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WO2009055068A1 - Adaptateur macromoléculaire de streptavidine et complexes de celui-ci - Google Patents

Adaptateur macromoléculaire de streptavidine et complexes de celui-ci Download PDF

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Publication number
WO2009055068A1
WO2009055068A1 PCT/US2008/012174 US2008012174W WO2009055068A1 WO 2009055068 A1 WO2009055068 A1 WO 2009055068A1 US 2008012174 W US2008012174 W US 2008012174W WO 2009055068 A1 WO2009055068 A1 WO 2009055068A1
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WO
WIPO (PCT)
Prior art keywords
sama
biotin
protein
streptavidin
functionalized
Prior art date
Application number
PCT/US2008/012174
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English (en)
Inventor
F. Raymond Salemme
Patricia C. Weber
Original Assignee
Imiplex Llc
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Publication date
Application filed by Imiplex Llc filed Critical Imiplex Llc
Publication of WO2009055068A1 publication Critical patent/WO2009055068A1/fr
Priority to US12/766,658 priority Critical patent/US8993714B2/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • a biotin-residue functional ized streptavidin macromolecular adaptor (SAMA) protein may include two designated surface amino acid residues and two biotin or biotin derivative groups. Each biotin or biotin derivative group can be covalently bonded to a designated surface amino acid residue, and each biotin or biotin derivative group can be positioned to bind with a separate biotin binding site of a pair of biotin binding sites on a streptavidin tetramer.
  • the SAMA protein can be a protein that was not previously known.
  • a biotin-nucleotide functionalized streptavidin macromolecular adaptor (SAMA) protein includes two binding sites and two bifunctional crosslinking reagents.
  • Each bifunctional crosslinking reagent can include a first moiety and a second moiety.
  • the first moiety can be biotin, iminobiotin, derivatives of these, or chemical analogs of these.
  • the second moiety can be a nucleotide, an enzyme inhibitor, an enzyme substrate, an enzyme cofactor, derivatives of these, or chemical analogs of these.
  • a biotin-ligand crosslinking reagent can include a biotin-type moiety and a ligand moiety.
  • Each first moiety can be positioned to bind with a separate biotin binding site of a pair of biotin binding sites on a streptavidin tetramer.
  • Each second moiety can be bound to a binding site of the SAMA protein.
  • the binding sites can, for example, be separated by a distance of about 20.5 Angstroms.
  • a binding site can be, for example, an adenosine triphosphate (ATP) binding site.
  • a streptavidin macromolecular adaptor (SAMA) can be formed of two subunits, and the designated surface amino acid residue on the first subunit, the designated surface amino acid residue on the second subunit, the binding site on the first subunit, and the binding site on the second unit can lie in about the same plane.
  • a biotin-residue, biotin- nucleotide functionalized streptavidin macromolecular adaptor (SAMA) protein includes two binding sites, at least two designated surface amino acid residues, two biotin or biotin derivative groups, and two bifunctional crosslinking reagents.
  • Each bifunctional crosslinking reagent includes a first moiety and a second moiety.
  • Each biotin or biotin derivative group is covalently bonded to a designated surface amino acid residue.
  • Each biotin or biotin derivative group is positioned to bind with a separate biotin binding site of a pair of biotin binding sites on a streptavidin tetramer.
  • the first moiety can be biotin, iminobiotin, derivatives of these, or chemical analogs of these.
  • the second moiety can be a nucleotide, an enzyme inhibitor, an enzyme substrate, an enzyme cofactor, derivatives of these, or chemical analogs of these.
  • Each first moiety can be positioned to bind with a separate biotin binding site of a pair of biotin binding sites on a streptavidin tetramer.
  • Each second moiety can be bound to a binding site of the SAMA protein.
  • a biotin-residue linked 1 :1 streptavidin:SAMA complex includes a streptavidin tetramer having biotin binding sites, a SAMA protein having two binding sites and comprising at least two designated surface amino acid residues, and two biotin or biotin derivative groups.
  • Each biotin or biotin derivative group can be covalently bonded to a designated surface amino acid residue.
  • Each biotin or biotin derivative group can be bound to a separate biotin binding site of a pair of biotin binding sites on the streptavidin tetramer.
  • a biotin-nucleotide linked 1:1 streptavidin: SAMA complex includes a streptavidin tetramer having biotin binding sites, a SAMA protein having at least two binding sites and comprising at least two designated surface amino acid residues, and two bifunctional crosslinking reagents, each comprising a first moiety and a second moiety.
  • the first moiety can be selected from the group consisting of biotin, iminobiotin, derivatives of these, and chemical analogs of these.
  • the second moiety can be selected from the group consisting of a nucleotide, an enzyme inhibitor, an enzyme substrate, an enzyme cofactor, derivatives of these, and chemical analogs of these.
  • Each first moiety can be bound to a separate biotin binding site of a pair of biotin binding sites on the streptavidin tetramer.
  • Each second moiety can be bound to a binding site of the SAMA protein.
  • a strut includes at least two biotin-residue linked 1 :1 streptavidin: SAMA complexes.
  • Each biotin-residue linked 1 :1 streptavidin: SAMA complex can be attached to at least one and at most two 1 :1 streptavidin: SAMA complexes.
  • a first and second attached biotin-residue linked 1 :1 streptavidin: SAMA complex can includes two bifunctional crosslinking reagents, each comprising a first moiety and a second moiety.
  • the first moiety can be biotin, iminobiotin, derivatives of these, or chemical analogs of these.
  • the second moiety can be a nucleotide, an enzyme inhibitor, an enzyme substrate, an enzyme cofactor, derivatives of these, or chemical analogs of these.
  • Each first moiety can be bound to a separate biotin binding site of a pair of biotin binding sites on the streptavidin tetramer of the first biotin-residue linked 1:1 streptavidin:SAMA complex.
  • Each second moiety can be bound to a separate binding site of a pair of binding sites on the SAMA protein of the second biotin-residue linked 1:1 streptavidin:SAMA complex.
  • a strut includes at least two proteins or protein multimers. Each of the at least two proteins or protein multimers can be linked to at least one and at most two of the at least two proteins or protein multimers.
  • the dissociation constant for two linked proteins or protein multimers can be less than about 10 " " M.
  • the linked proteins can lie along a common axis, and the linked proteins can be substantially rigid.
  • a nucleotide-linked antibody biosensor includes a substrate functionalized with biotin or biotin derivative groups, a strut, and an antibody having 2 Fc chain termini.
  • Two biotin binding sites of a streptavidin of the strut can be bound with the biotin or biotin derivative groups with which the substrate is functionalized.
  • Each Fc chain terminus can be functionalized with a nucleotide or nucleotide derivative.
  • Each nucleotide or nucleotide derivative with which an Fc chain terminus is functionalized can be bound to a binding site of a pair of binding sites on a SAMA of the strut.
  • a biotin-linked antibody biosensor includes a substrate functionalized with nucleotides or nucleotide derivatives, a strut, and an antibody having two Fc chain termini.
  • Two binding sites of a SAMA of the strut can be bound with the nucleotide or nucleotide derivative groups with which the substrate is functionalized.
  • Each Fc chain terminus can be functionalized with a biotin or biotin derivative.
  • Each biotin or biotin derivative with which an Fc chain terminus is functionalized can be bound to a biotin binding site of a pair of biotin binding sites on a streptavidin of the strut.
  • a method according to the invention includes providing a SAMA protein having at least two designated surface amino acid residues, for example, a SAMA protein that includes a dimer having two subunits, each subunit having a designated surface amino acid residue, mixing the SAMA protein with a thiol-reactive biotinylation reagent to form a reaction solution, allowing the SAMA protein and the thiol-reactive biotinylation reagent to react to form a biotin-residue functionalized SAMA protein; and purifying the reaction solution to obtain a substantially pure biotin-residue functionalized SAMA protein.
  • Each biotin of the biotin-residue functionalized SAMA protein can be positioned to bind with a separate biotin binding site of a pair of biotin binding sites on a streptavidin tetramer.
  • the SAMA protein provided can include a pair of binding sites, a pair of designated surface amino acid residues, and a first end and a second end. The second end can be opposed to the first end, and a dyad axis can span from the first end to the second end.
  • Each member of the pair of binding sites can be symmetric about the dyad axis at the first end, and each member of the pair of designated surface amino acid residues can be symmetric about the dyad axis at the second end.
  • the thiol-reactive biotinylation reagent can be capable of bonding with the designated surface amino acid residue.
  • a method according to the invention includes providing a SAMA protein having two binding sites, mixing the SAMA protein with a bifunctional crosslinking reagent having a first moiety and a second moiety to form a reaction solution, allowing the SAMA protein and the bifunctional crosslinking reagent to react to form a biotin-nucleotide functionalized SAMA protein, and purifying the reaction solution to obtain a substantially pure biotin-nucleotide functionalized SAMA protein.
  • the first moiety can be biotin, iminobiotin, derivatives of these, or chemical analogs of these.
  • the second moiety can be a nucleotide, an enzyme inhibitor, an enzyme substrate, an enzyme cofactor, derivatives of these, or chemical analogs of these.
  • a binding sequence linked antibody sensor includes a substrate functionalized with nucleotides or nucleotide derivatives, a SAMA protein, and an antibody.
  • the SAMA protein can include a symmetric dimer, a binding domain comprised of a binding polypeptide chain, and a linker peptide.
  • the dimer can include two polypeptide chains.
  • the binding polypeptide chain can be covalently bonded to the linker peptide, and the linker peptide can be covalently bonded to the polypeptide chain.
  • the binding polypeptide chain of the biotin-residue functionalized SAMA protein can be an antibody binding polypeptide.
  • the biotin-residue functionalized SAMA protein can comprise at least two binding sites. The at least two binding sites of the biotin-residue functionalized SAMA protein can be bound with the nucleotide or nucleotide derivatives with which the substrate is functionalized.
  • the antibody can be bound to the antibody binding polypeptide.
  • a binding sequence linked antibody sensor can include a substrate functionalized with biotin or biotin derivative groups, a biotin-residue functionalized SAMA protein, a streptavidin tetramer having biotin binding sites, and an antibody.
  • the binding polypeptide chain of the biotin-residue fiinctionalized SAMA protein can be an antibody binding polypeptide.
  • the biotin or biotin derivative group of the biotin-residue fiinctionalized SAMA protein can be bound to a separate biotin binding site of a pair of biotin binding sites on the streptavidin tetramer.
  • a pair of biotin binding sites on the streptavidin tetramer can be bound with the biotin or biotin derivative groups with which the substrate is fiinctionalized,
  • the antibody can be bound to the antibody binding polypeptide.
  • a biotin binding site exposed assembly includes a substrate functionalized with nucleotides or nucleotide derivatives, a first SAMA protein having two binding sites and two designated surface amino acid residues with a nucleotide or nucleotide derivative bound to each designated surface amino acid residue, and a biotin-residue linked 1 :1 streptavidin:SAMA complex.
  • the nucleotides or nucleotide derivatives of the substrate can be bound to each binding site of the first SAMA protein.
  • Each nucleotide or nucleotide derivative of the first SAMA protein can be bound to a binding site of the SAMA protein of the biotin-residue linked 1:1 streptavidin:SAMA complex.
  • a binding site exposed assembly includes a substrate functionalized with nucleotides or nucleotide derivatives, a first SAMA protein having two binding sites and two designated surface amino acid residues with a biotin or biotin derivative bound to each designated surface amino acid residue, and a biotin-residue linked 1:1 streptavidin: SAMA complex.
  • the nucleotides or nucleotide derivatives of the substrate can be bound to each binding site of the first SAMA protein.
  • an iminobiotin exposed assembly includes a substrate functionalized with nucleotides or nucleotide derivatives and a SAMA protein having two binding sites and two designated surface amino acid residues with an iminobiotin linked to each designated surface amino acid residue.
  • a streptavidin macromolecular adaptor (SAMA) protein includes a dimer having two polypeptide chains.
  • Each polypeptide chain can include a designated surface amino acid residue that is a cysteine residue.
  • the designated surface amino acid residue can be located such that when a biotin or biotin derivative group is covalently bonded to the designated surface amino acid residue, each biotin or biotin derivative group on the dimer is positioned to bind with a separate biotin binding site of a pair of biotin binding sites on a streptavidin tetramer.
  • the SAMA protein can be a protein whose amino acid sequence is not identical to a previously known protein.
  • the SAMA protein may be a natural sequence that has been modified to substitute any cysteine residues in the natural sequence by an alternative amino acid.
  • a streptavidin macromolecular adaptor (SAMA) protein includes a dimer having 2 polypeptide chains. Each polypeptide chain includes a designated surface amino acid residue that can be, for example, cysteine, lysine, histidine, arginine, methionine, tyrosine, serine, or threonine.
  • the designated surface amino acid residue can be located such that when a biotin or biotin derivative group is covalently bonded to the designated surface amino acid residue, each biotin or biotin derivative group on the dimer is positioned to bind with a separate biotin binding site of a pair of biotin binding sites on a streptavidin tetramer.
  • the SAMA protein can be a protein whose amino acid sequence is not identical to a previously known protein.
  • the SAMA protein may be a natural sequence that has been modified to substitute any cysteines, lysine, histidine, arginine, methionine, tyrosine, serine, or threonine residues in the natural sequence by an alternative amino acid.
  • a method according to the invention of identifying a streptavidin macromolecular adaptor (SAMA) framework protein includes analyzing protein coordinate sets from at least one publicly available database and/or the Protein Data Bank and identifying protein dimers that are 2-fold symmetric and have two ligand-binding pockets.
  • the two ligand-binding pockets can be separated by a distance within ⁇ 10 Angstroms of the distance between two biotin binding sites on a streptavidin tetramer.
  • the two ligand-binding pockets are separated by a distance of from about 10 Angstroms to about 30 Angstroms.
  • the two ligand-binding pockets are separated by a distance of from about 15 Angstroms to about 25 Angstroms.
  • a streptavidin:SAMA complex can include a streptavidin tetramer having a pair of biotin binding sites and a SAMA protein having a pair of binding sites and having a pair of designated surface amino acid residues.
  • Two biotin-type groups can be covalently bonded to a designated surface amino acid residue and can be bound to a biotin binding site of the pair of biotin binding sites of the streptavidin tetramer to link the streptavidin and SAMA proteins together.
  • Two bifunctional crosslinking reagents each comprising a biotin-type moiety bound to a biotin binding site of the pair of biotin binding sites of the streptavidin tetramer and a second moiety bound to a binding site of the pair of binding sites of the SAMA protein can be used to link the streptavidin and SAMA proteins together.
  • the SAMA protein can have a dyad axis that spans from a first end of the SAMA protein to a second end of the SAMA protein. The second end of the SAMA protein can be opposed to the first end of the SAMA protein.
  • Each member of the pair of binding sites on the SAMA protein can be symmetric about the dyad axis at the first end of the SAMA protein, and each member of the pair of designated surface amino acid residues on the SAMA protein can be symmetric about the dyad axis at the second end of the SAMA protein.
  • the second moiety can include, for example, a nucleotide, an enzyme inhibitor, an enzyme substrate, an enzyme cofactor, derivatives of these, and/or chemical analogs of these.
  • the streptavidin tetramer can have a dyad axis. Each member of a pair of biotin binding sites on the streptavidin can be symmetric about the dyad axis.
  • a strut can include at least two proteins or protein multimers.
  • Each of the at least two proteins or protein multimers can be linked to at least one and at most two of the at least two proteins or protein multimers.
  • the dissociation constant for two linked proteins or protein multimers can be less than about 10 " " M.
  • the at least two proteins can lie along a common axis.
  • the linked at least two proteins can be substantially rigid.
  • an iminobiotin exposed assembly can include a substrate functionalized with nucleotides or nucleotide derivatives and a SAMA protein having two binding sites and two designated surface amino acid residues with an iminobiotin linked to each designated surface amino acid residue.
  • a SAMA protein can include a functional polypeptide sequence.
  • the functional polypeptide sequence can be covalently bound to an amino or carboxy terminus of the subunit.
  • the functional polypeptide sequence can be within a surface loop of one or two polypeptide chains.
  • the functional polypeptide sequence can be an Fab sequence (a sequence from or similar to a fragment antigen binding (Fab) region of an antibody).
  • a binding site exposed assembly includes a substrate functionalized with nucleotides or nucleotide derivatives, a first SAMA protein having two binding sites and two designated surface amino acid residues with a biotin or biotin derivative bound to each designated surface amino acid residue, and a biotin-residue linked 1 :1 streptavidin:SAMA complex.
  • the nucleotides or nucleotide derivatives of the substrate can be bound to each binding site of the first SAMA protein.
  • an iminobiotin exposed assembly includes a substrate functionalized with nucleotides or nucleotide derivatives and a SAMA protein having two binding sites and two designated surface amino acid residues with an iminobiotin linked to each designated surface amino acid residue.
  • a kit includes a nanostructure building block and a linking compound.
  • the linking compound can be a biotin- ligand crosslinking reagent, such as a biotin-nucleotide crosslinking reagent (e.g., Figs. 23cl- 23c2 and Fig. 23g), a biotin-biotin crosslinking reagent (e.g., Figs. 2311-230), a ligand- ligand crosslinking reagent (e.g., Fig. 23h), can be a biotinylation reagent (e.g., the compounds of Figs. 23al-23a5 and Figs.
  • 23bl-23b5 such as a thiol-reactive biotinylation reagent (e.g., Figs. 23al and 23bl), or can be a ligand functional ization reagent, such as a thiol reactive - nucleotide reagent (e.g., Figs. 23dl-23d2).
  • a thiol-reactive biotinylation reagent e.g., Figs. 23al and 23bl
  • a ligand functional ization reagent such as a thiol reactive - nucleotide reagent (e.g., Figs. 23dl-23d2).
  • Figure 1 shows a cartoon and a molecular model of the streptavidin tetramer indicating biotin ligand binding sites.
  • Figure 2 shows the reaction of protein cysteine sulfhydryl groups with biotinylation reagents.
  • Figure 3 presents cartoons of two-dimensional lattices formed using threefold and four-fold symmetric nodes.
  • Figure 4 presents cartoons of various three-fold symmetric nodes.
  • Figure 5 presents cartoons of various four-fold symmetric nodes.
  • Figure 6 presents steps in the assembly of a biotin-residue linked 1 :1 streptavidin: SAMA complex.
  • Figure 7 presents steps in the formation and assembly of functionalized SAMA proteins.
  • Figure 8 presents a SAMA cartoon and computer representation of the
  • Figure 9 presents a computer model showing the MJ0577 protein dimer and the streptavidin tetramer in apposition.
  • Figure 10 presents SAMA amino acid sequences based on the MJ0577 protein.
  • Figure 11 presents a cartoon and a molecular model of a biotinylated SAMA based on the MJ0577 protein.
  • Figure 12 presents a cartoon and a molecular model of a biotin-linked 1 : 1
  • SAMA streptavidin complex based on the MJ0577 protein, and illustrates regeneration of binding capability to the complex.
  • Figure 13 presents a cartoon and a molecular model of a biotin-photo-ATP crosslinked 1 :1 SAMA: streptavidin complex based on the MJ0577 protein, and illustrates regeneration of binding capability to the complex.
  • Figure 14 presents a cartoon and a molecular model of a SAMA based on the
  • Figure 15 presents a cartoon outlining steps in assembling a 3:4 streptavidin:SAMA strut in solution.
  • Figure 16 presents cartoons of various streptavidin:SAMA complexes.
  • Figure 17 presents methods of assembly of various biosensors incorporating antibodies.
  • Figure 18 presents methods for altering the assembly polarity of structures assembled from SAMA and streptavidin.
  • Figure 19 presents methods for altering the assembly polarity of structures assembled from SAMA and streptavidin using SS-linked crosslinking reagents.
  • Figure 20 presents steps in assembling a biotin-residue linked 1 : 1 streptavidin: SAMA complex using a support matrix.
  • Figure 21 presents steps in assembling a biotin-photo-ATP linked 1 :1 streptavidin:SAMA complex using a support matrix.
  • Figure 22 presents steps in assembling a 4:4 streptavidin: SAMA strut using a support matrix.
  • Figure 23 shows chemical structures and schematic illustrations of reagents used in streptavidin:SAMA nanostructure assembly.
  • Figure 24 illustrates the structures of the expression vectors for expression of
  • Figure 25 shows Polyacrylamide Gel Electrophoresis (PAGE) separations of
  • Figure 26 shows Polyacrylamide Gel Electrophoresis (PAGE) separations of
  • SAMA streptavidin complexes.
  • the biomolecular components can include molecular-scale “struts” and
  • nodes are components that basically function as linear structural elements or linear connectors. Different struts or arrays of struts can be used to establish predetermined distances in a structure. Nodes are connectors that have multiple, for example, three or more, attachment points with defined geometry. Nodes can be linked together, for example, by struts, to establish the topology of a structure.
  • lattices can have utility themselves and/or can be further functionalized through chemical modification or the incorporation of additional specific binding proteins.
  • compositions and methods discussed herein apply the philosophies of interchangeable parts and mass production, which drove unprecedented economic expansion in the last two centuries, to the nanoscale.
  • Providing such a "parts box" of biomolecular components allows users to experiment with a range of structures and thereby facilitates the development of a new generation of functional nanodevices, biosensors, and biomaterials, potentially finding broad application in areas as diverse as biomedical devices and nanoelectronic applications.
  • Proteins have a number of advantages for use as biomolecular components, including, but not limited to the following. Proteins already exist in nature as functional polypeptide units with well-defined three-dimensional 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, and certain proteins, with minimal tailoring, can perform as a desired building block.
  • Naturally occurring proteins have diverse and sophisticated functionality. They can show high interaction specificity and manifest catalytic properties. They can exhibit interesting and useful optical, magnetic, and redox properties, for example, by incorporating metal centers and a wide variety of prosthetic groups. Such metal centers and prosthetic groups can, as well as the polypeptide sequence itself, be tailored to produce a protein having a desired functionality.
  • DNA encodes a polypeptide sequence that spontaneously and reproducibly folds to form a predetermined three-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.
  • 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 three- dimensional structures formed from struts and nodes, as described herein, may be applied in the fields of biosensors and diagnostics.
  • strut-node technology presented herein can enable the development of new kinds of sensors incorporating, for example, multiple antibodies specifically immobilized in patterned arrays.
  • Other applications may not have direct natural analogs, but are intended to interact with natural biological systems.
  • 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 for prostheses.
  • biomolecular components that described herein are based on proteins of thermostable bacteria of known three-dimensional crystal structure.
  • the proteins provide several advantages in node production, handling and purification.
  • the enzymatic binding sites of proteins used as nodes can provide additional sites for functionalization of the nanostructure through covalent binding of inhibitors linked to other chemical moieties or proteins.
  • struts Two fundamental nanoscale biomolecular components of a "parts box” from which a structure, for example, a device, can be assembled are “struts” and “nodes”. Struts are molecular components that function as linear connectors. Nodes connect struts and orient them with defined geometries.
  • a strut can be formed from streptavidin (Fig. 1), a tetrameric protein of
  • FIG. 1 shows a cartoon and a molecular model of the streptavidin tetramer indicating biotin ligand binding sites.
  • Part a shows a schematic of a streptavidin tetramer (streptavidin) which has binding sites for 4 biotin groups.
  • Part b designates the location of a pair of biotin binding sites on the same "side" of the tetramer that are spaced approximately 20.5 Angstroms apart.
  • Part c shows an all atom (excluding hydrogen atoms) stick bond representation of the streptavidin tetramer, including four bound biotin molecules in space-filling representation.
  • Part d designates two of the four bound biotins on the same "side" of the tetramer in space filling representation.
  • Part e is a representation of the streptavidin tetramer surface showing overall molecular shape. Weber et al. (1989) determined the X-ray structure of streptavidin and described the origins of its ability to bind the vitamin biotin.
  • biotin:streptavidin interaction is non-covalent
  • 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.
  • the biotin-binding sites are arranged as two pairs in an "H" orientation that facilitates specific pairwise binding.
  • the biotin binding sites are arranged with D2 symmetry.
  • the biotin molecules When bound to the streptavidin biotin-binding sites, the biotin molecules have their terminal valeric acid chains (which are the usual chemical modification sites for generating biotin conjugated reagents) in extended conformation and oriented approximately parallel to one of the diad axes of the streptavidin tetramer. The distance between the two closest and roughly parallel pair of bound biotin chain termini is about 20.5 Angstroms.
  • a streptavidin tetramer when serving as a strut, can be linked to two other biomolecular components, such as nodes, through biotin molecules.
  • the streptavidin tetramer is approximately 60 Angstroms (6 nanometers) wide by 45 Angstroms (4.5 nanometers) deep by 50 Angstroms (5.0 nanometers) long in the direction that facilitates pairwise biotin interactions.
  • several related proteins are known (e.g., egg white avidin) that have similar amino acid sequence, structure, and biotin binding properties as streptavidin.
  • such a protein may have greater than about 80%, 90%, 95%, 98%, or 99% protein sequence similarity (homology) with streptavidin.
  • protein sequence similarity can refer to an amino acid composition similarity by relative proportion of amino acid composition.
  • the applications pertaining to "streptavidin” shall generally be construed to apply to all homologues or recombinantly produced variants of the naturally occurring streptavidin protein, or its homolog avidin, that incorporate 4 biotin binding sites arranged with same geometry as the native streptavidin or avidin tetramer.
  • Variants include shortened or modified versions of the protein (Kopetzki, 1987, Cantor 1989, Goshorn et al.
  • a node can connect three or more struts with predefined orientation of each strut with respect to the other connected struts.
  • a node can be a symmetric protein multimer.
  • a node can be an enzyme that has catalytic binding sites with high binding specificity for certain substrates and cofactors.
  • a naturally occurring protein can be used in its native state, or can be engineered, for example, using site-specific modification techniques, to render it suitable or optimal for an intended function as a node. Selection of a naturally occurring protein for use as a node can be made from the large number of X-ray crystal structures of stable protein multimers having different symmetries available.
  • selection can be made from protein sequences that have over 70% sequence homology with sequences with known X-ray structures, since it is known that homologous protein sequences also have similar three-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.
  • suitable natural symmetric protein multimers are available having 2-, 3-, 4-, 5-, 6-, 7-, and higher-fold symmetry useful for forming finite or extended planar nanoassemblies organized in two dimensions, as well as multimers having tetrahedral, octahedral, and other symmetries useful for forming three- dimensional nanoassemblies.
  • 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 three-dimensional geometry, such as lattices.
  • 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.
  • a node of defined geometry can be formed.
  • biotinylating reagents that can modify protein cysteine sulfhydryl groups are presented in Fig. 2.
  • Part a shows a free sulfhydryl group on a protein.
  • Part b shows the biotinylation reagent Sulfosuccinimidyl 2-(biotinamido)-ethyl-l,3-dithiopropionate (EZ-Link Sulfo-NHS-SS-Biotin: Pierce).
  • Part c shows the reaction product after biotinylation.
  • Part d 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.
  • Part e shows the imino-biotin reaction product.
  • Part f shows the reaction schemes schematically using the schema of Figure 23. Additional chemical reagents useful in nanostructure assembly are presented in Figure 23.
  • Fig. 3 shows two-dimensional lattices formed using nodes with three-fold (C3) and four-fold (C4) rotational symmetry.
  • Two types of symmetric 2D lattice structures that can be assembled through the association of biotin-modified symmetric node structures and streptavidin are illustrated.
  • Part a shows part of a square 2D lattice incorporating tetrameric nodes b and connected through streptavidin tetramers c.
  • Part d shows part of an hexagonal 2D lattice incorporating trimeric nodes e.
  • Part f shows the hexagonal lattice of Part d that has been functionalized through specific attachment of an additional protein g such as an immunoglobulin.
  • Biotin linking reagents can be covalently bound to engineered sites on the node proteins, so that they make rigid pairwise interactions with tetrameric streptavidin struts.
  • Single chain constructs of a node protein can be formed.
  • these fused protein multimers can be constructed by incorporating a DNA sequence coding for a polypeptide linker connecting the C-terminus of a first multimer gene to the N-terminus of a second multimer, and so on, to create a single contiguous gene coding for the complete multimer.
  • This approach can allow for the subunits of a multimeric protein to be non- identical.
  • surface cysteine residues for biotinylation can be included in some subunits, but not in other subunits, so that struts can be attached at certain faces of the multimeric protein, but not at others.
  • a protein having multiple subunits that are formed from a single polypeptide chain is termed a multimer, as is a protein having multiple subunits with each subunit formed from a separate polypeptide chain.
  • Figures 4a-4d show nodes based on a protein trimer having three-fold (C3) rotational symmetry.
  • 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 as formed from three independent, identical chains that are not covalently associated. Two biotins are bound to each chain, so that a streptavidin strut can bind to each subunit.
  • Figure 4b 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 polypeptide chain. Two biotins are bound to each chain, so that a streptavidin strut can bind to each subunit.
  • the structure shown in Fig. 4b is termed a protein trimer herein.
  • Figure 4c 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 4d 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.
  • Figs. 4b through 4d illustrate nodes with various streptavidin binding geometry and valency.
  • Figures 5a through 5f show nodes based on a protein tetramer having fourfold (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 5a shows a node that is a tetramer as formed from four independent, identical chains that are not covalently associated. 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.
  • Figure 5b 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 Fig. 5b the individual chains of the non-covalently associated tetramer are covalently connected together in a single polypeptide chain, i.e., a linear amino acid sequence.
  • FIG. 5b The structure shown in Fig. 5b is termed a protein tetramer herein.
  • Figure 5c 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 5d shows a node based on a protein tetramer formed from a single chain construct.
  • FIG. 5e 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.
  • 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 5f 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.
  • tetramer can serve as a terminator of a strut, and cannot serve as a connector or branch point between struts.
  • Figs. 5c through 5f 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.
  • Nodes can also be functional ized by making a gene fusion between a node protein and a specific protein binding domain.
  • a gene fusion can be made between a node protein and a Protein A or Protein G domain that binds with high affinity to Immunoglobin Fc (fragment crystallizable) regions.
  • a method of using a template multimeric protein as a nanostructure node can include the following.
  • a template multimeric protein can be connected with a nanostructure strut.
  • the template multimeric protein can have a known 3- dimensional structure.
  • the template multimeric protein can be derived from a thermostable microorganism.
  • the template multimeric protein can have Cn, Dn, or higher symmetry.
  • the template multimeric protein can incorporate a specific binding site for the attachment of at least one nanostructure strut with predefined stoichiometry and orientation.
  • methods of producing nanostructure nodes and nanostructure assemblies can include the following.
  • a mathematical and/or computer graphic representation of the 3-dimensional molecular structure of a template multimeric protein and a streptavidin tetramer can be generated.
  • Each surface cysteine residue of the template multimeric protein can be replaced with an alternative amino acid in the representation.
  • Several spatial configurations of the streptavidin tetramer relative to the template multimeric protein can be iterated through in the representation, with the streptavidin tetramer in approximate Van der Waals contact with the template multimeric protein.
  • cysteine can be assigned 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.
  • biotin e.g., the biotin valeric acid carbon atom
  • a measure of quality can be assigned to each spatial configuration (e.g., 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/or the potential energy of electrostatic interaction between the nanostructure node multimeric protein and the streptavidin tetramer).
  • Each spatial configuration and associated nanostructure node multimeric protein can be stored.
  • An optimal nanostructure node multimeric protein can be selected for production (for example, based on the measure of quality associated with a spatial configuration of the optimal nanostructure node multimeric protein).
  • a template multimeric protein with Cn subunit symmetry can be used 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.
  • a mathematical and/or computer graphic representation of the 3-dimensional molecular structure of the Cn symmetric template multimeric protein and a streptavidin tetramer can be generated.
  • a computer graphics and/or mathematical method can be used to identify surface cysteine residues on the surface of the template multimeric protein.
  • An alternative amino acid(s) (e.g., Ala, Serine, Asp, etc.) can be assigned to replace the identified surface cysteine residues in the template sequence.
  • the mathematical and/or computer graphic representation can be used to initially position the 3-dimensional coordinates of the template multimeric protein and streptavidin tetramer, so that the Cn symmetry (or z) axis of the template multimeric protein is parallel to the streptavidin tetramer z-dyad axis or y-dyad axis, the centers of mass of the template multimeric protein and streptavidin coordinates have the same or nearly the same z coordinate, and the molecules do not physically intersect.
  • the mathematical and/or computer graphic representation can be used 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.
  • the computational and/or computer graphics method can be used 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.
  • a cysteine can be assigned 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.
  • a computational and/or computer graphics method can be used to create 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 quality (e.g., 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).
  • rms root-mean-square
  • a computational and/or computer graphics method can be used 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.
  • a measure of complementarity of fit and/or energetic stability can be assigned based on, e.g., steric and electrostatic complementarity of amino acid residues at the interface, maintenance of preferred amino acid side chain rotomer conformations, low potential energy as estimated using a computational method such as molecular mechanics, quantum mechanics, or potential energy calculations, or through experimental methods of measuring complex stability, including affinity measurements, calorimetry, or other experimental methods.
  • the 3-dimensional coordinates of the nanostructure node multimeric protein : streptavidin complex can be stored along with quality measures in a database.
  • a rotation of the template multimeric protein about the Cn axis can be incremented.
  • the steps of positioning the 3-dimensional coordinates of the template multimeric protein and streptavidin tetramer through incrementing the rotation of the template multimeric protein can be repeated over an angular increment of at least 360/n degrees, where n defines the foldedness of the multimeric protein symmetry axis.
  • Quality measures of stored nanostructure node multimeric protein : streptavidin complexes can be ranked and/or coordinates of stored nanostructure node multimeric protein : streptavidin complexes can be examined in selecting an optimal nanostructure node multimeric protein for production.
  • Modifications of this approach can be used, for example, to design and produce nodes for use as an apex in a polyhedron or other geometrical structure; the nodes, for example, attached to each other by nanostructure struts.
  • the Cn symmetry (or z) axis of the template multimeric protein and streptavidin tetramer z- dyad axis or y-dyad axis can be oriented at an angle corresponding to a polyhedral node apex angle.
  • a nanostructure node can include a nanostructure node multimeric protein comprising at least one polypeptide chain.
  • the nanostructure node multimeric protein can have one or more of the following: a known 3-dimensional structure; essentially a Cn, Dn, or higher symmetry with a number of subunits; stability at a temperature of 70 0 C or greater; an amino acid sequence not found in nature; and/or 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. Each specific amino acid reactive residue can have a covalently attached biotin group. For example, two or more subunits can be covalently interconnected with a polypeptide linker.
  • the nanostructure node can be a planar node, and/or the nanostructure strut can be a streptavidin strut.
  • the nanostructure node multimeric protein can include one polypeptide chain.
  • the nanostructure node multimeric protein can have an amino acid sequence with greater than 80 per cent sequence identity with the amino acid sequence of a pdb code:lthj protein trimer.
  • Precision nanostructure assembly requires a much higher level of specific control over successive steps in the assembly process than can be achieved by a simple strategy of forming streptavidin-biotin-protein multimer links.
  • a controllable component adaptor can act as a protecting group for the end of a strut, to either allow or prevent the strut from linking to a node or another strut.
  • natural proteins which can be further tailored, can be considered.
  • a computational approach e.g., an algorithm, manual inspection, or a combination of automated and manual techniques can be used to analyze protein coordinate sets downloaded from the Protein Data Bank (http://www.rcsb.org) or another publicly available database to identify a suitable protein for use as a controllable component adaptor.
  • a suitable protein can have surface amino acids to which a linking molecule that can link to a strut can be bound.
  • a protein can be tailored, for example, through genetic engineering techniques, to have surface amino acids to which a linking molecule that can link to a strut can be bound.
  • a suitable protein can have the surface amino acids located such that each bound linking molecule will bond to a site on the strut.
  • a streptavidin macromolecular adaptor (SAMA) protein serves as a controllable component adaptor for a streptavidin strut.
  • the SAMA can act as a reversible protecting group for pairs of streptavidin binding sites, and provide the required precise control over nanostructure assembly.
  • the SAMA can provide key advantages known from solid-phase chemical synthesis (Merrifield & Stewart 1965; Merrifield et al.
  • Figure 6c presents a cartoon of a 1:1 di-biotin linked streptavidin:SAMA complex.
  • This complex can serve as a basic building block enabling the controlled assembly of nanostructures based on strut-node architecture.
  • this complex can serve in streptav id in-based immobilization applications where improved control over immobilization chemistry is desired.
  • a SAMA can function both as a protecting group and as an immobilization agent.
  • Figure 6a through 6c present steps in the assembly of the biotin- residue linked 1 :1 streptavidin:SAMA complex shown in Fig. 6c. The molecules are in the complex linked through biotins occupying two of the four streptavidin biotin binding sites.
  • Part a shows a schematic of the SAMA
  • part b shows the tetrameric protein streptavidin
  • part c shows the 1 :1 biotin-linked streptavidin: SAMA complex.
  • This is an example of a basic nanostructure building block.
  • Other examples of nanostructure building blocks, such as struts, are presented herein.
  • Chemical structures for schematic representations of cross-link chemistry are given in Figure 23.
  • Figure 7 presents steps in the formation and assembly of several functionalized SAMA proteins.
  • Chemical structures for schematic representations of crosslink chemistry are given in Figure 23.
  • Figure 7 part a shows a schematic of a representative SAMA template protein, in this case a symmetric dimer with two ligand binding sites.
  • Part b shows the introduction of a reactive surface amino acid residue into each monomer of the dimer so that the sites are separated by approximately 10.0 to 35.0 Angstroms and lie approximately in the same plane as two ligand binding sites on the dimer.
  • the modified SAMA can be reacted with a biotinylating reagent c (e.g.
  • Figure 23al through 23a4) capable of reacting with the surface reactive amino acids introduced through site-specific modification of the native protein, producing the di-biotin-substituted SAMA d.
  • the SAMA of part d may be further reacted with a bifunctional crosslinking reagent.
  • a bifunctional crossl inker e incorporating biotin on one end and a photo-reactive analog of the SAMA ligand on the other binds to the protein and then becomes covalently attached through photo-crosslinking (e.g. Figure 23cl and c2). This produces the modified SAMA f with four covalently bound biotin groups.
  • the SAMA of part b may also be reacted with other reagents such as a reagent g ( Figure 23bl through 23b4) that modifies the protein with 2-iminobiotin groups, allowing reversible, pH-dependent binding, e.g., between the MJ0577 SAMA embodiment and streptavidin.
  • a ligand interaction can be one in which a ligand moiety on the crosslinking reagent binds to a site on the SAMA, for example, wherein a nucleotide binds to a nucleotide binding site, or an enzyme inhibitor, substrate, or cofactor binds to an enzyme active site, or an antigen binds to an antibody domain.
  • the crosslinking reagent includes a moiety that is a nucleotide, an enzyme inhibitor, a substrate, a cofactor, or an antigen, respectively, or chemical analogs or derivatives of these.
  • the SAMA protein binding site comprises a nucleotide binding site, an enzyme active site, or an antibody domain, respectively.
  • the crosslinking reagent may comprise a derivative or chemical analog of the ligand moiety.
  • thermostable protein may be selected as a SAMA, so that the SAMA has a denaturation temperature in aqueous solution of at least about 60 0 C and/or maintains secondary, tertiary, and quaternary structure in a solvent having a dielectric constant of at least about 15.
  • a SAMA used as a biomolecular component can have a protein sequence homology (similarity) of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% with a sequence derived from a thermophilic organism, or can have complete sequence homology (similarity) with a sequence derived from a thermophilic organism except for one, two, or four amino acid residues at suitable positions on the surface of the SAMA to serve as designated amino acid residues for biotin binding sites.
  • a suitable protein for a SAMA can have a longest dimension greater than about 20.5 Angstroms.
  • a suitable protein for a SAMA can have at least two designated surface amino acid residues located 10 and 40 Angstroms apart, so that biotin linking molecules bound to these have a spacing sufficiently similar to that of biotin binding sites on streptavidin.
  • a designated surface amino acid is an amino acid on the exterior of a protein, such that the amino acid contacts the environment surrounding the protein, and that is intended to be reacted with a chemical group to impart a chemical functionality to the protein.
  • a cysteine on the exterior of a protein may serve as a designated surface amino acid, with which a thiol-reactive biotinylating reagent may react, so that biotin functionality is imparted to the protein.
  • a cysteine on the exterior of a protein may serve as a designated surface amino acid, with which a thiol-reactive ATP photo label, such as a 2-azido or 8-azido adenosine photo-crosslinking reagent functional ized to form S-S bonds with free sulfhydryl groups ( Figure 23dl or 23d2) may react, so that ATP photo label functionality is imparted to the protein.
  • a thiol-reactive ATP photo label such as a 2-azido or 8-azido adenosine photo-crosslinking reagent functional ized to form S-S bonds with free sulfhydryl groups ( Figure 23dl or 23d2) may react, so that ATP photo label functionality is imparted to the protein.
  • designated surface amino acid residues include cysteine, lysine, histidine, arginine, methionine, and tyrosine.
  • a designated surface amino acid residue can be the only amino acid of its type, for example, a cysteine, in a polypeptide chain or in a polypeptide chain that is on the surface of the protein.
  • the two designated surface amino acid surface residues can be the same residue or different residues.
  • a designated surface amino acid surface residue can include side chain atoms.
  • the location of these designated surface amino acid surface residues can be termed a surface reactive region.
  • a sulfur-sulfur bond can be formed between the biotin-containing moiety of a biotinylation reagent and a surface cysteine on a SAMA protein, concurrent with the release of a chromogenic leaving group from the biotinylation reagent.
  • a water-soluble biotin-derivative can be mixed with the SAMA protein solution and the time course of reaction followed colorimetrically by monitoring the release of the chromogenic group attached to the derivatized biotin (Green, 1975). Excess biotinylation reagent and/or multiple reaction cycles can be used to drive complete derivatization of the SAMA.
  • a schematic diagram outlining the steps of site specific modification and biotinylation at the modified sites are illustrated in Figs. 7a through 7d.
  • a size exclusion chromatography (SEC) column can be used to completely remove any unreacted biotin reagent, and the extent of SAMA biotinylation can be measured by titration of any remaining free SAMA sulfhydryl groups with Ellman's Reagent (5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB).
  • Ellman's Reagent (5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB).
  • DTNB readily forms a mixed disulfide with thiols to liberate 5-mercapto-2-nitrobenzoic acid, a chromophore with absorption maximum at 410 nm and extinction coefficient -13,600 Cm 1 M ' '.
  • an additional purification step involving passage of the biotinylated SAMA over a free thiol affinity column to remove any unreacted or mono-biotinylated SAMA can be performed for further purification to obtain di-biotinylated SAMA.
  • a suitable protein for a SAMA can be a dimer of two subunits.
  • the dimer can be formed of one or more polypeptide chains, for example, one or two polypeptide chains. Each subunit can be formed of a separate polypeptide chain, or the subunits can be formed of a single polypeptide chain.
  • the dimer can be symmetric and the polypeptide chains can have the same amino acid sequence.
  • the two subunits forming the dimer can be substantially structurally identical and/or substantially sequentially identical.
  • substantially structurally identical can mean that the secondary and tertiary structure of one subunit is similar to that of the other subunit, although there may be small differences, for example, in the position of secondary structures such as alpha helices and beta sheets.
  • substantially sequentially identical can mean that the amino acid sequence of the polypeptide forming one subunit is similar to that of the polypeptide forming the other subunit, although there may be small differences, for example, the addition (insertion) of one or a few amino acids, the deletion of one or a few amino acids, and/or the substitution of one or a few amino acids in a polypeptide.
  • the polypeptide chains can be covalently linked.
  • a polypeptide chain forming a first subunit can be covalently linked to a polypeptide chain forming a second subunit.
  • each member of a pair of designated surface amino acid residues can be related by, e.g., be symmetric about, a dyad symmetry axis.
  • the surface reactive amino acids can be introduced into the polypeptide chain at suitable positions determined through molecular modeling using methods of site-specific mutagenesis. Surface reactive amino acids can vary according to the chemistry used to introduce covalently bound biotin groups.
  • useful reactive amino acids include lysine, arginine, tyrosine, histidine, serine, and threonine, as well as the free amino terminus of the polypeptide chain.
  • Each member of a pair of binding sites on the SAMA can be symmetric about a dyad symmetry axis, for example, about the same dyad symmetry axis about which each member of a pair of designated surface amino acid residues is symmetric.
  • such a dyad axis can span from a first end of the SAMA protein to a second end of the SAMA protein, with the second end being opposed to the first end, that is, with the first end on one side of the SAMA protein and the second end on the opposite side of the SAMA protein.
  • each member of a pair of binding sites can be symmetric about the dyad axis at the first end of the SAMA protein, and each member of a pair of designated surface amino acid residues can be symmetric about the dyad axis at the second end of the SAMA protein.
  • biotin groups on a SAMA bind to two biotin binding sites on streptavidin, the two binding sites are effectively "capped", in that they cannot react with any other available biotin molecules. That is, the SAMA serves as a protecting group, and can prevent the uncontrolled polymerization between streptavidin and biotin functionalized proteins observed by Ringler & Schulz (2003).
  • biotin-type groups can be covalently bonded to each of a pair of designated surface amino acid residues on a SAMA protein.
  • a biotin-type group can include a biotin, an iminobiotin, a portion of a biotin or an iminobiotin, or a derivative or chemical analog of biotin or iminobiotin.
  • a biotin-type group is capable of bonding with a biotin binding site on a streptavidin tetramer.
  • a biotin-type group can also include a group capable of binding with another molecule, for example, a thiol group capable of covalently binding with a cysteine used as a designated surface amino acid on a protein, such as a SAMA protein.
  • Each biotin-type group can be bound to a biotin binding site of a pair of biotin binding sites of a streptavidin tetramer.
  • two bifunctional crosslinking reagents each comprising a biotin-type moiety and a second moiety can be used to link a SAMA protein to a streptavidin tetramer.
  • the biotin-type moiety can be bound to a biotin binding site of the pair of biotin binding sites of a streptavidin tetramer, and the second moiety can be bound to a binding site of the pair of binding sites of a SAMA protein.
  • the second moiety can include a nucleotide, an enzyme inhibitor, an enzyme substrate, an enzyme cofactor, derivatives of these, and/or chemical analogs of these.
  • Each member of a pair of biotin binding sites can be symmetric about a dyad axis of a streptavidin tetramer.
  • a dyad axis of the SAMA protein about which each member of a pair of designated surface amino acid residues and/or a pair of binding sites are symmetric can be colinear with a dyad axis of the streptavidin tetramer.
  • the biotin binding capability of the streptavidin should be able to be regenerated.
  • Regeneration means that the capped binding sites can be linked through another protein to empty, available biotin binding sites.
  • the SAMA can include two or more binding sites. These binding sites can be, for example, separated from each other by from about 10 Angstroms to about 30 Angstroms. Each binding site can lie within about 8 Angstroms of a plane in which a side chain atom of each designated surface amino acid residue and the other binding site lie.
  • the binding sites and the bifunctional crosslinking reagents which can bind to them are further discussed below.
  • MJ0577 protein dimer isolated from the thermostable bacterium Methanococcus jannaschii can serve as a SAMA (see Fig. 8).
  • MJ0577 belongs to a large family of proteins involved in stress responses, termed universal stress response proteins (Usp) (Sousa & McKay (2001); Saveanu et al. (2002)).
  • Usp universal stress response proteins
  • the widespread occurrence of Usp domains either isolated or as parts of larger proteins suggests additional ligand- activated roles (Siegele 2005).
  • other universal stress response proteins or universal stress response domains in large proteins may be used as SAMAs.
  • a SAMA can have an amino acid composition homology (similarity) by relative proportion of amino acid composition of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% to MJ0577 or to another naturally occurring protein, or can have complete sequence homology (similarity) with MJ0577 or another naturally occurring protein except for one, two, or four amino acid residues at suitable positions on the surface of the SAMA to serve as designated amino acid residues for biotin binding sites.
  • Figure 8 presents a cartoon representation of a SAMA and a computer representation of the MJ0577 protein dimer structure.
  • the streptavidin macromolecular adaptor (SAMA) embodiment, MJ0577 is a dimeric protein with C2 symmetry.
  • Part a shows a cartoon of a SAMA.
  • Part b designates binding sites for two specific binding ligands, such as nucleotides or nucleotide derivatives, related by a dyad axis of symmetry and spaced approximately 24.5 Angstroms apart.
  • Part c shows an all atom (excluding hydrogen atoms) stick bond representation of an embodiment of a SAMA based on a modified form of the MJ0577 protein.
  • Part d shows the positions of two surface reactive groups on the SAMA dimer allowing the covalent attachment of biotin groups through reaction with an appropriate chemical reagent.
  • Part e shows two adenosine triphosphate (ATP) molecules, shown in space filling representation, bound to the MJ0577 protein and spaced approximately 24.5 Angstroms apart.
  • Part f shows a surface representation of the MJ0577 SAMA embodiment showing overall shape. Chemical structures for schematic representations of cross-link chemistry are given in Figure 23.
  • MJ0577 has 162 residues with no cysteines, so that minimal tailoring or engineering is required and no stabilizing disulfides need to be removed.
  • MJ0577 is thermostable to at least 80 0 C (Zarembinski et al. 1998), and this thermostability implies the chemical stability required for chemical derivatization.
  • the structure of MJ0577 has been determined to 1.7 Angstrom resolution (Zarembinski et al. 1998), and exhibits two-fold symmetry, and appropriate overall molecular dimensions and shape for a bidentate interaction with streptavidin. MJ0577 has been expressed in E. coli.
  • the protein was purified by incubating the soluble protein extract at 80 0 C, and then removing denatured proteins by centrifugation. Subsequent anion exchange chromatography over DEAE Sepharose produced protein that readily crystallized. The protocol yielded about 2.5 mg of purified protein per liter of cell culture (Zarembinski et al. 1998). The expression and purification protocol used standard methods, so that MJ0577 can be routinely produced.
  • MJ0577 The structure of MJ0577 is such that an ATP (adenosine triphosphate) molecule can be fit to electron density at each ligand-binding pocket of the dimer (Zarembinski et al. 1998). Such binding to ATP can make MJ0577 suitable as an ATP-dependent molecular switch or ATPase. These ligand-binding pockets of MJ0577 can serve as binding sites so that the biotin binding capability of a streptavidin "capped" by MJ0577 can be regenerated.
  • ATP adenosine triphosphate
  • MJ0577 the high intrinsic stability, ease of production and purification, and ligand-binding capabilities of MJ0577 suggest its use as an integral biomolecular component of nanostructures and suggest its use in the solid-state synthesis of nanostructures.
  • the SAMA based on MJ0577 is approximately 80 Angstroms (8 nanometers) wide by 50 Angstroms deep (5 nanometers) by 45 Angstroms (4.5 nanometers) long in the direction through which it is connected to streptavidin.
  • Figure 9 presents a computer model showing apposition of the MJ0577 dimer
  • MJ0577 can be engineered using site-specific modification of the native
  • MJ0577 gene to add surface cysteine residues.
  • cysteine residues There are no cysteine residues present in the native MJ0577 structure, so there is no necessity to replace any cysteine residues in the native protein sequence.
  • One surface cysteine residue can be placed in each monomer of the dimer.
  • the cysteine residues can be placed to allow covalent attachment of biotin groups such that they uniquely can occupy only the closest pair of the streptavidin biotin binding sites, separated by about 20.5 Angstroms (the second possible pair of streptavidin biotin binding sites are separated by about 33.5 Angstroms).
  • each surface cysteine residue can react with a thiol-reactive biotinylating reagent and thus serve as a covalent attachment site for a biotin alkylating reagent ( Figure 2).
  • Figure 2 use of computer modeling methods suggests several alternative positions in the native MJ0577 protein sequence where an existing surface amino acid residue can be replaced by an amino acid with a reactive side chain allowing the chemical attachment of biotin. Examples of specific embodiments that are capable of biotinylation with the sulfhydryl reactive reagents shown in Fig.
  • 2b include the following substitutions: K29 to C29, L31 to C31, K32 to C32, A33 to C33, E93 to C93, D94 to C94, V95 to C95, or G96 to C96, where a single residue in the native chain may be substituted by cysteine to provide for covalent attachment of one biotin group per SAMA subunit (Fig. 10).
  • the letter is the standard abbreviation for an amino acid, and the number is the location of the amino acid in the protein sequence.
  • “L31” indicates the leucine at the 31 position.
  • Figure 10 presents SAMA amino acid sequences based on the MJ0577 protein.
  • Sequence A is the native sequence of the MJ0577 protein, a polypeptide containing 162 amino acid residues.
  • Sequence(s) in C are variants of sequences B where either or both the amino and carboxy terminus of the polypeptide chains have been extended by the incorporation of functional polypeptide sequences (Sequences D and E) to aid in isolation, immobilization, or additional chemical functional ization of the SAMA.
  • the functional polypeptide sequences are connected to the SAMA or to each other by short linking sequences that may for example incorporate serine (S) and glycine (G) amino acid residues.
  • functional amino terminus extension Sequence Dl is a poly-histidine tag that binds to metals for immobilization or isolation
  • D2 is a polypeptide tag that binds to streptavidin
  • sequences D3, D4, and D5 are streptococcal protein A or G sequences that bind to immunoglobulins.
  • the sequence DO is a specific protease cleavage site for the endoprotease Factor Xa. Inclusion of a protease site in the sequence allows controlled release or removal through proteolysis of one or more of the functional domains bound to SAMA.
  • E Sequences are the same as D sequences, but are attached via linkers to the carboxy terminus of the SAMA polypeptide chain.
  • Part a shows a cartoon or schematic (used in other diagrams)
  • part b shows an all atom representation of the SAMA embodiment MJ0577 in stick bond representation.
  • Part c shows the covalently attached biotin moieties in schematic and in space filling representation
  • part d shows the SAMA ATP binding sites in schematic and in space filling representation.
  • Chemical structures for schematic representations of cross-link chemistry are given in Figure 23. [001 13] Although the site-specific modification of individual residues is not expected to alter global properties of the native MJ0577 dimer, any such alterations can be monitored. Molecular properties of the engineered MJ0577 SAMA and the native MJ0577 from which it was derived can be monitored by SDS PAGE during the purification and verified by electrospray ionization mass spectroscopy (ESI MS) of the purified proteins.
  • ESI MS electrospray ionization mass spectroscopy
  • the various SAMAs made from these proteins can have a range of different types of ligand binding sites, so that the structures can be linked together with protein nodes or streptavidin using different types of bi-functional cross-linking chemistry.
  • Each of these various SAMAs with different linking chemistry can be used independently of other types of SAMAs or in combination with other types of SAMA to expand the diversity and complexity of structures that can be assembled from protein building blocks.
  • the Universal Stress Protein from Aquifex aeolicus (Protein Data Bank (PDB) code Iq77) is an example of another dimeric protein besides MJ0577 that binds ATP ligands and can be engineered to produce a SAMA.
  • the amino acid sequence is as follows: SNAMKVLLVLTDAYSDCEKAITYAVNFSEKLGAELDILAVLEDVYNLERANVTFGLP FPPEIKEESKKRIERRLREVWEKLTGSTEIPGVEYRIGPLSEEVKKFVEGKGYELVVW ACYPSAYLCKVIDGLNLASLIVK. (SEQ. ID NO. 1)
  • the Tryptophanyl-tRNA synthetase (EC 6.1.1.2) (tmO492) from Thermotoga maritima (PDB code 2g36) is an example of a dimeric protein that can be engineered to produce a SAMA.
  • This protein has specific binding sites for the amino acid tryptophan, chemical derivatives of which can, for example, form one end of a SAMA bifunctional linking reagent.
  • the amino acid sequence (including an N-terminal His tag incorporated for ease of isolation) is as follows:
  • Thermotoga maritima (PDB code 2ftz) is an example of a dimeric protein that can be engineered to produce a SAMA.
  • This protein has specific binding sites for various hydrocarbon chains, chemical derivatives of which can, for example, form one end of a SAMA bifunctional linking reagent.
  • the amino acid sequence (including an N-terminal His tag incorporated for ease of isolation) is as follows:
  • the 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase enzyme from Thermotoga maritima (PDB code 1Q8A) is an example of a dimeric protein that can be engineered to produce a SAMA.
  • This protein has specific binding sites for various folate and folate analogs, chemical derivatives of which can, for example, form one end of a SAMA bifunctional linking reagent.
  • sequence of the first 566 residues of the chain that form an intact structural domain suitable for a SAMA is as follows: MRNRREVSKLLSERVLLLDGAYGTEFMKYGYDDLPEELNIKAPDVVLKVHRSYIESG SDVILTNTFGATRMKLRKHGLEDKLDPIVRNAVRIARRAAGEKLVFGDIGPTGELPYP LGSTLFEEFYENFRETVEIMVEEGVDGIIFETFSDILELKAAVLAAREVSRDVFLIAHM TFDEKGRSLTGTDPANFAITFDELDIDALGINCSLGPEEILPIFQELSQYTDKFLVVEPN AGKPIVENGKTVYPLKPHDFAVHIDSYYELGVNIFGGCCGTTPEHVKLFRKVLGNRK PLQRKKKRIFAVSSPSKLVTFDHFVVIGERINPAGRKKLWAEMQKGNEEIVIKEAKTQ VEKGAEVLDVNFGIESQIDVRYVEKIVQTLPYVSNV
  • Thermotoga maritima (PDB code 2glu) is an example of a dimeric protein that can be engineered to produce a SAMA.
  • This protein has specific binding sites for adenosine monophosphate, chemical derivatives of which can, for example, form one end of a SAMA bifunctional linking reagent.
  • the amino acid sequence (including an N-terminal His tag incorporated for ease of isolation) is as follows:
  • a biotinylation reagent can be reacted with a designated surface amino acid residue on a SAMA to bond, for example, to covalently bond, a biotin group or biotin derivative group to the surface residue.
  • a "biotin-type group” refers to a biotin group or a biotin derivative group that can include biotin, iminobiotin, derivatives of biotin and/or iminobiotin, and/or chemical analogs of biotin and/or iminobiotin.
  • Biotinylation reagents can vary, for example, in the length of the chemical linker and chemical structure of the chromophore ( Figure 23).
  • a thiol-reactive biotinylation reagent can consist of biotin or a biotin derivative group, such as iminobiotin ( Figure 23bl), attached via an amide linkage to an aliphatic hydrocarbon chain linked to a chromophore by a sulfur-sulfur bond.
  • a range of thiol-reactive biotinylating reagents is commercially available. Additional variations are known or can be readily synthesized (e.g., Figures 23b2 through b4).
  • Such reagents can vary, for example, in the chemical structure of the leaving group, nature of the chemical groups forming the linker, number of methylene carbons between amide and S-S bond, and chemical structure of the biotin derivative.
  • sulfosuccinimidyl 2-biotinamido- ethyl-l,3-dithiopropionate EZ-Link sulfo-NHS-SS-biotin, Pierce, Rockford IL
  • Figure 2c schematically depicts the S-S linkage formed between biotin and a
  • a biotinylation reagent with a chemical linker sufficiently long enough to bridge between a SAMA, such as MJ0577, and streptavidin should be selected. If the chemical linker is too short, then the SAMA and streptavidin cannot make a binary attachment. On the other hand, if the chemical linker is too long, only monovalent attachment of the SAMA to the streptavidin may occur and/or unintended high order aggregates may be formed.
  • a SAMA such as MJ0577
  • biotinylated with a thiol-reactive biotinylating reagent at two selected surface residues, such as cysteine residues can be linked to a streptavidin, in that each biotin group on the SAMA is bonded with a biotin binding site on streptavidin.
  • Such a complex can be termed a biotin-residue linked 1:1 streptavidin complex. This complex is shown schematically and as a molecular model in Fig. 12.
  • the streptavidin spans a distance of about 60 Angstroms along the common molecular two-fold axis and the SAMA spans a distance of about 50 Angstroms along the axis, so that the complex as a whole spans a distance of about 110 Angstroms along the axis.
  • Figure 12 presents a cartoon and a molecular model of a biotin-linked 1 :1 SAMA:streptavidin complex based on the MJ0577 protein, and illustrates regeneration of binding capability to the complex.
  • Part a shows a schematic view of the SAMA.
  • Part b shows a schematic view of streptavidin.
  • Part c shows a stick bond representation of the SAMA.
  • Part d shows a stick bond representation of the streptavidin.
  • Part e designates the covalently linked biotins linking the SAMA dimer and streptavidin tetramer in schematic and space filling representation.
  • Part f shows the reaction of the 1:1 SAMA: streptavidin complex with a bifunctional linking reagent, producing the SAMA product g with regenerated ability to bind additional streptavidin tetramers h.
  • Association of the streptavidin with the 1:1 SAMA:streptavidin produces a 1:2 SAMA:streptavidin complex i, that regenerates the biotin binding functionality lost on formation of the initial streptavidin: SAMA complex.
  • Chemical structures for schematic representations of cross-link chemistry are given in Figure 23.
  • specialized surrogate ligands for biotin can be developed to act as temporary linking ligands for the biotin binding sites on streptavidin.
  • a thiol-reactive biotinylating reagent such as the sulfosuccinimidyl 2- biotinamido-ethyl-l,3-dithiopropionate mentioned above, is a bifunctional crosslinking agent.
  • the thiol-reactive group can covalently bond with the thiol group of a surface cysteine residue, such as on an engineered SAMA, and the biotin group can react with a biotin binding site, such as on streptavidin. However, once the covalent bond with the surface cysteine and the bond with the biotin binding site are formed, these are essentially irreversible.
  • Controllable bifunctional crosslinking agents can facilitate the isolation and assembly of biomolecular components and building blocks of several biomolecular components into complex nanostructures.
  • controllable bifunctional crosslinking agents can be designed to fit into binding sites in order to provide biotin functionality or regenerate the biotin binding capacity of a SAMA-capped streptavidin.
  • the controllable bifunctional crosslinking agent can be selected to undergo a chemical change when subjected to an external stimulus applied by a user.
  • controllable bifunctional crosslinking agent can be selected to covalently bond to the ligand binding site of a SAMA when irradiated with light.
  • controllable bifunctional crosslinking agent can be selected to release from a streptavidin binding site when the pH drops below a predetermined value.
  • a controllable bifunctional crosslinking reagent can include a first moiety and a second moiety.
  • the first and/or the second moiety can be selected to bind only upon the application of an external stimulus or change in an environmental condition, such as irradiation with light, or to release binding upon the application of an external stimulus or change in an environmental condition, such as a decrease in ambient pH.
  • Figure 23 shows chemical structures and schematic illustrations of reagents
  • Parts al through a4 of Figure 23 show a variety of biotinylation reagents that reacts with free sulfhydryl groups.
  • Part a5 shows a biotin derivative functionalized by incorporation of the linker H 2 N((CH 2 ) 2 )SH to provide a free SH group for subsequent reaction.
  • Linker length can be varied by extension of the thiol alkyl amine moiety (e.g. H 2 N((CH 2 ) 2 )SH moiety can be lengthened to H 2 N((CH 2 ) n )SH as required).
  • Parts bl through b4 of Figure 23 show a variety of biotinylation reagents that reacts with free sulfhydryl groups.
  • Part b5 shows an iminobiotin derivative functionalized by incorporation of the linker H 2 N((CH 2 ) 2 )SH to provide a free SH group for subsequent reaction.
  • Linker length can be varied by extension of the thiol alkyl amine moiety (e.g. H 2 N((CH 2 ) 2 )SH moiety can be lengthened to H 2 N((CH 2 ) n )SH as required).
  • Part cl shows a bifunctional biotin-ATP photo crosslinking reagent incorporating the photo-crossl inking group 2-azido adenosine.
  • Part c2 shows an alternate bifunctional biotin-ATP photo crosslinking reagent incorporating the photo-crosslinking group 8-azido adenosine.
  • Part dl shows a 2-azido adenosine photo-crosslinking reagent functionalized to form S-S bonds with free sulfhydryl groups.
  • Part d2 shows an 8-azido adenosine photo- crosslinking reagent functionalized to form S-S bonds with free sulfhydryl groups.
  • Part el shows a 2-azido adenosine photo-crosslinking reagent functionalized by incorporation of the linker H 2 N((CH 2 ) 2 )SH to provide a free SH group for subsequent reaction.
  • Linker length can be varied by extension of the thiol alkyl amine moiety (e.g. H 2 N((CH 2 ) 2 )SH moiety can be lengthened to H 2 N((CH 2 ) n )SH as required).
  • Part e2 shows an 8-azido adenosine photo- crosslinking reagent functionalized by incorporation of the linker H 2 N((CH 2 ) 2 )SH to provide a free SH group for subsequent reaction.
  • Linker length can be varied by extension of the thiol alkyl amine moiety (e.g. H 2 N((CH 2 ) 2 )SH moiety can be lengthened to H 2 N((CH 2 ) n )SH as required).
  • Part fl shows a bifunctional di-biotin crosslinker formed by reaction of reagents al and a5.
  • Part f2 shows a bifunctional di-biotin crosslinker with terminal groups interconnected by a polyethylene oxide linker.
  • Part g shows a bifunctional biotin-photo-ATP crosslinker formed through reaction of reagents a5 and dl.
  • An alternative structure can be formed by reaction of reagents a5 and d2.
  • Part h shows a bifunctional photo-ATP crosslinker, formed by reaction of reagents dl and el, dl and e2, or d2 and e2.
  • Part i shows HABA, a dye that absorbs light when bound to streptavidin and which is useful in spectrophotometric monitoring of the assembly of biotin-linked streptavidin: SAMA complexes.
  • HABA derivatives are also useful in this application.
  • Part j shows an ATP derivative labeled with a fluorescent dye, which is useful in spectrophotometric monitoring of the assembly of ATP- linked streptavidin: SAMA complexes.
  • the first and/or the second moiety can be an iminobiotin group.
  • iminobiotin strongly binds to a streptavidin binding site, with an iminobiotin dissociation constant of about 10 "11 M (Hoffmann et al. 1980). Although this value is 3 orders of magnitude greater than the biotin-streptavidin dissociation constant, the binding of iminobiotin to streptavidin is nearly irreversible in the usual context of biochemical interactions. However, at pH values of less than or equal to about 4, the imino group on iminobiotin becomes charged, so that it is displaced from the streptavidin binding site.
  • a structure can be temporarily formed using an iminobiotin-functionalized crosslinking reagent.
  • a temporary structure can, for example, guide the formation of a permanent structure using a biotin-functionalized crosslinking reagent. Then, by lowering the pH below 4, the iminobiotin bonds can release, and components of the temporary structure can be washed away.
  • a bifunctional crosslinking reagent with an iminobiotin group as the first and/or second moiety can be used to form a temporary scaffold, which assists in the construction of a permanent nanostructure.
  • annealing is known to be an important step in forming high quality crystals with a low density of defects. In the context of complex and extensive nanoassemblies, such "annealing” can be useful in "healing” defects arising during construction of the nanoassembly.
  • controllable bifunctional crosslinking reagent that can provide such reversible binding functionality is sulfosuccinimidyl 2-iminobiotinamido-ethyl- 1,3-dithiopropionate, a reagent where the usual biotin group ( Figure 23al through a4 and Fig. 2b) is replaced by a 2-iminobiotin group ( Figure 23bl through b4 and Fig. 2d).
  • This chemical structure and others shown in Figure 23 are either commercially available (Pierce, Rockford IL) or are readily synthesized using conventional organic chemistry methods by one skilled in the art.
  • an iminobiotinylated SAMA can be formed by reacting the sulfosuccinimidyl leaving group to bind the iminobiotinylation agent with, for example, the thiol group of a surface cysteine residue, such as on an engineered SAMA.
  • the iminobiotin group now covalently attached to the SAMA, can then bind with the binding site on streptavidin.
  • the result is an iminobiotin- residue linked streptavidin:SAMA complex.
  • Figure 7h shows a schematic of an iminobiotinylated SAMA.
  • the first and/or the second moiety of a controllable bifunctional crosslinking reagent can be selected to have binding with an intended site that is activated by an external stimulus or change in an environmental condition.
  • the first and/or the second moiety can be a photoactivated group, such as an azidoadenosine triphosphate group.
  • the photoactivated group can form a covalent bond with a corresponding binding site.
  • an azidoadenosine triphosphate group can react to form a covalent bond with an ATP binding site, such as found on MJ0577.
  • Such binding that is activated by an external stimulus or environmental condition can be exploited, for example, as follows.
  • a set of reactions can be carried out in darkness.
  • the photoactive groups of controllable bifunctional crosslinking agents do not form bonds in the dark.
  • reagents present may or may not be washed away, and additional reagents may or may not be introduced.
  • the reaction system can be irradiated with light.
  • the photoactive groups can react with binding sites to form bonds and form a predetermined second structure. In this way, construction of a nanostructure can be carried out in defined, discrete stages controlled by exposure to light.
  • adenosine triphosphate (ATP) crosslinking reagents can be used as controllable bifunctional crosslinking agents ( Figure 23cl and c2).
  • ATP crosslinking reagents can use an alkyl diamine to couple the biotin valerate (forming an amide bond) and the gamma ATP phosphate group (forming an N-linked phosphamide).
  • An example of a controllable bifunctional crosslinking agent whose binding is activated by exposure to light is 2-azidoadenosine 5 '-triphosphate [g]-5(biotinamido)pentylamine ( Figure 23cl) (Affinity Labeling Technologies, Lexington KY).
  • the biotin group of this agent can bind with the streptavidin biotin binding site, and the 2-azidoadenosine 5'-triphosphate group can bind with the MJ0577 binding site when activated by light.
  • Another example ( Figure 23c2) is 8- azidoadenosine 5'-triphosphate [g]-5(biotinamido)pentylamine (Affinity Labeling Technologies, Lexington KY).
  • These two controllable bifunctional crosslinking agents differ with respect to the position of the photo-active azido group on the adenine ring.
  • One or the other of these reagents may be more suitable, for example, depending on the steric and electrostatic interactions with the selected binding site.
  • one of these reagents may form a bond more readily with the ATP binding site of MJ0577 when irradiated with light than the other reagent.
  • an energetic analysis of the binding can be performed or experiments can be conducted.
  • the biotin group of the bifunctional crosslinking agent can bind with the streptavidin biotin binding site, and the azidoadenosine triphosphate group can bind with the MJ0577 binding site when activated by light.
  • a complex can be formed in which streptavidin is linked to a SAMA through a bifunctional crosslinking agent of which one end is a biotin bound to a binding site on streptavidin, and of which another end is a ligand bound to a binding site on the SAMA, for example, an adenosine triphosphate group bound to the ATP binding site on MJ0577.
  • Figure 23 illustrates several reagents that can be reacted to form bifunctional linkers that are linked through S-S bonds.
  • the S-S linked bifunctional reagents are assembled through reaction of a reagent with a functional group bearing a free sulfhydryl group (e.g. Figure 23 a5,el,e2) and second reagent bearing a functional group and a thiol reactive leaving group (Figure 23 al through a4, bl through b4, dl and d2).
  • linkers can be generated using commercially available reagents (Affinity Labeling Technologies, Lexington KY; Pierce, Rockford IL) or compounds readily synthesized by one with skill in the art.
  • Bifunctional crosslinking agents linked through S-S bonds provide additional flexibility in the assembly of nanostructures, both by providing a greater variety of crosslinking functionality, such as Figure 23 fl, ⁇ ,g, and h,, and also as a means of changing the direction of assembly or polarity of a long assembly of SAMA:streptavidin complexes during the assembly process.
  • Numerous products can be made from reaction of reagents Figure 23al through a4, bl through 4, dl and d2 with the reactants of Figure 23a5,el, and e2.
  • MJ0577 SAMA embodiment has a binding site that precisely fits the adenine ring of ATP and ATP analogs and binds them with high specificity and reasonable (dissociation coefficient estimated to be approximately 10 "5 M) affinity.
  • the relatively weak binding of the ATP group and its azido-analogs are useful as this allows some degree of binding reversibility and "annealing" when assembling complex structures.
  • irradiation of the azido-ATP reagent produces a covalent bond between the reagent and the protein, rendering the binding irreversible. Consequently, such a controllable bifunctional crosslinking agent can link two biomolecular components together upon exposure to light, and then release the two biomolecular components from each other when the pH is decreased below 4.
  • Such an agent can be used, for example, to construct a complex, but still temporary scaffold that guides the construction of a permanent nanostructure.
  • Additional bifunctional crosslinking agents can be developed that enable functionalization of assemblies built using strut-node architecture. These agents incorporate a 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 one end of the linker and a protein-specific reactive agent on the other end.
  • a specific protein-reacting group for example, a group able to react with cysteine side chain thiol group or a polypeptide chain terminal amine group
  • the aforementioned azido-ATP analogs represent 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.
  • di- or multimeric strut or node proteins can be modified forms of enzymes that carry out specific catalytic processes on biochemical substrates, these enzymes will have generally active sites that bind substrates and catalyze reactions with great specificity.
  • covalent inhibitors or suicide substrates are known that irreversibly inhibit the enzyme activity by forming a highly specific covalent bond with the catalytic amino acid side chain groups in the enzyme's active site. These agents are generally termed suicide substrates or covalent inhibitors of enzyme activity.
  • These agents when connected to one end of a bifunctional crosslinking reagent as described above, can provide a means of specific immobilization of a protein to an underlying strut-node architecture.
  • immunoglobulins, lectins, or other specific binding molecules could be linked to nanostructures constructed of struts and nodes using this means, as outlined in Fig.3f.
  • Such functionalization can be used in nanostructures intended to serve in, for example, filters, diagnostics, or biological sensing applications.
  • controllable bifunctional crosslinking reagents that can be activated to bind and/or be induced to release from binding can be used.
  • the binding and/or release from binding can be triggered by one or more external stimuli or changes in environmental conditions, including, for example, temperature, visible light, ultraviolet light, change in pH, change in concentration of ionic species other than H + or OH " , temperature, binding of specific molecules, and other conditions.
  • external stimuli or changes in environmental conditions including, for example, temperature, visible light, ultraviolet light, change in pH, change in concentration of ionic species other than H + or OH " , temperature, binding of specific molecules, and other conditions.
  • the changes, for example, binding or release from binding, exhibited by controllable bifunctional crosslinking reagents can be themselves used for sensing or transduction applications.
  • binding sequences include immunoglobulin domains, polyhistidine sequences, polypeptide sequences that bind to streptavidin, for example, StreptagTM (Skerra & Schmidt, 2000), for example, the polypeptide sequences WSHPQFEK (SEQ ID NO: 6) or AWRHPQFGG (SEQ ID NO: 7), Staphylococcus Protein-A, Staphylococcus Protein-G, and others together with sequences designed to be linkers with greater or lesser conformational flexibility (Fig 10).
  • Figures 7i and 7j show a cartoon of SAMAs where each chain of the SAMA dimer has been extended at its amino terminus through a polypeptide linker with a specific binding sequence.
  • Figure 7 presents steps in the formation and assembly of several fiinctionalized SAMA proteins.
  • Part a shows a schematic of a representative SAMA template protein, in this case a symmetric dimer with two ligand binding sites.
  • Part b shows the introduction of a reactive surface amino acid residue into each monomer of the dimer so that the sites are separated by approximately 10.0 to 35.0 Angstroms and lie approximately in the same plane as two ligand binding sites on the dimer.
  • the modified SAMA can be reacted with a biotinylating reagent c (e.g., Figure 23al through a4) capable of reacting with the surface reactive amino acids introduced through site-specific modification of the native protein, producing the di-biotin-substituted SAMA d.
  • a biotinylating reagent c e.g., Figure 23al through a4
  • the SAMA of part d may be further reacted with a bifunctional crosslinking reagent.
  • a bifunctional crosslinker e incorporating biotin on one end and a photo-reactive analog of the SAMA ligand on the other binds to the protein and then becomes covalently attached through photo- crosslinking (e.g., Figure 23cl,c2). This produces the modified SAMA f with 4 covalently bound biotin groups.
  • the SAMA of part b may also be reacted with other reagents such as a reagent g ( Figure 23b through b4) that modifies the protein with 2-iminobiotin groups, allowing reversible, pH-dependent binding, e.g., between the MJ0577 SAMA embodiment and streptavidin.
  • a reagent g Figure 23b through b4 that modifies the protein with 2-iminobiotin groups, allowing reversible, pH-dependent binding, e.g., between the MJ0577 SAMA embodiment and streptavidin.
  • Parts i and j schematically show engineered forms of SAMA where the SAMA gene has been extended at either end with a gene coding for a linker sequence and a polypeptide sequence corresponding to a binding domain.
  • binding sequences include immunoglobulin domains, polyhistidine sequences, polypeptide sequences that bind to streptavidin (StreptagTM), Staphylococcus Protein A, and Staphylococcus Protein G. Depending on the length of the linkers either or both of the binding functions shown in part f can be preserved.
  • Chemical structures for schematic representations of cross-link chemistry are given in Figure 23.
  • Agents may serve to provide information to the user on the state of a nanoassembly. Such agents could, for example, serve to indicate the arrangement of a nanoassembly under construction and thus guide the subsequent steps a user takes during construction.
  • such agents could serve to indicate the arrangement of a nanoassembly whose structure is sensitive to an environmental condition and thus provide a readout for a nanoassembly intended as a sensor.
  • 4'-hydroxyazobenzene-2- carboxylic acid (HABA) and derivatives thereof can serve as biotin displacement detection dyes. These dyes absorb light and/or fluoresce when bound to the biotin binding site in streptavidin. Absorption and/or fluorescence is diminished or abolished when HABA is displaced by biotin.
  • HABA 4'-hydroxyazobenzene-2- carboxylic acid
  • the biotin or biotin derivative of the bifunctional crosslinking agent can bind with streptavidin and the nucleotide or nucleotide derivative can bind with a binding site on the SAMA.
  • the resulting complex can be termed a biotin-nucleotide linked 1:1 streptavidin:SAMA complex.
  • the SAMA can be selected or engineered, so that it is sterically complementary to streptavidin at the streptavidin:SAMA interface. Furthermore, the SAMA can be so selected or engineered, so that ligand binding pockets for a nucleotide or a nucleotide derivative on the SAMA have a favorable position and orientation with respect to the biotin binding sites on streptavidin.
  • a bifunctional crosslinking agent can be selected, for example, to have the appropriate length, so that its biotin group binds with the binding site on streptavidin and its nucleotide or nucleotide derivative group binds with the binding site on the SAMA.
  • the bifunctional crosslinking agent can include a photo-crosslinkable adenosine triphosphate derivative.
  • the bifunctional crosslinking agent can include a moiety having a binding property that varies with pH, such as iminobiotin.
  • iminobiotin adenosine triphosphate (ATP) binding sites. When ATP molecules are bound to these sites, the ATP terminal phosphate groups are spaced approximately 24.5 Angstroms apart. This is similar to the spacing between the termini of two biotin molecules bound to the closest sites on streptavidin, about 20.5 Angstroms.
  • the biotin "tails" are oriented in an approximately horizontal direction in the reference frame of the picture as are the phosphate terminal chains of the ATPs.
  • the bifunctional crosslinking agent can be selected to have a length sufficiently long to bridge the space between the areas of attachment on each biomolecular component, but not so long that aggregation results or, if a link through multiple bifunctional crosslinking agents is intended, only a link through a single bifunctional crosslinking agent results.
  • the bifunctional crosslinking agent can be selected to accommodate SAMA:streptavidin steric interactions and the approximately 4 Angstroms spacing difference between biotin binding sites on streptavidin and ATP binding sites on an MJ0577 SAMA.
  • Both 2-azidoadenosine 5 '-triphosphate [g]-5(biotinamido)pentylamine or 8-azidoadenosine 5 '-triphosphate [g]-5(biotinamido)pentylamine have an appropriate length to link MJ0577 at the ATP binding site with streptavidin at the biotin binding site and form a tight connection between MJ0577 and streptavidin to obtain a rigid complex after the photo ATP reagent is irradiated and forms a covalent bond with MJ0577.
  • the two crosslinking agents differ with respect to the position of the photo-active azido group on the adenine ring.
  • One or the other of these agents may be more suitable, for example, may form a more favorable bonding arrangement with residues in the ATP binding site of MJ0577 when irradiated with light than the other agent.
  • an energetic analysis of the binding can be performed or experiments can be conducted. Purification steps can be used to obtain functional ized, ATP-free SAMA.
  • Figure 13 illustrates the biotin-photo-ATP crosslinker bound to the MJ0577 SAMA embodiment together with the 1 :1 complex formed with streptavidin with a cartoon and a molecular model and illustrates regeneration of binding capability to the complex.
  • Part a shows a schematic of the SAMA with two bound biotin- photo-ATP crosslinks b.
  • Part c shows a stick bond representation of the SAMA structure with the crosslink shown in space-filling representation.
  • Part d shows a schematic of the 1 :1 streptavidin:SAMA biotin-photo-ATP-linked complex.
  • Part e shows a stick bond representation of the complex with the crosslink shown in space-filling representation.
  • Part f shows the reaction of the 1 :1 SAMA:streptavidin complex with a biotin linking reagent, producing the SAMA product g with regenerated ability to bind additional streptavidin tetramers h.
  • a SAMA can prevent the uncontrolled polymerization between streptavidin and biotin functional ized proteins by "capping" the biotin-binding sites on streptavidin.
  • a SAMA should allow for regeneration of the biotin-binding capability of a capped SAMA.
  • a SAMA can be functional ized both by reaction of surface reactive amino acid residues with a biotinylating reagent, providing two biotin binding groups at one side of the SAMA, and by reaction with a bifunctional crosslinker that binds to specific binding sites on the SAMA, providing two additional biotin groups at the opposite side of the SAMA.
  • a thiol-reactive biotinylation reagent having a biotin group at one end and a thiol- reactive group at the other end, can be covalently bonded by the thiol-reactive group to a designated surface amino acid residue on the SAMA.
  • a bifunctional crosslinking reagent having a biotin group at one end and a nucleotide, nucleotide derivative, or ligand at the other end can be bound by the nucleotide, nucleotide derivative, or ligand binding site on the SAMA. All four of the biotin groups of the fully biotinylated SAMA can be coplanar. This can be useful in creating extended structures of defined geometry.
  • a thiol-reactive biotinylation reagent can be covalently bonded to a surface cysteine residue on an engineered MJ0577 protein that serves as a SAMA, and a controllable bifunctional crosslinking reagent having a biotin group on one end and a photoactivated group, such as an azidoadenosine triphosphate group, on the other end can be bound by the photoactivated group to the binding site on MJ0577.
  • Steps that can be used to form such a SAMA bearing two pairs of biotin groups are illustrated in Figs. 7a through 7f.
  • Such a fully biotinylated SAMA is illustrated in Fig.
  • FIG. 14 both in cartoon form in part a and as a molecular model of the SAMA embodiment MJ0577 in part b.
  • the fully biotinylated SAMA shown in Fig. 14 incorporates four biotin groups introduced through reaction with crosslinking reagents.
  • the SAMA is reacted with a biotin-photo-ATP crosslinking agent c at two ligand binding sites and biotinylated on two specific surface sites d. Because the chemistry involved in the introduction of the two pairs of biotins on the SAMA are different, the scheme can be used for the controlled sequential assembly of biotin- streptavidin linked structures. Chemical structures for schematic representations of cross-link chemistry are given in Figure 23.
  • a streptavidin can be "capped" by a SAMA, where two biotin binding sites on a side of the streptavidin tetramer are occupied by biotin groups of a biotinylation reagent covalently bonded to a surface residue on the SAMA.
  • the biotinylation reagent can be covalently bonded to suitable surface amino acids on the SAMA. For example, cysteine surface residues on an engineered SAMA can be reacted to covalently bind with a thiol- reactive biotinylating reagent.
  • sulfosuccinimidyl 2-biotinamido-ethyl-l,3- dithiopropionate can be the thiol-reactive biotinylation reagent.
  • the streptavidin and the SAMA can have steric complementarity at the interface where they meet.
  • the surface residues to which the biotinylation reagent is bound and the biotin binding sites on streptavidin can be located such that there is a unique pairwise interaction between the biotins presented on the functionalized SAMA surface and two of the most closely spaced biotin binding sites on streptavidin.
  • the ability of the "capped" streptavidin to form additional biotin-linked interactions on the same side of the complex occupied by the SAMA can be regenerated as follows (Fig. 12 f,g,h,i). Exposed binding sites on the face of the SAMA opposite the SAMA-streptavidin interface can be reacted with a bifunctional crosslinking agent of which one end is a ligand (such as an ATP group) that binds to the exposed binding site on the SAMA and the other end is a biotin or biotin derivative group.
  • the exposed biotin groups of the bifunctional crosslinking agent bound to the SAMA can then react with two biotin- binding sites on one side of an additional streptavidin tetramer.
  • the two biotin-binding sites on the other side of the additional streptavidin tetramer are then available for receiving biotin groups, so that the biotin-streptavidin binding capability of the streptavidin has been regenerated.
  • a streptavidin can also be "capped" by a SAMA, through an interaction where two biotin binding sites on a side of the streptavidin are occupied by biotin groups of a bifunctional crosslinking reagent, the other end of which is a nucleotide (such as ATP), nucleotide derivative, or ligand bound to a binding site on the SAMA.
  • a nucleotide such as ATP
  • nucleotide derivative such as ATP
  • ligand bound to a binding site on the SAMA ligand bound to a binding site on the SAMA.
  • 2- azidoadenosine 5 '-triphosphate [g]-5(biotinamido)pentylamine
  • 8-azidoadenosine 5'- triphosphate [g]-5(biotinamido)pentylamine can be the bifunctional crosslinking reagent.
  • Figs. 13d and 13e Such a configuration is illustrated in Figs. 13d and 13e.
  • the streptavidin and the SAMA can have steric complementarity at the interface where they meet.
  • the SAMA ligand binding site shown as an ATP binding site
  • the biotin binding sites on streptavidin can be located such that there is a unique pairwise interaction between the biotins presented on the functionalized SAMA surface and two of the most closely spaced biotin binding sites on streptavidin.
  • the ability of the "capped" streptavidin to form additional biotin-linked interactions on the same side of the complex occupied by the SAMA can be regenerated as follows (Fig 13 f,g,h,i).
  • a biotinylation reagent can be covalently bonded to suitable surface amino acids on the face of the SAMA opposite the SAMA-streptavidin interface.
  • the exposed biotin groups linked through the biotinylation reagent to the SAMA can then react with two biotin-binding sites on one side of an additional streptavidin tetramer.
  • the two biotin-binding sites on the other side of the additional streptavidin tetramer are then available for receiving biotin groups, so that the biotin-binding capability of the streptavidin has been regenerated.
  • a SAMA can have an asymmetrical structure or a symmetrical structure.
  • the MJ0577 protein dimer can be viewed as a template for an asymmetrical SAMA.
  • An asymmetric non-biotinylated SAMA is shown in Fig. 8
  • an asymmetric biotinylated SAMA is shown in Fig. 11.
  • Such an asymmetrical SAMA can have chemical groups with different binding properties at opposite ends.
  • the asymmetric SAMA of Fig. 8 has ligand binding sites, e.g., nucleotide binding sites (such as adenosine triphosphate (ATP) binding sites), on its left side (Fig.
  • nucleotide binding sites such as adenosine triphosphate (ATP) binding sites
  • asymmetric SAMA of Fig. 11 has ligand binding sites, e.g., nucleotide binding sites (such as adenosine triphosphate (ATP) binding sites), on its left side (Fig. lid) and has biotin or biotin-type groups, on its right side (Fig. lie).
  • asymmetrical SAMA can be viewed as having a chemical functional "polarity”.
  • a SAMA can have a symmetrical structure.
  • a SAMA with a symmetrical structure can be formed of a single protein subunit or of a single protein multimer.
  • a SAMA with a symmetrical structure can be formed of subcomponents; such subcomponents can include symmetric or asymmetric SAMAs.
  • subcomponents can include symmetric or asymmetric SAMAs.
  • two asymmetric SAMAs such as shown in Fig. 11, can be linked to each other at ends having the same type of chemical functionality, e.g., the same type of ligand binding sites (Fig. lid) can be linked to each other by a bifunctional crosslinking reagent, such as the di-photo ATP bifunctional reagent of Fig. 23h.
  • the resultant symmetric SAMA can have two biotin or biotin-type groups on each of two opposite ends.
  • the resultant symmetric SAMA can have two ligand binding sites, e.g., nucleotide binding sites (such as ATP binding sites) on each of two opposite ends.
  • Two or more biomolecular components can be linked together to form a nanostructure building block.
  • a biotin-residue linked 1 :1 streptavidin:SAMA complex in which two biotins are covalently bonded to surface residues of the SAMA and are bound to binding sites on the streptavidin, such as illustrated by the cartoon of Fig. 6c, can serve as a nanostructure building block.
  • Such a biotin-residue linked 1:1 streptavidin:SAMA building block can be used to form, for example, longer struts of predetermined length having alternating streptavidin and SAMA biomolecular components, as discussed below.
  • a 4:5 streptavidin: SAMA strut as illustrated in Fig. 15, can be formed.
  • SAMA strut can have a defined "polarity". That is, one end of the strut can include a streptavidin with a biotin binding site. The other end of the strut can include a SAMA, either not biotinylated (and thus not “primed” to bind to another streptavidin) or biotinylated (and thus “primed” to bind to another streptavidin).
  • SAMA cyclotavidin
  • a streptavidin can only bind to a biotinylated SAMA.
  • the biotin-nucleotide linked 1: 1 streptavidin: SAMA illustrated in the cartoon Fig. 13d can also be considered as a nanostructure building block.
  • a streptavidin is capped at one end by a SAMA in which the biotins are part of a bifunctional crosslinking reagent bound through an ATP group to the binding site on the SAMA.
  • Figure 16 presents cartoons of some 1 :2 streptavidin:SAMA building blocks.
  • Figure 16a illustrates a streptavidin capped at both ends by biotin-residue linked SAMAs in which the biotins are covalently bound to the SAMA designated surface amino acid residues, for example, cysteine groups.
  • This symmetric complex can be termed a biotin-residue-linked 1 :2 streptavidin:SAMA complex.
  • the biotin- residue-linked 1:2 streptavidin:SAMA complex Fig. 16a can be formed from reaction of a biotinylated SAMA Fig. 6a with a 1 :1 di-biotin linked streptavidin: SAMA complex Fig. 6c.
  • Figure 16b illustrates a streptavidin capped at both ends by SAMAs in which the biotins are part of a bifunctional crosslinking reagent bound through a group, for example, a nucleotide or nucleotide derivative, such as an ATP group, to the binding site on a SAMA.
  • This symmetric complex can be termed a biotin-nucleotide-linked 1 :2 streptavidin:SAMA complex, and can be, for example, a streptavidin:SAMA 1 :2 biotin-photo-ATP linked complex.
  • 16b can be formed from reaction of a SAMA biotinylated through interactions at nucleotide binding sites Fig. 13a with a biotin-nucleotide linked 1 :1 streptavidin: SAMA Fig. 13d.
  • Chemical structures for schematic representations of cross-link chemistry are given in Figure 23. Longer struts can themselves serve as building blocks.
  • Fig. 17 presents synthetic steps for generating an antibody-based biosensor.
  • Figure 17a shows the irradiation induced reaction of a biotin- residue linked 1:1 streptavidin:SAMA complex
  • Fig. 17c with a substrate
  • Fig. 17b that has been functionalized by reaction with a reagent that can bind to a binding site on a SAMA and form a photo-crosslink on irradiation (for example, an ATP photo label, such as the azo-ATP derivatives of Figure 23el and e2).
  • the resultant streptavidin:SAMA complex is bound to the substrate as an immobilized streptavidin: SAMA complex Fig. 17d.
  • An immunoglobulin or antibody Fig. 17e labeled, for example, covalently functionalized, on its Fc region, e.g., the Fc chain termini, with biotin groups, can then be bound to the immobilized streptavidin, to form the biosensor construct Fig. 17f, an immobilized and oriented immunoglobulin complex.
  • FIG. 17g shows the reaction of a biotin-residue linked 1:1 streptavidin: SAMA complex Fig. 17i with a substrate Fig. 17h that has been functionalized by reaction with a biotinylation reagent (for example, Figure 23a5).
  • the complex binds with the functional ized substrate to form the substrate-immobilized streptavidin:SAMA complex Fig. 17j.
  • a substrate can be patterned to be functionalized with biotin in certain areas and functionalized with a ligand for a SAMA (for example, ATP or an ATP derivative) in other areas.
  • the antibody shown in Fig. 17e can be a first antibody type different from a second antibody type shown in Fig. 17k, so that when the antibodies are reacted with the patterned, functionalized substrate, the first and second types of antibodies are bound to different areas on the substrate.
  • FIG. 17m shows the reaction of a SAMA protein Fig. 17o that has been modified through incorporation of an antibody binding sequence, for example, a Staphylococcus Protein-A or Protein-G domain Fig. 10, with a substrate Fig. 17n that has been functionalized by reaction with a reagent (for example, the azo-ATP derivatives of Figure 23el and e2) that can bind to a binding site on a SAMA and form a photo-crosslink on irradiation.
  • a reagent for example, the azo-ATP derivatives of Figure 23el and e2
  • a Protein-A or Protein-G domain can be, for example, covalently incorporated into a SAMA through fusion of the SAMA gene and genes coding for the Protein-A or Protein-G domains.
  • Protein-A and Protein-G domains can fold spontaneously into compact domains that bind strongly to the Fc regions of immunoglobulins.
  • the modified SAMA is bound to the substrate to form the immobilized complex Fig. 17p.
  • An immunoglobulin or antibody Fig. 17q can then be bound to the immobilized SAMA via the specific interactions made between the antibody Fc region and the SAMA bound protein-A or Protein-G domains to form the biosensor construct Fig. 17r, an immobilized and oriented immunoglobulin complex.
  • FIG. 17s through 17x An alternative approach to construction of a biosensor is shown in Fig. 17s through 17x.
  • a substrate Fig. 17t that has been functionalized by reaction with a biotinylation reagent.
  • Figure 17u shows a biotin-linked streptavidin:SAMA complex formed between the biotinylated SAMA of Fig. 7j and streptavidin. The complex binds with the functionalized substrate to form a substrate-immobilized streptavidin:SAMA complex Fig. 17v.An immunoglobulin or antibody Fig. 17w, can then be bound to the immobilized SAMA via interactions between the antibody Fc region and the SAMA bound protein-A or Protein-G domains to form the biosensor construct Fig.
  • FIG. 17x an immobilized and oriented immunoglobulin complex.
  • Chemical structures for schematic representations of cross-link chemistry are given in Figure 23.
  • An advantage of the approaches shown in Fig. 17m and in Fig. 17s, in which an antibody or immunoglobulin is bound by an antibody binding sequence, such as a Staphylococcus Protein-A or Protein-G domain, is that there is no need to chemically modify the antibody itself.
  • the antibody or immunoglobulin must be modified by first binding to it a biotin or biotin derivative or a nucleotide or nucleotide derivative.
  • the substrate can be a metal.
  • the substrate can be a noble metal, such as gold, silver, platinum, palladium, or rhodium.
  • the substrate can be a base metal, such as iron, copper, nickel, zinc, or lead.
  • the substrate can be a metal alloy.
  • the substrate can be a non-metal, such as carbon, e.g., graphite or diamond.
  • the substrate can be a metalloid, such as boron, silicon, or germanium.
  • the substrate can be a metal oxide, such as copper oxide, or a metalloid oxide, such as silicon oxide.
  • the substrate can be a ceramic.
  • the substrate can be a compound of nonmetallic elements or a compound of nonmetallic and metallic elements.
  • the substrate can be a liquid crystal.
  • a substrate can be patterned to be functionalized with biotin in certain areas and functionalized with a ligand for a SAMA (for example, ATP or an ATP derivative) in other areas.
  • SAMA for example, ATP or an ATP derivative
  • the antibodies shown in Figs. 17e, 17k, 17q, and 17w can be antibody types with different recognition specificities, so that when the antibodies are reacted with the patterned, functionalized substrate, the different types of antibodies are bound to different areas on the substrate.
  • FIG. 18 schematically illustrates a method of altering the polarity of a streptavidin:SAMA nanostructure during its assembly that depends upon modification of an immobilized SAMA that is subsequently modified using the S-S linked crosslink chemistry described in Figure 23.
  • Figure 18a shows a substrate functionalized by reaction with a nucleotide, such as an ATP photo label.
  • a nucleotide such as an ATP photo label.
  • Combination with the SAMA of Fig. 18b (Fig. 7b) produces the immobilized SAMA complex Fig. 18c having designated surface amino acid residues, such as free cysteine sulfhydryl groups.
  • Chemical modification of the immobilized SAMA Fig. 18c with different reagents can produce streptavidin:SAMA complexes with different exposed binding functionality.
  • reaction of the immobilized SAMA Fig. 18c with a photo-ATP reagent Fig. 18d produces the immobilized complex Fig. 18e.
  • the immobilized complex Fig. 18e is combined with a 1:1 biotin-linked streptavidin:SAMA complex (Fig. 18f) and irradiated, a structure is produced (Fig. 18g) where the biotin binding sites (Fig. 18f) on the immobilized streptavidin:SAMA complex are exposed for further reaction.
  • Fig. 18i produces the immobilized complex Fig. 18j.
  • Fig. 18j is combined with a 1 :1 biotin-linked streptavidin:SAMA complex Fig. 18k, an immobilized structure Fig. 181 is produced where the photo-ATP binding sites Fig. 18m on the immobilized streptavidin:SAMA complex are exposed for further reaction.
  • the structure, or strut, formed from SAMA and streptavidin shown in Fig. 181 can be further extended.
  • a bifunctional crosslinking reagent having a nucleotide on each end such as the S-S linked di-photo ATP (2-azido ATP) shown in Figure 23h, can be bound at the ATP binding sites Fig. 18m of the SAMA farthest from the substrate Fig. 18a.
  • a SAMA:streptavidin:SAMA structure, with the binding sites of the SAMAs facing away from the streptavidin in the center of the structure, can then be bound to the exposed nucleotide of the bifunctional crosslinking reagent linked to the ATP binding site Fig. 18m.
  • SAMA(d) indicates a SAMA with the designated surface amino acid residues, to which biotins or iminobiotins are bound in Fig. 18, facing away from the substrate Fig. 18a.
  • SAMA(n) indicates a SAMA with the ATP binding sites facing away from the substrate Fig. 18a.
  • a SAMA in the SAMA(d) orientation can be viewed as having a polarity opposite to that of a SAMA in the SAMA(n) orientation.
  • Such a SAMA(d):streptavidin:SAMA(n):SAMA(d):streptavidin: SAMA(n) structure may be advantageous in certain situations.
  • the location of the designated surface amino acid residues on the SAMA can be selected so that their separation is close to 20.5 A, which is the separation between biotin binding sites on streptavidin. Therefore, the distance between, for example, a thiol group and a biotin group on a reagent that links the SAMA to the streptavidin, for example, the thiol-functionalized biotin linking reagent shown in Figure 23aS, can be minimized.
  • the location of the designated surface amino acid residues on the SAMA can be selected so that the spacing between the designated surface amino acid residues is optimal.
  • the location may be selected so that the spacing is such that the distance between the thiol group and the biotin group of the selected reagent is long enough to span between the designated surface amino acid residue on the SAMA and the biotin binding site on the streptavidin, but short enough so that there is not excessive "play" in the selected reagent and the SAMA and streptavidin are locked rigidly together.
  • SAMA(n) blocks will have their ends with the ATP binding sites facing each other.
  • the separation between the ATP binding site on one subunit and the ATP binding site of the other subunit on a given SAMA (SAMA(d) or SAMA(n)) can necessarily be the same as the separation between ATP binding sites on the opposing SAMA (SAMA(n) or SAMA(d)). Therefore, the distance between nucleotide ends on a bifunctional crosslinking, such as shown in Figure 23h can be minimized.
  • Reaction of Fig. 18c with a 2-imino-biotinylation reagent Fig. 18n produces the immobilized complex Fig. 18o.
  • This substrate provides a pH-dependent binding function for the assembly of streptavidin: SAMA nanoassemblies.
  • Figure 19 presents methods for altering the assembly polarity of structures assembled from SAMA and streptavidin using sulfur-sulfur-linked crosslinking reagents (SS- linked crosslinking reagents).
  • Figure 19a shows the immobilized complex of Fig. 18g.
  • a biotin derivative functional ized with a reactive linking group Fig. 19b such as the thiol substituted biotin reagent of Figure 23a5
  • an immobilized complex Fig. 19c is produced with two reactive linking groups.
  • Reaction of the product Fig. 19c with the reagent of Fig. 19d (such as the thiol-reactive photo-ATP reagents of Figure 23dl,d2) produces the immobilized complex Fig.
  • an immobilized complex Fig. 19j is produced with two reactive linking groups.
  • Reaction of the product Fig. 19j with the reagent Fig. 19k (such as the thiol-reactive photo-ATP reagents of Figure 23dl,d2) produces the immobilized complex Fig. 191, with, e.g., photo-ATP groups exposed to allow additional specific structure assembly or immobilization.
  • FIG. 11 Generation of a SAMA biotinylated at surface residues (Fig. 11) can be carried out as follows (Fig. 7b,c,d).
  • a SAMA protein having two designated reactive surface amino acid residues separated by from about 10 Angstroms to about 35 Angstroms can be formed by site specific modification techniques.
  • the reactive amino acids are cysteine residues that have been introduced alternatively at sequence positions 29, 31, 32, 33, 93, 94, 95, or 96, preferably at positions 31 or 33.
  • the SAMA protein can be mixed with at least 2 molar equivalents of a thiol- reactive biotinylation reagent, such as sulfosuccinimidyl-2-biotinamido-ethyl-l,3- dithiopropionate ( Figure 23al) to form a reaction solution.
  • a thiol- reactive biotinylation reagent such as sulfosuccinimidyl-2-biotinamido-ethyl-l,3- dithiopropionate ( Figure 23al) to form a reaction solution.
  • the SAMA protein and the thiol- reactive biotinylation reagent can be allowed to react to form a biotinylated SAMA protein.
  • size exclusion chromatography can be used to completely remove any unreacted biotin reagent, and the extent of SAMA biotinylation measured by removal of an aliquot of the reaction product solution and titration of any remaining free SAMA sulfhydryl groups with Ellman's Reagent (5,5'-dithiobis-(2- nitrobenzoic acid) or DTNB).
  • Ellman's Reagent (5,5'-dithiobis-(2- nitrobenzoic acid) or DTNB).
  • DTNB readily forms a mixed disulfide with thiols to liberate 5-mercapto-2-nitrobenzoic acid, a chromophore with absorption maximum at 410 nm and extinction coefficient -13,600 Cm -1 M '1 .
  • an additional reaction step and/or purification step involving passage of the biotinylated SAMA over a free thiol affinity column to remove any unreacted or mono- biotinylated SAMA will be performed to obtain purified, di-biotinylated SAMA.
  • a SAMA protein having two nucleotide binding sites, such as binding sites for ATP, can be selected or engineered.
  • the SAMA protein can be mixed with at least about 2 molar equivalents of the bifunctional crosslinking reagent to form a reaction solution.
  • the bifunctional crosslinking reagent can include a biotin moiety and a photo-reactive nucleotide moiety, for example the reagents of Figure 23cl and c2.
  • the SAMA protein and the bifunctional crosslinking reagent can be allowed to associate and are irradiated to induce the formation of photo cross- links between the azido- ATP moiety of the cross-linking reagent and the SAMA protein to form a biotin-nucleotide functionalized SAMA protein.
  • Conditions for the reaction can be optimized by performing test reactions in the presence of an ATP analog that binds to the crosslinking ATP binding site and is displaced during the cross-linking reaction.
  • FIG. 23j An example of such a reagent ( Figure 23j) is the fluorescently tagged ATP reagent Adenosine 5'-triphosphate [ ⁇ ]-l- Naphthalenesulfonic acid-5(2-Aminoethylamide) (ATP[ ⁇ ]-l,5-EDANS).
  • the reaction solution can be purified to obtain a substantially pure biotin-nucleotide functionalized SAMA protein. Purification can include, for example, subjecting the reaction solution to one or more of the techniques of size exclusion chromatography, ion exchange chromatography, electrophoresis, or free thiol affinity column chromatography.
  • a biotin-residue linked 1 :1 streptavidin:SAMA complex (Fig. 12a, b) can be formed in solution as follows.
  • a SAMA protein in which biotin or biotin derivative groups are covalently bonded to designated surface amino acid residues can be provided in solution.
  • This biotinylated SAMA protein can be mixed with about a molar equivalent of streptavidin tetramer in solution to form a reaction solution.
  • the biotinylated SAMA protein and the streptavidin tetramer can be allowed to react to form a biotin-residue linked 1 :1 streptavidin:SAMA complex.
  • the procedure may result in the formation of the biotin-residue linked 1:2 streptavidin:SAMA complex (Fig. 16a) as well.
  • the reaction solution can be purified to obtain a substantially pure biotin-residue linked 1:1 streptavidin:SAMA complex. Purifying can include, for example, subjecting the reaction solution to one or more of the techniques of electrophoresis, size exclusion chromatography, ion exchange chromatography, and high performance liquid chromatography.
  • the dyes can be used as spectroscopic probes to directly and quantitatively measure biotin binding to streptavidin (Green 1965). More generally, the dyes can be used to monitor the assembly of streptavidin-linked nanostructures.
  • Several derivatives of the prototype diazo reporter dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA) are available commercially.
  • HABA 2-(4'-hydroxyazobenzene) benzoic acid
  • SAMA complex can then be monitored spectrophotometrically by measuring the release of HABA.
  • reaction products Before or after purification, reaction products can be analyzed by SDS PAGE
  • Products isolated by size exclusion chromatography or high performance liquid chromatography can be analyzed using DLS (dynamic light scattering), ESI MS (electrospray ionization mass spectroscopy), ultraviolet (UV) light detection, refractive index, and/or viscosity measurements to verify molecular weights and characterize the structural integrity and molecular weight dispersity of products.
  • DLS dynamic light scattering
  • ESI MS electrospray ionization mass spectroscopy
  • UV light detection ultraviolet
  • refractive index refractive index
  • viscosity measurements to verify molecular weights and characterize the structural integrity and molecular weight dispersity of products.
  • a biotin-residue linked 1:1 streptavidin:SAMA complex can be formed on an immobilized resin serving as a support matrix as follows. This procedure may produce a higher fraction of 1 :1 streptavidin:SAMA complex than the solution procedure.
  • the complex is immobilized on particles of a support resin that can be present, for example, as a slurry or as a bed in a flow-through reaction column. In either case, the support matrix resin is easily washed, so that any unreacted or excess reagents can be removed, and additional increments of reagents may be added or reaction steps repeated to obtain high product yields.
  • the procedure is illustrated in Fig. 20.
  • Figure 20a shows a schematic of a solid matrix (such as a surface or resin) that has been derivatized with a SAMA (Fig. 18o) that has been functionalized with, for example, 2-iminobiotin.
  • a solid matrix such as a surface or resin
  • SAMA Fig. 18o
  • the solution of streptavidin can be saturated with HABA (Figure 23i).
  • HABA binds 5 orders of magnitude more weakly to streptavidin than does iminobiotin and binds 8 orders of magnitude more weakly to streptavidin than does biotin.
  • Addition of at least one molar equivalent of a SAMA protein Fig. 2Od in which biotin or biotin derivative groups are covalently bonded to designated surface amino acid residues results in formation of the immobilized complex Fig. 2Oe.
  • the biotinylated SAMA protein solution can be flowed over the column to form an immobilized complex, the resin bound biotin-residue linked 1:1 streptavidin:SAMA complex Fig. 2Oe.
  • the biotinylated SAMA protein Fig. 2Od will essentially not displace the substrate bound SAMA functionalized with iminobiotin Fig. 20a from the streptavidin Fig. 20b. This is because for such a displacement to occur, both iminobiotins would have to dissociate from the biotin binding sites on the streptavidin.
  • the eluted solution can be purified to obtain a substantially pure biotin-residue linked 1 :1 streptavidin:SAMA complex.
  • Purifying can include, for example, subjecting the reaction solution to one or more of the techniques of electrophoresis, size exclusion chromatography, ion exchange chromatography and high performance liquid chromatography.
  • the analytical techniques used with the solution reaction can also be used with this immobilized resin reaction. Steps can be carried out in a different order than presented above. Chemical structures for schematic representations of cross-link chemistry are given in Figure 23.
  • a biotin-nucleotide linked 1 :1 streptavidin:SAMA complex (Fig. 13d) can be formed in solution as follows.
  • a SAMA protein that has been functionalized through reaction with a biotin-nucleotide photo-crosslink (Fig. 13a and synthesis described above), and having 2 attached biotin groups geometrically situated to react with one pair of binding sites on streptavidin can be mixed with a solution containing about a molar equivalent of streptavidin tetramer to form a reaction solution.
  • the progress of the association reaction can be monitored by displacement of the dye HABA ( Figure 23i) from the streptavidin binding biotin sites when the SAMA biotin groups bind.
  • 1 :2 streptavidin: SAMA biotin-nucleotide linked complexes may be formed.
  • the reaction solution can be purified to obtain a substantially pure biotin-nucleotide linked 1:1 streptavidin: SAMA complex.
  • Purifying can include, for example, subjecting the reaction solution to one or more of the techniques of electrophoresis, size exclusion chromatography, ion exchange chromatography, and high performance liquid chromatography.
  • a biotin-nucleotide linked 1 :1 streptavidin:SAMA complex can be formed on a support matrix or immobilized resin as follows. This procedure may produce a higher fraction of 1 :1 streptavidin: SAMA complex than the solution procedure. In this resin-based scheme the complex is immobilized on particles of a support resin that can be present as a slurry or as a bed in a flow-through reaction column. In either case, the support matrix resin is easily washed, so that any unreacted or excess reagents can be removed, and additional increments of reagents may be added or reaction steps repeated to obtain high product yields. The procedure is illustrated in Fig. 21.
  • Figure 21a shows a schematic of a solid matrix, such as a surface or resin, that has been derivatized with a SAMA (Fig. 18o) that has been functionalized with 2-iminobiotin.
  • a SAMA Fig. 18o
  • 2-iminobiotin Reaction of about 1 molar equivalent of streptavidin tetramer Fig. 21b with the solid matrix at a pH greater than about 6.5 produces the immobilized streptavidin complex Fig 21c. Because of the "opposite facing" orientation of the pairs of biotin binding sites on the streptavidin tetramer, and the geometrical alignment of the biotin binding sites with the matrix-bound SAMA, the streptavidin is expected to bind to the column resin predominantly through a single pair of the biotin binding sites.
  • the solution of streptavidin can be saturated with HABA (Figure 23i).
  • HABA binds 5 orders of magnitude more weakly to streptavidin than does iminobiotin and 8 orders of magnitude more weakly to streptavidin than does biotin.
  • the solution of SAMA protein functionalized with the covalently bound nucleotide crosslink can be flowed over the column to form the resin bound 1 :1 streptavidin: SAMA complex Fig. 21e.
  • a solution having pH of less than about 4 can be flowed over the column to release the biotin-nucleotide linked 1 :1 streptavidin:SAMA complex into an eluted solution (Figs. 21f and 2Ig).
  • iminobiotin becomes charged, releasing the biotin- nucleotide linked 1:1 streptavidin:SAMA complex Fig. 21g.
  • the eluted solution can be purified to obtain a substantially pure biotin-residue linked 1:1 streptavidin:SAMA complex.
  • Purifying can include, for example, subjecting the reaction solution to one or more of the techniques of electrophoresis, size exclusion chromatography, ion exchange chromatography and high performance liquid chromatography.
  • the analytical techniques used with the solution reaction can also be used with this immobilized resin reaction. This process can also be performed with some steps carried out in a different order.
  • Chemical structures for schematic representations of cross-link chemistry are given in Figure 23. Solution Synthesis of 4:5 streptavidin:SAM ⁇ Strut
  • Fig. 15 illustrates the formation of a 4:5 streptavidin:SAMA strut formed in solution.
  • This structural assembly uses the biotin-residue linked 1:1 streptavidin:SAMA complex as a basic building block.
  • Figure 15a shows a cartoon of a SAMA whose binding sites have been modified by reaction with a bifunctional crosslinking agent having a biotin moiety and a ligand (for example, a nucleotide moiety) attached.
  • Fig. 15a can be the biotin-ATP SAMA construct of Fig. 13a.
  • Fig. 15c can be reacted in solution with at least 2 molar equivalents of a bifunctional crosslinking agent having a biotin moiety and a ligand (for example, a photoactivated-nucleotide moiety, such as the biotin-ATP crosslinking agent shown in Figure 23cl,c2) (Fig. 15d), and the solution irradiated with light to functionalize the available binding sites on a SAMA with biotins and form a reactivated complex, the biotin functionalized 1:2 streptavidin:SAMA complex shown in Fig. 15e.
  • a bifunctional crosslinking agent having a biotin moiety and a ligand for example, a photoactivated-nucleotide moiety, such as the biotin-ATP crosslinking agent shown in Figure 23cl,c2
  • Fig. 15d the solution irradiated with light to functionalize the available binding sites on a SAMA with biotins and form a reactivated
  • Fig. 15e may be monitored using ATP[ ⁇ ]-l,5-EDANS ( Figure 23j) as described above, and the resulting solution may be subjected to purification to separate the pure complex Fig. 15e from any unreacted materials.
  • the steps of reaction with a biotin-residue linked 1 :1 streptavidin complex and functionalization of binding sites on the SAMA with the photo- activated bifunctional crosslinking reagent can be repeated as desired to form a polar strut of predetermined length.
  • Fig. 15f a 4:5 streptavidin:SAMA strut is shown.
  • the strut has additional binding sites for functionalization at both ends enabling connection of the strut to nodes or other biomolecules in a directional and controlled manner.
  • the polar strut Fig. 15f has different chemical binding reactivity sites 15g and 15h at each end of the strut. Chemical structures for schematic representations of cross-link chemistry are given in Figure 23.
  • Figure 22 illustrates the formation of a 4:4 streptavidin:SAMA strut by a solid support matrix or an immobilized resin technique.
  • the growing strut is immobilized on particles of a support resin that can be present as a slurry or as a bed in a flow-through reaction column.
  • the support matrix resin is easily washed, so that any unreacted or excess reagents can be removed, and additional increments of reagents may be added or reaction steps repeated to obtain high product yields.
  • Figure 22a shows a schematic of a solid matrix (such as a surface or resin) that has been derivatized with a SAMA (Fig.
  • a bifunctional crosslinking agent having a biotin moiety and a ligand Fig. 22d for example, a photoactivated-nucleotide moiety Figure 23cl,c2
  • a biotin-photo-ATP crosslinking reagent such as a biotin-photo-ATP crosslinking reagent
  • the reaction mixture is irradiated with light to functionalize the available binding sites on a SAMA with biotins and form the immobilized biotin-nucleotide functionalized 1 :2 streptavidin:SAMA complex Fig. 22e.
  • the newly incorporated biotin binding sites allow for the addition of another 1 :1 biotin-linked streptavidin:SAMA complex.
  • ATP[ ⁇ ]- 1,5-ED ANS Figure 23j
  • the steps of reaction with a biotin-residue linked 1 :1 streptavidin complex and functional ization of binding sites on the SAMA with a photo-activated bifunctional crosslinking reagent can be repeated as desired to form a strut of predetermined length Fig. 22f.
  • a solution having pH of less than about 4 can then be flowed over the column or slurry to release the strut complex from the support matrix into an eluted solution (Figs. 22g and 22h).
  • the eluted solution can be purified to obtain a substantially pure 4:4 streptavidin:SAMA complex.
  • Purifying can include, for example, subjecting the eluted solution to one or more of the techniques of electrophoresis, size exclusion chromatography, ion exchange chromatography and high performance liquid chromatography. This process can also be performed if some steps are carried out in different order.
  • the analytical techniques used with the solution reaction can also be used with this immobilized resin reaction. Chemical structures for schematic representations of cross-link chemistry are given in Figure 23.
  • the location of designated surface amino acid residues on a SAMA can be selected so as to preserve or induce a change in orientation of the SAMA and streptavidin components as a strut is traversed along its length.
  • a line joining the two biotin binding sites on one end of the streptavidin is at a relative angle of about 36 degrees with respect to a line joining the biotin binding sites on the opposite end of the streptavidin. That is, in considering Fig.
  • an observer looking in a direction extending from left to right through the streptavidin would see that the relative orientation of the two biotin binding sites on the left side of the streptavidin differed by 36 degrees from the relative orientation of the two biotin binding sites on the right side of the streptavidin.
  • the binding sites Fig. 8b e.g., a nucleotide binding site, such as an ATP binding site
  • Fig. 8d e.g., a nucleotide binding site, such as an ATP binding site
  • the location of the designated surface amino acid residues Fig. 8d can be selected, so that the relative orientation of the binding sites on the left side is the same as or similar to the relative orientation of the designated surface amino acid residues on the right side. That is, the location of the designated surface amino acid residues on the right side can be selected so that a line connecting them would be parallel or nearly parallel to a line connecting the binding sites on the left side.
  • the location of the designated surface amino acid residues Fig. 8d can be selected, so that the relative orientation of the binding sites on the left side is different from the relative orientation of the designated surface amino acid residues on the right side. That is, an observer looking left to right through the SAMA of Fig.
  • biotin binding sites 8a would see a line connecting the biotin binding sites at an angle with respect to a line connecting the designated surface amino acid residues.
  • the location of the biotin binding sites can be selected, so that this angle is or approximates a predetermined, desired value.
  • an observer looking left to right through the strut of Fig. 22h will see that the relative orientation of the biotin binding sites on the right side (for the observer, the far side) of the first streptavidin is about 36 degrees clockwise with respect to the relative orientation of the biotin binding sites on the left side (for the observer, the near side) of the streptavidin.
  • the location of the designated surface amino acid residues on the left side (for the observer, the near side) of the first SAMA can be selected so that the relative orientation of the binding sites (e.g., nucleotide binding site, such as an ATP binding site) on the right side (for the observer, the far side) of the SAMA is about 36 degrees counterclockwise with respect to the designated surface amino acid residues on the left side (for the observer, the near side) of the SAMA. That is, the location of the designated surface amino acid residues on the SAMA can be selected so that, from the perspective of an observer traveling from left to right through the strut of Fig.
  • the binding sites e.g., nucleotide binding site, such as an ATP binding site
  • the relative rotation of 36 degrees clockwise from the orientation of the near to the far biotin binding sites on the streptavidin is canceled by the relative rotation of 36 degrees counterclockwise from the orientation of the near designated surface amino acid residues to the far binding sites on the subsequent SAMA.
  • the net twist of the relative orientation of the left (for the observer, the near) biotin binding sites of the first streptavidin, through a single streptavidin:SAMA repeating unit, to the relative orientation of the left biotin binding sites of the second, subsequent streptavidin of the next repeating unit can be zero or approximately zero. That is, the relative orientation of streptavidin: SAMA repeating units along the strut can be the same.
  • the location of the designated surface amino acid residues on the left side (for the observer, the near side) of the SAMA can be selected so that the relative orientation of the binding sites (e.g., nucleotide binding site, such as an ATP binding site) on the right side (for the observer, the far side) of the SAMA is zero (or approximately zero) degrees with respect to the designated surface amino acid residues on the left side (for the observer, the near side) of the SAMA. That is, the location of the designated surface amino acid residues on the left side of the SAMA can be selected so that their relative orientation is parallel to the relative orientation of the binding sites on the right side of the SAMA.
  • the binding sites e.g., nucleotide binding site, such as an ATP binding site
  • the net twist of the relative orientation of the left (for the observer, the near) biotin binding sites of the first streptavidin, through a single streptavidin:SAMA repeating unit, to the relative orientation of the left biotin binding sites of the second, subsequent streptavidin of the next repeating unit of the Fig. 22h strut can be 36 degrees (or approximately 36 degrees) clockwise.
  • the net twist can be 180 degrees (or approximately 180 degrees) clockwise).
  • the streptavidin tetramer is D2 symmetric
  • the orientation of the sixth streptavidin repeating unit will be the same as the orientation of the first streptavidin repeating unit.
  • subsequent orientation of the repeating units in the strut lends the strut the appearance of a right-handed helix.
  • SAMA(d):streptavidin:SAMA(n), SAMA(d):SAMA(d): streptavidin, or others, with appropriate reagents to link the SAMA and streptavidin components to each other and the repeating units to each other, along with appropriate selection of the locations of designated surface amino acid residues on the SAMAs, can be made to obtain a strut that preserves orientation among all repeating units (i.e., all repeating units have the same orientation) or can be made to obtain a strut that has essentially any desired rate of twist from one repeating unit to the next with the strut having the form of a right-handed or left-handed helix.
  • struts can be formed from streptavidin(s) and SAMA(s) arranged in a quasiperiodic or an aperiodic order.
  • Such struts formed from streptavidin(s) and SAMA(s) arranged in a quasiperiodic or an aperiodic order can be designed, so that streptavidins and/or SAMAs at selected portions of the chain have a predetermined orientation with respect to each other.
  • Detection methods can include ultraviolet (UV), ultraviolet-visible (UV-Vis), refractive index (RI), and viscosity methods.
  • the extent and uniformity of the structures produced may be determined through direct visualization, for example, with atomic force microscopy (AFM) and electron microscopy (EM).
  • EM electron microscopy
  • a 4:4 streptavidin:SAMA strut has dimensions of about 400 Angstroms (40 nanometers) long by about 80 Angstroms (8 nanometers) in broadest cross section, so that it should be clearly visible with either atomic force microscopy or electron microscopy (see Cherny et al. 1998).
  • biomolecular components and building blocks of several biomolecular components described herein can be functionalized with chemical and/or biochemical groups.
  • the SAMA and/or the streptavidin component of a 1 :1 SAMA:streptavidin complex can be functionalized with biocompounds, inorganic compounds, organic compounds, and/or organometallic compounds.
  • a biomolecular component can be functionalized with one or more organometallic compounds, such as chelate complexes, porphyrins, hemes, chlorophylls, and ferrocene.
  • a biomolecular component can be functionalized with one or more metalloproteins, such as metalloenzymes, iron-sulfur proteins, e.g., ferredoxin, hemoproteins, e.g., cytochrome and hemoglobin.
  • a biomolecular component can be functionalized with inorganic compounds, such as metals, semiconductors, iron-sulfur compounds, and metal and semiconductor nanostructures, such as quantum dots.
  • a biomolecular component can be functionalized with organic compounds.
  • a biomolecular component can be functionalized with organic nanostructures, such as fullerenes and carbon nanotubes and with organometallic nanostructures.
  • a biomolecular component can be functionalized with organic biocompounds, such as proteins, carbohydrates, and glycoproteins.
  • a biomolecular component can be functionalized with a compound, group, or structure that exhibits useful electrical, optical, chemoelectrical, or chemooptical properties.
  • a biomolecular component can be functionalized with a compound, group, or structure to make the biomolecular component useful as a sensor.
  • a biomolecular component can be functionalized, so that its electrical and/or optical properties change in the presence or absence of particular chemical species.
  • a functional group may be placed on each SAMA and each streptavidin of a SAMA:streptavidin strut, only on the SAMAs, or only on the streptavidins.
  • a functional group may be placed only on every other 1:1 SAMArstreptavidin building block.
  • Two different functional groups may alternate on consecutive 1 :1 SAMA:streptavidin building blocks.
  • More complex periodic or aperiodic patterns of one or multiple types of functional groups along a structure, such as a SAMA:streptavidin strut can be made.
  • the control over the spacing between one or multiple types of functional groups on a structure, such as a SAMA:streptavidin strut can be used to control emergent properties arising from interactions among individual functional groups, such as quantum tunneling effects.
  • the MJ0577 wt gene sequence (with the open reading frame in upper case, the ribosome binding site (RBS) in lower case and italics, initiating methionine codon in bold and stop codon in bold) follows: g ⁇ gg ⁇ g ⁇ tatac ⁇ fATGAGCGTCATGTATAAAAAAATCCTGTATCCGACCGACTTTAGC GAAACCGCCGAAATTGCACTGAAACATGTTAAAGCATTTAAAACCCTGAAAGCC GAAGAAGTGATCCTGCTGCATGTCATCGACGAACGCGAAATTAAAAAACGTGAT ATTTTTAGCCTGCTGCTGGGTGTTGCCGGTCTGAACAAAAGCGTGGAAGAATTCG AAAATGAACTGAAAAATAAACTGACCGAAGAAGCGAAAAATAAAATGGAAAAT ATTAAAAGAACTGGAAGACGTGGGCTTTAAAGTCAAGGATATTATTGTTGTG GGCATTCCGCATGAAGAAATTGTTAAAATTGCAGAAGATGAAGGCGTGGATATTATATTATTGTG
  • the MJ0577wt amino acid sequence in standard 1 -letter code and also shown in Figure 10, is: MSVMYKKILYPTDFSETAEIALKHVKAFKTLKAEEVILLHVIDEREIKKRDIFSLLLGV AGLNKSVEEFENELKNKLTEEAKNKMENIKKELEDVGFKVKDIIVVGIPHEEIVKIAE DEGVDIIIMGSHGKTNLKEILLGSVTENVIKKSNKPVLVVKRKNS. (SEQ ID NO: 1 1).
  • the L31C amino acid sequence in standard 1 -letter code and also shown in Figure 10, is MSVMYKKILYPTDFSETAEIALKHVKAFKTCKAEEVILLHVIDEREIKKRDIFSLLLGV AGLNKSVEEFENELKNKLTEEAKNKMENIKKELEDVGFKVKDIIVVGIPHEEIVKIAE DEGVDIIIMGSHGKTNLKEILLGSVTENVIKKSNKPVLVVKRKNS.
  • V95C amino acid sequence in standard 1 -letter code and also shown in Figure 10, is MSVMYKKILYPTDFSETAEIALKHVKAFKTLKAEEVILLHVIDEREIKKRDIFSLLLGV AGLNKSVEEFENELKNKLTEEAKNKMENIKKELEDCGFKVKDIIVVGIPHEEIVKIAE DEGVDIIIMGSHGKTNLKEILLGSVTENVIKKSNKPVLVVKRKNS.
  • SEQ ID NO: 13 Expression vectors for MJ0577wt, L31C and V95C are diagrammed in Figure 24.
  • MJ0577wt was expressed in bacterial cells.
  • MJ0577 wt was induced by addition of 1 mM isopropyl ⁇ -D-1- thiogalactopyranoside (IPTG) when the culture temperature was 27.7 0 C and cell culture optical density OD 6 oo was 1.239. Cells were harvested by centrifugation after 20 hours of growth, and at that time the cell density OD 6 Oo was 1.36. The yield of cells as a wet paste was 48.6 g. The wet paste was frozen and stored at -8O 0 C.
  • IPTG isopropyl ⁇ -D-1- thiogalactopyranoside
  • L31C was expressed in bacterial cells.
  • MJ0577 L31C was induced by addition of 1 mM isopropyl ⁇ -D-1- thiogalactopyranoside (IPTG) when the culture temperature was 27.9 0 C and cell culture optical density OD 600 was 1.1 13. Cells were harvested by centrifugation after 20 hours of growth and at that time the cell density OD 600 was 1.1 1. The yield of cells as a wet paste was 35.7 g. The wet paste was frozen and stored at -80 0 C. Expression of V95C SAMA variant based on MJ0577
  • V95C was expressed in bacterial cells.
  • MJ0577wt and SAMA variants were purified to at least
  • the whole cell lysate was stirred and heated to a temperature between 50 and 7O 0 C for 30 min.
  • the lysate was clarified by centrifugation at 12 000 x g for 15 minutes.
  • the supernatant was made 0.7M in ammonium sulfate by addition of solid salt and applied to a 10 mL Butyl Sepharose Fast Flow column (Pharmacia) previously equilibrated with 25 mM sodium phosphate buffer pH 7.0, 5 mM DTT, 0.5M (NH 4 ) 2 SO 4 .
  • the column was washed with 3 column volumes of the equilibrating buffer, then washed with 3 column volumes of 10 mM sodium phosphate buffer pH 7.0, 0.2M (NH 4 ) 2 SO 4 .
  • MJ0577wt, L31 C or V95C were eluted from the column with a 4-column volume linear gradient starting with 10 mM sodium phosphate buffer pH 7.0, 5 mM DTT, 0.2M (NH 4 ) 2 SO 4 and ending with 5 mM DTT.
  • MJ0577wt-, L31C- or V95C-containing fractions as determined by uv absorbance at 280 nm and polyacrylamide gel electrophoresis (PAGE), eluted near the end of the gradient.
  • PAGE polyacrylamide gel electrophoresis
  • Biotin- and iminobiotin-containing reagents were covalently linked to free cysteine residues on L31C or V95C using the following procedure.
  • the protein was equilibrated in 20 mM sodium phosphate buffer pH 6.8 for reaction with biotin-linking reagents MAL PEO3 ( Figure 23 a3) and MAL PEOl 1 ( Figure 23 a4) or iminobiotin-linking reagent MAL PEO3 ( Figure 23 b3) or equilibrated in 20 mM sodium phosphate buffer pH 7.6 for reaction with biotin-linking reagent EZ-Link HPDP ( Figure 23 a2) by either dialysis (Spectra/Por, 5 000 mW cutoff dialysis tubing, Cole-Palmer) or by using centrifugal protein concentrators (PierceNet) to concentrate the protein to about 10 ⁇ L followed by adding 2 mL of the appropriate buffer.
  • PierceNet centrifugal protein concentrators
  • Protein 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 extinction coefficient of 2980 M "1 cm “1 .
  • Solutions of biotin- and iminobiotin-containing reagents were prepared by adding solid reagent to an appropriate buffer.
  • maleimide-reactive reagents such as MAL PEO3 ( Figure 23 a3 and Figure 23 b3) and MAL PEOl 1 ( Figure 23 a4)
  • the buffer was 20 mM sodium phosphate buffer pH 6.8
  • the sulfur-reactive biotin-linking reagent EZ- Link HPDP Figure 23 a2
  • the dissolving solution was dimethyl sulfoxide (DMSO).
  • Maleimide-reactive reagents were added to the L31C or V95C solutions immediately after dissolution of the solid reagent. The molar concentrations of reagent solutions were at least 20 times that of L31C or V95C.
  • FIG 23 shows the chemical structures of the maleimide-reactive biotin-linking reagents MAL PEO3 ( Figure 23 a3) and MAL PEOl 1 ( Figure 23 a4), the maleimide-reactive iminobiotin-linking reagent MAL PEO3 ( Figure 23 b3), and the sulfur-reactive biotin-linking reagent (EZ-Link HPDP ( Figure 23 a2)).
  • the extent of derivatization was estimated by PAGE ( Figure 25).
  • FIG 25 a PAGE analysis using 12% Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis- Tris) polyacrylamide gel with 2-(N-Morpholino)ethanesulfonic acid (MES), sodium dodecyl sulfate (SDS) running buffer is shown. Lanes are numbered from left to right. Lane 1 shows purified and unreacted L31C. Lanes 2&8 show L31C reacted with biotin-linking reagent MAL PEOl 1 ( Figure 23 a4). Lane 3 is blank. Lanes 4&6 show L31C reacted with biotin- linking reagent MAL PEO3 ( Figure 23 a3).
  • MES 2-(N-Morpholino)ethanesulfonic acid
  • SDS sodium dodecyl sulfate
  • Lanes 5&7 show L31C reacted with biotin- linking reagent EZ-Link HPDP ( Figure 23 a2). Lane 9 shows V95C reacted with biotin- linking reagent MAL PEOl 1 ( Figure 23 a4).
  • Samples of L31C used in reactions 2, 4, and 5 were purified independently of those used to prepare samples shown in lanes 8, 6 and 7. An upward shift in protein band relative to the purified and unreacted L31C indicates derivatization.
  • Lane 10 shows molecular weight standards (NovexSharp Standards, Invitrogen) with MWs from top to bottom of 260 kDa, 160 kDa, 110 kDa, 80 kDa, 60 kDa, 50 kDa, 40 kDa, 30 kDa, 20 kDa, 15 kDa, 10 kDa and 3.5 kDa.
  • Underivatized L31C migrates between the 20 kDa and 15 kDa standards, as expected for a monomer MW of -18 330 kDa.
  • Streptavidin (herein, SAV) and SAMA solutions were mixed to allow formation of SAV:SAMA and SAV:2SAMA complexes. Streptavidin was added to the individual solutions of derivatized V95C and L31C in 2 to 4 aliquots until 2- to 3-fold molar excesses were achieved. Total reaction volumes ranged from 75 to 800 ⁇ L. Each mixture was allowed to react for at least 2 hours. Analyses by PAGE show formation of the SAV:SAMA and SAV:2SAMA complexes (Figure 26). Figure 26 shows a 4-12% Tris-glycine PAGE analysis of several mixtures where solutions of derivatized L31C and derivatized V95C were combined with solutions of SAV.
  • Tris-glycine gel running buffer (Invitrogen) contained 0.1% sodium dodecyl sulfate (SDS). No reducing agents were included in the running buffer.
  • SAV(B) streptavidin biotin tetramer
  • Uncomplexed SAMA is also dissociated under these conditions, as evidenced by a single band near 18 kDa in Figure 26 lane 1.
  • SAV SAMA complexes migrate as diffuse bands of molecular weights higher than that of the SAV(B) tetramer.
  • SAV:SAMA SAV:L31C derivatized with biotin-linking reagent MAL PEOI l ( Figure 23 a4)
  • SAV:2SAMA SAV:2L31C biotin-linking reagent MAL PEOl 1 ( Figure 23 a4)
  • Figure 26 lane 5 shows SAV in complex with V95C derivatized with biotin-linking reagent MAL PEOI l ( Figure 23 a4).
  • SAMA is bound to a solid matrix known to mimic nucleotides or a resin of immobilized ATP.
  • Resins such as Cibacron Blue (GE Life Sciences) and ProteoEnrich ATP-Binders (EMD Biochemicals) bind SAMA via the SAMA ATP binding sites.
  • the SAMA in complex with the solid matrix is separated from unbound SAMA by centrifugation.
  • the SAMA in complex with the solid matrix is resuspended in a solution of 50 mM sodium phosphate buffer pH 6.8. Streptavidin is then aliquoted into the suspension of SAMA in complex with the solid matrix and the reaction allowed to proceed for at least 2 hours.
  • SAV:SAMA complex bound to the solid matrix via the SAMA ATP binding sites is resuspended in a solution of 50 mM sodium phosphate buffer pH 6.8. Addition of ATP (or nucleotide analog) to the suspension releases the SAV:SAMA complex from the solid matrix.
  • Liu GY, Amro NA Partitioning protein molecules on surfaces: A nanoengineering approach to supramolecular chemistry" Proc Nat Acad Sci (2002)99:5165-5170.
  • Loo JA Edmonds CG, Udseth HR, Smith RD "Effect of reducing disulfide-cotaining proteins on electrospray ionization spectra” Anal Chem (1990)62:693-698.
  • Loo JA Kilby GW “Electrospray Ionization Mass Spectrometry of Peptides and Proteins” in Applied Electrospray Mass Spectrometry (BN Pramanik, AK Ganguly & ML Gross, eds.) Marcel Dekker, NY.
  • a streptavidin macromolecular adaptor (SAMA) protein may be used for the controlled assembly of nanostructure building blocks and struts including streptavidin:SAMA complexes.

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Abstract

L'invention concerne une protéine adaptateur macromoléculaire de streptavidine (SAMA) qui peut être utilisée pour l'assemblage contrôlé de blocs de construction et supports de nanostructures comprenant des complexes streptavidine:SAMA.
PCT/US2008/012174 2007-10-26 2008-10-27 Adaptateur macromoléculaire de streptavidine et complexes de celui-ci WO2009055068A1 (fr)

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US8993714B2 (en) 2007-10-26 2015-03-31 Imiplex Llc Streptavidin macromolecular adaptor and complexes thereof
US9102526B2 (en) 2008-08-12 2015-08-11 Imiplex Llc Node polypeptides for nanostructure assembly
US10481158B2 (en) 2015-06-01 2019-11-19 California Institute Of Technology Compositions and methods for screening T cells with antigens for specific populations
US12258613B2 (en) 2017-03-08 2025-03-25 California Institute Of Technology Pairing antigen specificity of a T cell with T cell receptor sequences

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WO2006058226A9 (fr) * 2004-11-24 2006-07-13 Univ Boston Streptavidines dimeres modifiees et leurs utilisations

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Cited By (6)

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Publication number Priority date Publication date Assignee Title
US8993714B2 (en) 2007-10-26 2015-03-31 Imiplex Llc Streptavidin macromolecular adaptor and complexes thereof
US9102526B2 (en) 2008-08-12 2015-08-11 Imiplex Llc Node polypeptides for nanostructure assembly
WO2010132363A1 (fr) * 2009-05-11 2010-11-18 Imiplex Llc Procédé de fabrication d'une nanostructure protéique
US9285363B2 (en) 2009-05-11 2016-03-15 Imiplex Llc Method of protein nanostructure fabrication
US10481158B2 (en) 2015-06-01 2019-11-19 California Institute Of Technology Compositions and methods for screening T cells with antigens for specific populations
US12258613B2 (en) 2017-03-08 2025-03-25 California Institute Of Technology Pairing antigen specificity of a T cell with T cell receptor sequences

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