PIFVGCRSNVQDGVVLHALETINEEGEPIEDNIVEVDGKEYAVYIGNNVSLAHQSQV HG
PAAVGDDTFIGMQAFVFKSKVGNNAVLEPRSAAIGVTIPDGRYIPAGMVVTSQAEAD K
LPEVTDDYAYSHTNEAVVCVNVHLAEGYKETGGSGGGDEFSNIRENPVTPWNPEPSA P
VIDPTAYIDPEASVIGEVTIGANVMVSPMASIRSDEGMPIFVGCRSNVQDGVVLHAL ETI
NEEGEPIEDNIVEVDGKEYAVYIGNNVSLAHQSQVHGPAAVGDDTFIGMQAFVFKSK V
GNNA VLEPRSAAIGVTIPDGRYIPAGMVVTSQAEADKLPEVTDDYA YSHTNEAVVCVN
VHLAEGYKETGGSGGGDEFSNIRENPVTPWNPEPSAPVIDPTAYIDPEASVIGEVTI GAN
VMVSPMASIRSDEGMPIFVGDRSNVQDGVVLHALETINEEGEPIEDNIVEVDGKEYA VY
IGNNVSLAHQSQVHGPAAVGDDTFIGMQAFVFKSKVGNNAVLEPRSAAIGVTIPDGR YI
PAGMVVTSQAEADKLPEVTDDYAYSHTNEAVVYVNVHLAEGYKETIEGRHHHHHH
[SEQ ID NO 27]
[00443] Thus, all of the proteins expressed by the vectors EXP14Q3193C2 (Example IA),
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.
Example 2: Expression of 4-Fold Symmetric Node
[00444] Synthetic genes and expression vectors for a 4-fold symmetric protein to be used as a node were constructed. The 4-fold node protein produced was IPP (isopentenyl pyrophosphate) isomerase with the following amino acid changes: the cysteine (C) at the 14 position was replaced by alanine; the serine (S) at the 44 position was replaced by cysteine (C); the threonine (T) at the 49 position was replaced by cysteine (C); and the cysteine (C) at the 237 position was replaced by serine (S). Furthermore, a histidine tag was added at the N-terminus. [00445] E. coli cells BL21 Star™ (DE3) pLysS with expression vector EXP14Q3164
(represented in Fig. 47) were cultured in 50 mL Terrific Broth supplemented with 0.1 mg/mL ampicillin. The culture was grown overnight at 37 0 C with rotation at 225 rpm. The culture was induced with 0.4 mM IPTG, then grown for 18.25 hours at 25.3 0 C to an OD 600 of 21.28. 2.1 g of cells were collected by low speed centrifugation.
[00446] In a second batch, E. coli cells BL21 Star™ (DE3) pLysS with expression vector
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 Star™ (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 ODβoo 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.
[00448] In a fourth batch, E. coli cells BL21 Star™ (DE3) pLysS 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 0.761 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.
[00449] In a fifth batch, E. coli cells BL21 Star™ (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.
[00450] In a sixth batch, E. coli cells BL21 Star™ (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.
[00451] In a seventh batch, E. coli cells BL21 Star™ (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 °C to an OD 6 oo of 14.06. 1.1 g of cells were collected by low speed centrifugation.
[00452] In an eighth batch, E. coli cells BL21 Star™ (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.
[00453] In a ninth batch, E. coli cells BL21 Star™ (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 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 Star™ (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.
[00455] In an eleventh batch, E. coli cells BL21 Star™ (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.
[00456] In a twelfth batch, E. coli cells BL21 Star™ (DE3) pLysS with expression vector
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.
[00457] 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 Star™ (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. 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. Following two 15 mL washes each of a solution of 50 mM sodium phosphate buffer at pH 8.0 and 0.5 M NaCl with 20 and 40 mM imidazole, 4-fold node protein was eluted from the Ni agarose using a solution of 250 mM imidazole, 0.5 M NaCl, and 50 mM sodium phosphate buffer at pH 8.0. Centrifugal concentrators were used to exchange the buffer to a solution of 25 mM sodium phosphate and 0.1 M NaCl at pH 7.4, and then concentrate the 4-fold node protein to ~ 5 mg/mL, as determined spectrophotometrically.
[00458] A second, alternative isolation procedure was carried out in a similar manner as the first isolation procedure, except that E. coli cells BL21 Star™ (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 HALT™ 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. [00459] 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. [00460] 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.
[00461] 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.
ATGAGCTATTATCACCATCATCATCATCATGACTATGATATCCCGACCACCGAAAAT
CTGTATTTCCAAGGTATGAACATCCGTGAACGCAAACGTAAACATCTGGAAGCGGC
CCTGGAAGGTGAAGTTGCATATCAAAAAACGACCACCGGTCTGGAAGGTTTCCGTC
TGCGCTATCAAGCCCTGGCCGGTCTGGCACTGTGTGAAGTCGATCTGTGTACCCCGT
TTCTGGGTAAAACGCTGAAAGCCCCGTTCCTGATTGGTGCAATGACGGGTGGTGAA
GAAAACGGTGAACGTATCAATCTGGCACTGGCCGAAGCCGCCGAAGCCCTGGGTGT
GGGTATGATGCTGGGTAGCGGCCGTATTCTGCTGGAGCGTCCGGAAGCCCTGCGTA
GCTTTCGTGTCCGTAAAGTTGCACCGAAAGCACTGCTGATTGCGAATCTGGGTCTGG
CACAACTGCGTCGTTATGGTCGCGATGATCTGCTGCGCCTGGTCGAAATGCTGGAA
GCCGATGCCCTGGCCTTTCATGTTAATCCGCTGCAAGAAGCAGTCCAACGTGGTGAT
ACCGATTTCCGTGGTCTGGTTGAACGTCTGGCCGAACTGCTGCCGCTGCCGTTCCCG
GTTATGGTCAAAGAAGTTGGCCACGGTCTGAGCCGTGAAGCAGCCCTGGCACTGCG
TGATCTGCCGCTGGCCGCAGTTGATGTTGCAGGTGCGGGTGGTACGAGCTGGGCAC
GTGTTGAAGAATGGGTTCGTTTTGGTGAAGTCCGCCACCCGGAACTGAGCGAAATT GGCATTCCGACGGCCCGCGCGATTCTGGAAGTTCGTGAAGTTCTGCCGCATCTGCCG
CTGGTTGCAAGCGGCGGTGTTTATACCGGCACCGATGGCGCAAAAGCACTGGCACT
GGGCGCCGATCTGCTGGCGGTTGCGCGTCCGCTGCTGCGCCCGGCCCTGGAAGGTG
CAGAACGTGTTGCGGCGTGGATTGGTGATTACCTGGAAGAACTGCGTACCGCACTG
TTTGCGATTGGTGCACGTAATCCGAAAGAAGCCCGCGGTCGTGTCGAACGCGTCTA
A [SEQ ID NO 28]
[00462] The corresponding amino acid sequence of the protein produced by the
EXP14Q3164 expression vector was as follows. The amino acid sequence is provided using the Standard one letter representation for each amino acid.
MSYYHHHHHHDYDIPTTENLYFQGMNIRERKRKHLEAALEGEVAYQKTTTGLEGFRL R
YQALAGLALCEVDLCTPFLGKTLKAPFLIGAMTGGEENGERINLALAEAAEALGVGM M
LGSGRILLERPEALRSFRVRKVAPKALLIANLGLAQLRRYGRDDLLRLVEMLEADAL AF
HVNPLQEAVQRGDTDFRGLVERLAELLPLPFPVMVKEVGHGLSREAALALRDLPLAA V
DVAGAGGTSWARVEEWVRFGEVRHPELSEIGIPTARAILEVREVLPHLPLVASGGVY TG
TDGAKALALGADLLAVARPLLRPALEGAERVAAWIGDYLEELRTALFAIGARNPKEA R
GRVERV [SEQ ID NO 29]
[00463] This amino acid sequence of the 4-fold node protein produced by the
EXP14Q3164 expression vector was confirmed by mass spectrometry.
Example 3: Biotinylation of a 4-Fold Symmetric Node
[00464] 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-dPEG™3-biotin, Quanta BioDesign, Powell OH) and N-d- biotinamido-N'-(3-maleimidopropionamido)-3,6,9,12,15,18,21,2 4,27,30,33- undecaoxapentatriacontane- 1,35 -diamine (MAL-dPEG™l l- biotin, Quanta BioDesign, Powell OH) or N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio)propionarnide (biotin-HPDP, PierceNet) by dialysis (Spectra/Por, 10 000 MW cutoff dialysis tubing, Cole-Palmer). 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 . 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. For maleimide-reactive reagents (MAL-dPEG™l l- biotin and MAL-dPEG™3-biotin) 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). For example, 2.7 mg MAL-dPEG™3 -biotin was dissolved in 0.05 niL PBS pH 7.4, 3.3 mg MAL-dPEG™l 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-dPEG™3-biotin:node were 2.5:1 and 3.6:1, and the molar ratios of MAL-dPEG™l 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).
Example 4: Formation of Node:Streptavidin (NODE:SAV) Complexes
[00465] 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 . Using this procedure, 0.2 mL of a 9.4 mg/mL SAV solution and 0.12 mL of a 28.5 mg/mL SAV solution were prepared. 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 μL 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).
[00466] 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-dPEG™3-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. 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. 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.
[00467] 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. For example, 0.30 mL of Ni-NTA agarose resin (Invitrogen) was equilibrated with PBS pH 7.4. After 0.9 mg MAL-dPEG™ l l-biotin derivatized node was added to the resin and allowed to equilibrate for 2 hrs, 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. In a separate reaction, 0.30 mL of Ni-NTA agarose resin (Invitrogen) was equilibrated with PBS pH 7.4. After 0.9 mg biotin-HPDP derivatized node was added to the resin and allowed to equilibrate for 2 hrs, 0.06 mg SAV in PBS pH 7.4 was added, and the resin with derivatized node and SAV 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, 0.37 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.
Example 5:
[00468] 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
(Invitrogen) molecular weight standards are in lanes 5 and 30, and correspond to standardized proteins of 260, 160, 110, 80, 60, 50, 40, 30 and 20 kDa.
[00469] In this study, the streptavidin tetramer subunits, which were obtained by fermentation from Streptomyces avidinii, had a range of molecular weights. For the purpose of this study, the average molecular weight of the streptavidin tetramer was understood to be approximately 52 kDa.
[00470] 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.
[00471] The samples were prepared under conditions where the streptavidinrbiotin complex is stable (Gonzalez M, Bagatolli LA, Echabe I, Arrondo JLR, Argarana CE, Cantor
CR, Fidelio GD "Interaction of Biotin with Streptavidin" J Biol Chem (1997) 272:112288-
1 1294). Other proteins under these conditions are not stable.
[00472] Samples were heated to at least 85 0 C for 10 min with SDS. In lanes 22, 24, 26,
32, 34, and 35 and lanes 10, 12, and 14, complexes eluted from a solid support were analyzed.
In 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. In lanes 1, 2, 3, 4, 6, 22, 24,
26, 29, 32, 34 and 35, samples were reacted with excess biotin for 15 min prior to the heating step. In lanes 2, 7, 14, 15, 22 and 35 complexes formed from the 4-fold node biotinylated with biotin-HPDP were analyzed. In lanes 1, 4, 9, 10, 11, 26 and 32 complexes formed from the 4- fold node biotinylated with MAL-dPEG™3 -biotin were analyzed. In lanes 3, 8, 12, 13, 24 and
34 complexes formed from the 4-fold node biotinylated with MAL-dPEG™ 11 -biotin were analyzed.
[00473] 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. The bands were not present in complexes prepared with newly prepared IPP isomerase. The degradation appears to occur at a specific site. In the initial stages, 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.
[00474] 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.
[00475] 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).
[00476] The lane 10 band at about 70 kDa (indicated by the double-ended arrow) 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. These analyses make use of the denaturation of all proteins at temperatures above 85 deg C, except the liganded streptavidin (which is stable to about 115 deg C). Activity assays of IPP isomerase show thermal denaturation within 15 minutes at 75 deg C. [00477] 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. The band at 110 kDa in lane 26, which analyzed the sample about 1 hour after elution from the resin, was smaller than the band at 110 kDa in lane 10, which analyzed the sample about 4 days after elution from the resin. These observations are consistent with the understanding that streptavidin linking two node chains is more likely to occur in solution than when the node is immobilized, and supports the conclusion that the 110 kDa band corresponds to the streptavidin tetramer bound to two node chains. By contrast, the 110 kDa band probably did not correspond to an aggregate of streptavidin:biotin tetramers, because the band for such aggregates was higher in molecular weight (compare lane 6). [00478] One node chain bound to the streptavidin tetramer would be about 88 kDa (52 +
36). Two node chains bound to the streptavidin tetramer would be about 124 kDa (52 + 36 + 36). The appearance of the one node chain:streptavidin complex at the position corresponding to about 70 kDa for the standard, and the appearance of the two node chain:streptavidin complex at the position corresponding to about 110 kDa suggests that both complexes migrated faster in the gel than the molecular weight standards of 70 and 110 kDa. It is well appreciated in the art that complex protein structures can exhibit different migration rates than standards. The mobility in an SDS PAGE gel represents that of an extended unfolded polypeptide. In the case of such an extended unfolded polypeptide, mobility scales with molecular weight, so that higher molecular weight (longer) polypeptide chains move more slowly in the gel matrix. However, in the complexes considered in the present case, it is understood that the mass of the migrating species is not contained in a single chain and one of the molecules (streptavidin) is folded. [00479] When a large quantity of the streptavidin:biotin complex was added to the lanes, higher aggregates that appeared as wide bands between the 110 kDa and 160 kDa markers were observed. For example, lane 6 was overloaded with streptavidin :biotin complex.
[00480] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Nothing in this specification should be considered as limiting the scope of the present invention. All examples presented are representative and non-limiting. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described. REFERENCES
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