DETAILED DESCRIPTION OF THE INVENTION

[0021] Definitions:

[0022] The terms in this application are generally used in a manner consistent with their ordinary meaning in the art. To provide clarity, however, in the event of a disagreement in the art, the following definitions control.

[0023] Assembly Unit: An assembly unit is an assemblage of atoms and/or molecules comprising structural elements, joining elements and/or functional elements. Preferably, an assembly unit is added to a nanostructure as a single unit through the formation of specific, non-covalent interactions. An assembly unit may two or more sub-assembly units. An assembly unit may comprise one or more structural elements, and may further comprise one or more functional elements and one or more joining elements. If an assembly unit comprises a functional element, that functional element may be attached to or incorporated within a joining element or, in certain embodiments, a structural element. Such an assembly unit, which may comprise a structural element and one or a plurality of non-interacting joining elements, may be, in certain embodiments, structurally rigid and have well-defined recognition and binding properties.

[0024] Assembly Unit, Initiator: An initiator assembly unit is the first assembly unit incorporated into a nanostructure that is formed by the staged assembly method of the invention. It may be attached, by covalent or non-covalent interactions, to a solid substrate or other matrix as the first step in a staged assembly process. An initiator assembly unit is also known as an “initiator unit.”

[0025] Bottom-up: Bottom-up assembly of a structure (e.g., a nanostructure) is formation of the structure through the joining together of substructures using, for example, self-assembly or staged assembly.

[0026] Capping Unit: A capping unit is an assembly unit that comprises at most one joining element. Additional assembly units cannot be incorporated into the nanostructure through interactions with the capping unit once the capping unit has been incorporated into the nanostructure.

[0027] Functional Element: A functional element is a moiety exhibiting any desirable physical, chemical or biological property that may be built into, bound or placed by specific covalent or non-covalent interactions, at well-defined sites in a nanostructure. Alternatively, a functional element can be used to provide an attachment site for a moiety with a desirable physical, chemical, or biological property. Examples of functional elements include, without limitation, a peptide, protein (e.g., enzyme), protein domain, small molecule, inorganic nanoparticle, atom, cluster of atoms, magnetic, photonic or electronic nanoparticles, or a marker such as a radioactive molecule, chromophore, fluorophore, chemiluminescent molecule, or enzymatic marker. Such functional elements can also be used for cross-linking linear, one-dimensional nanostructures to form two-dimensional and three-dimensional nanostructures.

[0028] Joining Element: A joining element is a portion of an assembly unit that confers binding properties on the unit, including, but not limited to: binding domain, hapten, antigen, peptide, PNA, DNA, RNA, aptamer, polymer or other moiety, or combination thereof, that can interact through specific, non-covalent interactions, with another joining element.

[0029] Joining Elements, Complementary: Complementary joining elements are two joining elements that interact with one another through specific, non-covalent interactions.

[0030] Joining Elements, Non-Complementary: Non-complementary joining elements are two joining elements that do not specifically interact with one another, nor demonstrate any tendency to specifically interact with one another.

[0031] Joining Pair: A joining pair is two complementary joining elements.

[0032] Nanomaterial: A nanomaterial is a material made up of a crystalline, partially crystalline or non-crystalline assemblage of nanoparticles.

[0033] Nanoparticle: A nanoparticle is an assemblage of atoms or molecules, bound together to form a structure with dimensions in the nanometer range (1-1000 nm). The particle may be homogeneous or heterogeneous. Nanoparticles that contain a single crystal domain are also called nanocrystals.

[0034] Nanostructure or Nanodevice: A nanostructure or nanodevice is an assemblage of atoms and/or molecules comprising assembly units, i.e., structural, functional and/or joining elements, the elements having at least one characteristic length (dimension) in the nanometer range, in which the positions of the assembly units relative to each other are established in a defined geometry. The nanostructure or nanodevice may also have functional substitutents attached to it to provide specific functionality.

[0035] Nanostructure intermediate: A nanostructure intermediate is an intermediate substructure created during the assembly of a nanostructure to which additional assembly units can be added. In the final step, the intermediate and the nanostructure are the same.

[0036] Non-covalent Interaction, Specific: A specific non-covalent interaction is, for example, an interaction that occurs between an assembly unit and a nanostructure intermediate.

[0037] Protein: In this application, the term “protein” is used generically to referred to peptides, polypeptides and proteins comprising a plurality of amino acids, and is not intended to imply any minimum number of amino acids.

[0038] Removing: Removing of unbound assembly units is accomplished when they are rendered unable to participate in further reactions with the growing nanostructure, whether or not they are physically removed.

[0039] Self-assembly: Self-assembly is spontaneous organization of components into an ordered structure. Also known as auto-assembly.

[0040] Staged Assembly of a Nanostructure: Staged assembly of a nanostructure is a process for the assembly of a nanostructure wherein a series of assembly units are added in a pre-designated order, starting with an initiator unit that is typically immobilized on a solid matrix or substrate. Each step results in the creation of an intermediate substructure, referred to as the nanostructure intermediate, to which additional assembly units can then be added. An assembly step comprises (i) a linking step, wherein an assembly unit is linked to an initiator unit or nanostructure intermediate through the incubation of the matrix or substrate with attached initiator unit or nanostructure intermediate in a solution comprising the next assembly units to be added; and (ii) a removal step, e.g., a washing step, in which excess assembly units are removed from the proximity of the intermediate structure or completed nanostructure. Staged assembly continues by repeating steps (i) and (ii) until all of the assembly units are incorporated into the nanostructure according to the desired design of the nanostructure. Assembly units bind to the initiator unit or nanostructure intermediate through the formation of specific, non-covalent bonds. The joining elements of the assembly units are chosen so that they attach only at pre-designated sites on the nanostructure intermediate. The geometry of the assembly units, the structural elements, and the relative placement of joining elements and functional elements, and the sequence by which assembly units are added to the nanostructure are all designed so that functional units are placed at pre-designated positions relative to one another in the structure, thereby conferring a desired function on the completely assembled nanostructure.

[0041] Stringency: The extent to which experimental conditions impose a high degree of complementarity on two nucleic acid sequences to achieve a stable hybridization interaction. Highly or moderately stringent conditions are commonly known in the art. By way of example and not limitation, exemplary conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6× SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C. in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×10 ⁶ cpm of ³² P-labeled probe. Washing of filters is done at 37° C. for 1 h in a solution containing 2× SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1× SSC at 50° C. for 45 min before autoradiography. Other conditions of high stringency that may be used are well known in the art. By way of further example and not limitation, exemplary conditions of moderate stringency are as follows: Filters containing DNA are pretreated for 6 h at 55° C. in a solution containing 6× SSC, 5× Denhart's solution, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution and 5-20×10 ⁶ cpm ³² P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 55° C., and then washed twice for 30 minutes at 60° C. in a solution containing 1× SSC and 0.1% SDS. Filters are blotted dry and exposed for autoradiography. Other conditions of moderate stringency that may be used are well-known in the art. Other conditions of high stringency that may be used are, in general, for probes between 14 and 70 nucleotides in length the melting temperature (TM) is calculated using the formula: Tm(° C.)=81.5+16.6(log[monovalent cations (molar)])+0.41 (% G+C)−(500/N) where N is the length of the probe. If the hybridization is carried out in a solution containing formamide, the melting temperature is calculated using the equation Tm(° C.)=81.5+16.6(log[monovalent cations (molar)])+0.41(% G+C)−(0.61% formamide)−(500/N) where N is the length of the probe. In general, hybridization is carried out at about 20-25 degrees below Tm (for DNA-DNA hybrids) or 10-15 degrees below Tm (for RNA-DNA hybrids).

[0042] Structural Element: A structural element is a portion of an assembly unit that provides a structural or geometric linkage between joining elements, thereby providing a geometric linkage between adjoining assembly units. Structural elements provide the structural framework for the nanostructure of which they are a part.

[0043] Subassembly: A subassembly is an assemblage of atoms or molecules consisting of multiple assembly units bound together and capable of being added as a whole to an assembly intermediate (e.g., a nanostructure intermediate). In many embodiments of the invention, structural elements also support the functional elements in the assembly unit.

[0044] Top-down: Top-down assembly of a structure (e.g., a nanostructure) is formation of a structure through the processing of a larger initial structure using, for example, lithographic techniques.

[0045] PNA Assembly Units

[0046] The present invention provides a new class of assembly units that can be used in production of nanostructures. These “PNA assembly units” contain at least one joining or functional element that is a PNA. In addition, the assembly unit may contain structural elements and/or other joining and functional elements.

[0047] PNA Joining Elements

[0048] In certain embodiments of the invention, a joining element comprises a peptide nucleic acid (PNA) and may have any of a number of general forms, such as that shown in FIG. 1 . PNA is a structural homologue of DNA that was first described by Nielsen et al. (1991, Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide, Science 254: 1497-1500) and has a neutral peptide or peptide-like backbone instead of a negatively-charged sugar-phosphate backbone ( FIG. 1 ). Therefore, a PNA may be viewed as a protein or oligopeptide in which the amino acid side chains have been replaced with the pyrimidine and purine bases of DNA. The same nitrogenous bases (i.e. adenine, guanine, cytosine and thymine) are used in PNAs as are found in DNA and RNA; PNAs bind to DNA and RNA molecules according to Watson-Crick and/or Hoogsteen base pairing rules. PNAs are not generally recognized as substrates by DNA polymerases, nucleic acid binding proteins, or other enzymes, including proteases and nucleases, although some exceptions do exist (see, e.g., Lutz et al., 1997, Recognition of uncharged polyamide-linked nucleic acid analogs by DNA polymerases and reverse transcriptases, J. Am. Chem. Soc. 119: 3177-78). The biology of PNAs has been reviewed extensively (see, e.g., Nielsen et al., 1992, Peptide nucleic acids (PNA). DNA analogues with a polyamide backbone, In Antisense Research and Applications, Crooke and Lebleu, eds., CRC Press, pp. 363-72; Nielsen et al., 1993, Peptide nucleic acids (PNAs): potential antisense and anti-gene agents, Anticancer Drug Des. 8(1): 53-63; Buchardt et al., 1993, Peptide nucleic acids and their potential applications in biotechnology, Trends Biotechnol. 11(9):384-86; Nielsen et al., 1994, Peptide nucleic acid (PNA). A DNA mimic with a peptide backbone, Bioconjug. Chem. 5(1): 3-7; Nielsen et al., 1996, Peptide nucleic acid (PNA): A lead for gene therapeutic drugs, in Antisense Therapeutics Vol. 4, Trainor, ed., SECOM Science Publishers B. V., Leiden, pp. 76-84; Nielsen, 1995, DNA analogues with nonphosphodiester backbones, Ann. Rev. Biophys. Biomol. Struct. 24: 167-83; Hyrup and Nielsen, 1996, Peptide nucleic acids (PNA): synthesis, properties and potential applications, Bioorg. Med. Chem. 4:5-23; De Mesmaeker et al., 1995, Backbone modifications in oligonucleotides and peptide nucleic acid systems, Curr. Opin. Struct. Biol. 5: 343-55; Dueholm and Nielsen, 1997, Chemical aspects of peptide nucleic acid, New J. Chem. 21: 19-31; Knudsen and Nielsen, 1997, Application of PNA in cancer therapy, Anti-Cancer Drug 8: 113-18; Nielsen, 1997, Design of Sequence Specific DNA Binding Ligands, Chemistry 3: 505-08; Corey, 1997, Peptide nucleic acids: expanding the scope of nucleic acid recognition. Trends Biotechnol. 15(6):224-29; Nielsen and Ørum, 1995, Peptide nucleic acid (PNA) as new biomolecular tools, in Molecular Biology: Current Innovations and Future Trends, Part 2, (Griffin, H., Ed.), Horizon Scientific Press, UK, pp. 73-86; Nielsen and Haaima, 1997, Peptide Nucleic Acid (PNA). A DNA Mimic with a Pseudopeptide Backbone, Chem. Soc. Rev.: 73-78).

[0049] In PNA, as shown in FIG. 1 , the phosphoribose backbone may be replaced, for example, by repeating units of N-(2-aminoethyl)-glycine linked by amide bonds (Egholm et al., 1992, Peptide nucleic acids (PNA), Oligonucleotide analogues with an achiral peptide backbone, J. Am. Chem. Soc. 114: 1895-97). Other substitutions in PNA of a neutral peptide or peptide-like backbone for a negatively-charged sugar-phosphate backbone are commonly known in the art and will be readily apparent to the skilled artisan. PNAs with modified polyamide backbones have been described, for example, in Hyrup et al. (1994, Structure-Activity studies of the binding modified Peptide Nucleic Acids, Journal of the American Chemical Society 116: 7964-70); Dueholm et al. (1994, Peptide Nucleic Acid (PNA) with a chiral backbone based on alanine, Bioorg. Med. Chem. Lett. 4: 1077-80); Peyman et al. (1996, Phosphonic Esters Nucleic Acids (PHONAs): Oligonucleotide Analogues with an Achiral Phosphonic Acid Ester Backbone, Angew. Chem. Int. Ed. Engl. 35: 2636-38); van der Laan et al. (1996, An approach towards the synthesis of oligomers containing a N-2-hydroxyethyl-aminomethylphosphonate backbone—A novel PNA analogue, Tetrahedron Letters 37: 7857-60); Jordan et al. (1997, Synthesis of new building blocks for peptide nucleic acids containing monomers with variations in the backbone, Bioorg. Med. Chem. Lett. 7: 681-86); Goodnow et al. (1997, Oligomer Synthesis and DNA/RNA Recognition Properties of a Novel Oligonucleotide Backbone Analog: Glucopyranosyl Nucleic Amide (GNA), Tetrahedron Lett. 38: 3199-3202); Zhang et al. (1999, Studies on the synthesis and properties of new PNA analogs consisting of L- and D-lysine backbones, Bioorg. Med. Chem. Lett. 9: 2903-08); Stammers et al. (1999, Synthesis of enantiomerically pure backbone alkyl substituted peptide nucleic acids utilizing the Et-DuPHOS-Rh+ hydrogenation of enamido esters, Tetrahedron Lett., 40, 3325-3328); Puschl et al. (2000, Pyrrolidine PNA: A Novel Conformationally Restricted PNA Analogue, Organic Letters 2: 4161-63); Vilaivan et al. (2000, Synthesis and properties of chiral peptide nucleic acids with a N-aminoethyl-D-proline backbone, Bioorg Med Chem Lett 10(22):2541-45); Yu et al., 2001, Synthesis and characterization of a tetranucleotide analogue containing alternating phosphonate-amide backbone linkages, Bioorg. Med. Chem. 9(1):107-19); Fader et al. (2001, Backbone modifications of aromatic peptide nucleic acid (APNA) monomers and their hybridization properties with DNA and RNA, J. Org. Chem. 66: 3372-79).

[0050] The nitrogenous bases of a PNA are attached to the neutral backbone by methylene carbonyl linkages. Because PNA does not have a highly-charged sugar-phosphate backbone, PNA binding to a target nucleic acid is stronger than with conventional nucleic acids, and that binding, once established, is virtually independent of salt concentration. This is reflected, quantitatively, by a high thermal stability of duplexes containing PNA.

[0051] Because the peptide backbone is uncharged, base-pairing between two complementary PNA molecules, or between, e.g., DNA and PNA in a DNA/PNA hybrid, is much stronger than in the corresponding DNA/DNA hybrid. Binding of a PNA to its complementary DNA or RNA target will occur more quickly than binding of the equivalent nucleic acid probe. The affinity of the PNA is so high that it can displace the corresponding strand in double stranded DNA (Nielsen et al., 1991, Sequence-selective recognition of DNA by strand displacement with a thymine substituted polyamide, Science 254: 1497-1500).

[0052] PNAs generally have a melting temperature that is higher than the corresponding DNA duplex, by approximately ⁻ 1° C. per base at moderate salt conditions (e.g., 100 mM NaCl) (Nielsen et al., 1991, Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide, Science 254: 1497-1500; Peffer et al., 1993, Strand-invasion of duplex DNA by peptide nucleic acid oligomers, Proc. Natl. Acad. Sci. USA 90: 10648-52; Demidov et al., 1995, Kinetics and mechanism of polyamide (“peptide”) nucleic acid binding to duplex DNA, Proc. Natl. Acad. Sci. USA 92: 2637-41). Thermal stability of a DNA-DNA duplex (as indicated by T _m ) is approximated using an estimate of 2° C. per AT base pair and 4° C. per GC base pair, whereby a 10 bp DNA duplex with 50% GC content would be estimated to have melting temperature of about 30° C. Accordingly, the corresponding PNA therefore would have a melting temperature of about 40° C. Similarly an 18 residue PNA duplex (50% GC) would be estimated to have a melting temperature of about 72° C. Therefore, in certain embodiments of the present invention a PNA joining element has about 8 residues to about 20 residues, about 10 residues to about 18 residues, or about 12 residues to about 16 residues.

[0053] In other embodiments, PNAs having fewer residues can be designed that have higher melting temperatures by taking advantage of the PNA's ability to form triple helices. In a specific embodiment, three PNA strands (two polypyrimidine, one polypurine) form this extremely stable structure. The structure can be further stabilized by using two PNA's such that one has two polypyrimidine PNA stretches separated by a glycine spacer, wherein the glycine spacer generally comprises three to five glycine residues. When mixed with the corresponding polypurine PNA, the two polypyrimidine PNA segments fold around the glycine space to form this triple helix. Having the “two” polypyrimidine strands on the same molecule raises the effective concentration and hence the rate of formation and strength of the triplex helix. For a staged assembly joining pair, one joining element of the joining pair would contain the polypurine strand while the other joining element of the joining pair is a double-length polypyrimidine PNA joining element.

[0054] PNAs may be synthesized by methods well known in the art using chemistries similar to those used for synthesis of nucleic acids and peptides. PNA monomers used in such syntheses are hybrids of nucleosides and amino acids. PNA products, services (such as custom-synthesis of PNA molecules), and technical support are commercially available from PerSeptive Biosystems, Inc. (a division of Applied Biosystems, Foster City, Calif.). PNA may be synthesized using commercially available reagents and equipment or can be purchased from contract manufacturers such as PerSeptive Biosystems, Inc. PNA oligomers may also be manually synthesized using either Fmoc or t-Boc protected monomers using standard peptide chemistry protocols. Similarly, standard peptide purification conditions may be used to purify PNA following synthesis.

[0055] In certain embodiments, a PNA used in the methods of the invention is a chimeric PNA or a binding derivative or modified version thereof, and references to PNA should be understood to encompass both PNSs and these variations. A chimeric PNA is a molecule that is modified at the base moiety or the peptide backbone, and that may include other appending groups or labels. A chimeric PNA also may be a molecule that comprises a PNA sequence linked by a covalent bond(s) to one or more amino acids or to a sequence of two or more contiguous amino acids.

[0056] For example, a chimeric or modified PNA may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil; 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

[0057] In a specific embodiment, a modified or chimeric PNA contains the “universal base” 3-nitropyrrole (Zhang et al., 2001, Peptide nucleic acid-DNA duplexes containing the universal base 3-nitropyrrole, Methods 23: 132-40).

[0058] Once a desired PNA is synthesized, it is cleaved from the solid support on which it was synthesized and treated, by methods known in the art, to remove any protecting groups present. The PNA may then be purified by any method known in the art, including extraction and gel purification. The concentration and purity of the PNA may be determined by examining PNA that has been separated on an acrylamide gel, or by measuring the optical density in a spectrophotometer.

[0059] In certain embodiments of the invention, a joining pair comprises a complementary pair of PNA joining elements that are capable of binding via standard Watson-Crick and/or Hoogsteen base-pairing. A PNA moiety can serve as a joining element, while an oligopeptide, protein, or protein fragment provides a small structural element and, in specific embodiments, the structural element further comprises a functional element, as depicted schematically in FIGS. 19 (A-B). As shown in FIGS. 19 (A-B), two PNA/oligopeptide units can dimerize to form a single assembly unit. The PNA portion provides joining elements A and B′, while the oligopeptide portion forms two coiled coil structural elements.

[0060] Like DNA, PNA/PNA molecules bind most stably in an antiparallel fashion (Wittung et al., 1994, DNA-like double helix formed by peptide nucleic acid, Nature 368: 561-63). For PNA molecules the amino terminus is equivalent to the 5′ end of a corresponding DNA sequence ( FIG. 1 ). Leucine zipper dimers normally bind in a parallel fashion (amino terminus adjacent to amino terminus) (Harbury et al., 1993, A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants, Science 262: 1401-07). Therefore, all the molecules depicted in the assembly units shown in FIG. 2 are shown in a parallel orientation (the amino terminals are the 5′ ends to the left and the carboxy terminals are the 3′ ends to the right).

[0061] In certain embodiments, the assembly unit can have a randomly coiled peptide that comprises a functional element, F, in the internal or center portion of the dimer ( FIG. 2A ) or at the end of the PNA molecule opposite the end comprising the joining element. The two functional elements may be the same or different. The joining elements are designed to obviate uncontrolled assembly to allow for staged assembly using such an assembly unit. In this illustration, at least two complementary pairs of PNA sequences are used. There must be no self-complementation or cross-complementation between the joining pairs.

[0062] FIG. 3 shows the order of elements of the upper synthetic protein monomer forming the staged assembly subunit shown in FIG. 2A . The order of the elements in the corresponding lower unit would be identical except that the PNA element is at the C-terminus. This reflects the parallel arrangement of the leucine zippers aligning the two units. The functionality sequence encodes the region at which a functional element may be added to the assembly subunit. Glycines separate each element to reduce steric interference between elements. Numbers below the line indicate the typical length in residues of each element.

[0063] Formation of a PNA/oligopeptide assembly unit structure may be monitored using the same methodologies commonly known in the art that are used for monitoring protein folding. For example, the oligopeptide portion can be modeled with software that predicts the formation of coiled-coils, e.g. Multicoil (Wolf et al., 1997, MultiCoil: A program for predicting two- and three-stranded coiled coils, Protein Science 6: 1179-89), Paircoil (Berger et al., 1995, Predicting coiled coils by use of pairwise residue correlations, Proc. Natl. Acad. Sci. USA, 92: 8259-63), COILS (Lupas et al., 1991, Predicting coiled coils from protein sequences, Science 252: 1162-64; Lupas, 1996, Prediction and analysis of coiled-coil structures, Meth. Enzymology 266: 513-25) and Macstripe (Lupas et al, 1991, Predicting Coiled Coils from Protein Sequences, Science 252: 1162-64). Standard techniques such as measurement of circular dichroism (CD), e.g., a CD spectrum, can also be used to monitor oligopeptide folding. Moreover, modeling of formation of a joining pair comprising PNA joining elements follows the same rules as DNA-DNA complementary pairing. PNA joining pairs are preferably evaluated using any of a variety of commercial software packages, e.g., Amplify (University of Wisconsin, Madison Wis.), Vector NTI (InforMax, Bethesda Md.), and GCG Wisconsin Package (Accelrys Inc., Burlington Mass.).

[0064] PNA/oligopeptide assembly units differ from other types of assembly units (such a pilin-based immunoglobulin-based assembly units) in several aspects. PNA/oligopeptide assembly units are hybrids of two different classes of biological molecules—PNA and oligopeptide—and are, therefore, chemically synthesized rather than biologically synthesized. Accordingly, a strict level of quality control and testing for each batch of such PNA-containing assembly units is required. These tests include, e.g., sandwich ELISAs and tests for circular dichroism for protein/protein interactions, evaluation of temperatures for PNA joining elements, and SDS-PAGE for determining the percent of full-length molecules.

[0065] The α-helical oligopeptide portion of an assembly unit is about 1 nm long per heptad repeat in embodiments where, for example, leucine zipper protein domains are used as structural elements in the construction of an assembly unit (Harbury et al., 1994, Crystal structure of an isoleucine-zipper trimer, Nature 371: 80-83). In embodiments in which an assembly unit has four to six heptads (28-42 amino acids), the structural element is about 4-6 nm long. The PNA joining element is structurally similar to DNA and has a length of about 0.34 nm/base. Therefore, in certain embodiments, a joining element of 10-18 residues will be about 3 to 6 nm in length and, therefore, such an assembly unit will be about 7-12 nm long.

[0066] PNA/oligopeptide assembly units also differ from other embodiments of the invention disclosed herein in that they are generally less rigid. In a specific embodiment, a PNA-peptide assembly unit has a structural element comprising a leucine zipper structure. Such a PNA-peptide assembly unit has an alpha helical portion that has some flexibility although, in certain embodiments, the presence of two or three helix bundles is not as flexible as an isolated a-helical coil. The PNA portion is relatively flexible, so that a structure assembled according to the staged assembly method of the invention from these units may be more analogous to a string of soft beads than to a rigid rod. In addition, a flexible domain (e.g., a tri-, tetra- or pentaglycine) which, in certain embodiments, links joining elements to structural elements, will add to the flexibility of the assembly unit and higher order structures. Two- and three-dimensional nanostructures made of these units are somewhat flexible as free units. However, upon attachment at multiple points to a solid support or matrix, the nanostructure can be made rigid by applying tension to the overall structure, in a manner analogous to the stiffening of a rope net or a spider web by application of a tensioning force.

[0067] The coiled coil structural elements also allow for flexibility in the design and construction of assembly units and the nanostructures fabricated from those assembly units. Generally, simple leucine zipper type coiled coils, as disclosed above, are not stable enough to hold the assembly units together by themselves but are stabilized by disulfide bridges (see above). Four helical bundles that are found, for example, in the Rop protein, are generally stable enough, at normal room temperature and can be lengthened, as needed, to provide the stability that is required for formation of assembly units. In addition, the distance between functional elements can be adjusted by changing the length of the coiled coils and by adding flexible peptide segments between, e.g., joining and functional elements. This would lead, in certain embodiments, to a flexible nanostructure more akin to a beads-on-a-string type of architecture.

[0068] Because the PNA/protein assembly molecule shares a common backbone, it can be synthesized as a single molecule. It is unnecessary to join the two components together after they are synthesized separately. Custom, contract PNA/protein synthesis is available commercially from PerSeptive Biosystems (division of Applied Biosystems, Framingham Mass.).

[0069] The sequence of each PNA joining element is critical to correct assembly. While designing complementary pairs is relatively easy to those skilled in the art, it is important to ascertain that there is no complementary base pairing between PNAs that will be part of the same assembly unit. There are a variety of DNA software packages known to skilled in the art, that can be used to analyze nucleotide sequences for complementarity, e.g., Amplify (University of Wisconsin, Madison Wis.), Vector NTI (InforMax, Bethesda Md.), and GCG Wisconsin Package (Accelrys Inc., Burlington Mass.). PNA segments that have internal complementarity can form hairpin loops and are preferably avoided according to the staged-assembly methods disclosed herein.

[0070] Table 1 below lists exemplary PNA sequences that can be comprised in joining elements in PNA/protein assembly units, and gives examples of usable and unusable sequences. In preferred embodiments, one member of the PNA joining pair is attached to a single assembly unit. The corresponding member of the joining pair is the direct complementary sequence, and is attached to another assembly unit. The sequences in Table 1 are listed in amino to carboxy (5′ to 3′) orientation. 1

[0071] Complementary binding pairs forming triple helices. “OOOO” represents residues with no base, essentially glycines that allow the PNA to fold back on itself to form the triple helix. 2

[0072] Sequences Unsuitable as Binding Elements

[0073] Sequences with cross-complementation (complementary sequences underlined) 3

[0074] Sequences forming hairpin loops 4

[0075] FIGS. 19 (A-B) contains line diagrams of two possible embodiments of synthetic molecules that can be used in the construction of an assembly unit useful for the present staged assembly methods. As shown in FIGS. 19 (A-B), two PNA/oligopeptide units can dimerize to form a single assembly unit. Two possible assembly units are shown in FIG. 2 A and FIG. 2B . The PNA portion provides joining elements A and B′, while the oligopeptide portion forms two coiled coil structural elements (S) stabilized by disulfide bonds at either end. One or more functional units (F), comprised of, e.g., protein segments, may also be incorporated into the assembly unit. In certain embodiments, the assembly unit can have a randomly coiled peptide that comprises a functional element, F, in the internal or center portion of the dimer ( FIG. 2A ) or at the end of the PNA molecule opposite the end comprising the joining element ( FIG. 2B ).

[0076] In this example, the order of elements (i.e., joining structural, and/or functional elements) in the corresponding next assembly unit (i.e., one to be added next during staged assembly) would be identical, except that the PNA element would be at the C-terminus. This reflects the parallel arrangement of the leucine zippers. Glycines separate each element to reduce steric interference between elements.

[0077] PNA Functional Elements

[0078] In another embodiment, functional elements (depicted as “F”) comprising peptide sequences are placed in two possible locations in an assembly unit formed by leucine zipper dimerization. Sequences can be added to the opposite end of the peptide from, e.g., a PNA, or can be inserted between two shorter α-helices, as shown in FIG. 2 .

[0079] Table 2 sets forth several non-limiting, illustrative examples of functional elements. 5

[0080] In one embodiment, the functional element comprises a PNA segment. Just as PNA can be placed at the end of the monomer during synthesis to serve as a joining element, a segment of PNA, comprising residues capable of base-paring, can be placed into the middle of a synthesized peptide subunit to serve as a functional element. This permits the fabrication of a precisely branched nanostructure, or a nanostructure comprising a PNA-conjugated joining element that is precisely attached to the nanostructure by base-pairing interactions with the structural element-embedded PNA functional element. In preferred embodiments, functional elements, and/or bridging cysteine residues, are generally separated from neighboring structural and/or joining elements by a peptide segment of about two to five glycine residues, so that the protein/peptide domains can form independently.

[0081] Other Elements

[0082] In certain embodiments of the present invention, an assembly unit comprises a structural element. As noted above, that structural element may be a leucine zipper. More generally, the structural element generally has a rigid structure (although in certain embodiments, described below, the structural element may be non-rigid). The structural element is preferably a defined peptide, protein or protein fragment of known size and structure that comprises at least about 50 amino acids and, generally, fewer than 2000 amino acids. Peptides, proteins and protein fragments are preferred since naturally-occurring peptides, proteins and protein fragments have well-defined structures, with structured cores that provide stable spatial relationships between and among the different faces of the protein. This property allows the structural element to maintain pre-designed geometric relationships between the joining elements and functional elements of the assembly unit, and the relative positions and stoichiometries of assembly units to which it is bound.

[0083] The use of proteins as structural elements has particular advantages over other choices such as inorganic nanoparticles. Most populations of inorganic nanoparticles are heterogeneous, making them unattractive scaffolds for the assembly of a nanostructure. In most populations, each inorganic nanoparticle is made up of a different number of atoms, with different geometric relationships between facets and crystal faces, as well as defects and impurities. A comparably sized population of proteins is, by contrast, very homogeneous, with each protein comprised of the same number of amino acids, each arranged in approximately the same way, differing in arrangement, for the most part, only through the effect of thermal fluctuations. Consequently, two proteins designed to interact with one another will always interact with the same geometry, resulting in the formation of a complex of predictable geometry and stoichiometry. This property is essential for massively parallel “bottom-up” assembly of nanostructures.

[0084] A structural element may be used to maintain the geometric relationships among the joining elements and functional elements of a nanostructure. As such, a rigid structural element is generally preferred for construction of nanostructures using the staged assembly methods described herein. This rigidity is typical of many proteins and may be conferred upon the protein through the properties of the secondary structural elements making up the protein, such as α-helices and β-sheets.

[0085] Structural elements may be based on the structure of proteins, protein fragments or peptides whose three-dimensional structure is known or may be designed ab initio. Examples of proteins or protein fragments that may be utilized as structural elements in an assembly unit include, but are not limited to, antibody domains, diabodies, single-chain antibody variable domains, and bacterial pilins.

[0086] In some embodiments, structural elements, joining elements and functional elements may be of well-defined extent, separated, for example, by glycine linkers. In other embodiments, joining elements may involve peptides or protein segments that are integral parts of a structural element, or may comprise multiple loops at one end of a structural element, such as in the case of the complementarity determining regions (CDRs) of antibody variable domains (Kabat et al., 1983, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services). A CDR is a joining element that is an integral part of the variable domain of an antibody. The variable domain represents a structural element and the boundary between the structural element and the CDR making up the joining element (although well-defined in the literature on the basis of the comparisons of many antibody sequences) may not always be completely unambiguous structurally. There may not always be a well-defined boundary between a structural element and a joining element, and the boundary between these domains, although well-defined on the basis of their respective utilities, may be ambiguous spatially.

[0087] Structural elements of the present invention comprise, e.g., core structural elements of naturally-occurring proteins that are then modified to incorporate joining elements, functional elements, and/or a flexible domain (e.g., a tri-, tetra- or pentaglycine), thereby providing useful assembly units. Consequently, in certain embodiments, structures of existing proteins are analyzed to identify those portions of the protein or part thereof that can be modified without substantially affecting the rigid structure of that protein or protein part.

[0088] For example, in certain embodiments, the amino acid sequence of surface loop regions of a protein or structural element are altered with little impact on the overall folding of the protein. The amino acid sequences of a surface loop of a protein are generally preferred as amino acid positions into which the additional amino acid sequence of a joining element, a functional element, and/or a flexible domain may be inserted, with the lowest probability of disrupting the protein structure. Determining the position of surface loops in a protein is carried out by examination of the three-dimensional structure of the protein or a homolog thereof, if three-dimensional atomic coordinates are available, using, for example, a public-domain protein visualization computer program such as RASMOL (Sayle et al., 1995, RasMol: Biomolecular graphics for all, Trends Biochem. Sci. (TIBS) 20(9): 374-376; Saqi et al., 1994, PdbMotif—a tool for the automatic identification and display of motifs in protein structures, Comput. Appl. Biosci. 10(5): 545-46). In this manner, amino acids included in surface loops, and the relative spatial locations of these surface loops, can be determined.

[0089] If the three-dimensional structure of the protein being engineered is not known, but that of a close homolog is known (as is the case, for example, for essentially all antibody molecules), the amino acid sequence of the molecule of interest, or a portion thereof, can be aligned with that of the molecule whose three-dimensional structure is known. This comparison (done, for example, using BLAST (Altschul et al., 1997, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res. 25: 3389-3402) or LALIGN (Huang and Miller, 1991, A time efficient, linear-space local similarity algorithm, Adv. Appl. Math. 12: 337-357) allows identification of all the amino acids in the protein of interest that correspond to amino acids that constitute surface loops (β-turns) in the protein of known three-dimensional structure. In regions in which there is high sequence similarity between the two proteins, this identification is carried out with a high level of certainty. Once a putative loop is identified and altered according to methods disclosed herein, the resultant construct is tested to determine if it has the expected properties. This analysis is performed even in those instances where identification of the loop is highly reliable, e.g. where that determination is based upon a known three-dimensional protein structure.

[0090] Structural elements comprising leucine zipper-type coiled coils can also be employed in assembly units in the nanostructures of the invention. In certain embodiments, the invention encompasses structural elements comprising leucine zipper-type coiled coils for use in the construction of nanostructures using the staged assembly methods of the invention. Leucine zippers are well-known, α-helical protein structures (Oas et al., 1994, Springs and hinges: dynamic coiled coils and discontinuities, TIBS 19: 51-54; Branden et al., 1999, Introduction to Protein Structure 2nd ed., Garland Publishing, Inc., New York) that are involved in the oligomerization of proteins or protein monomers into dimeric, trimeric, and tetrameric structures, depending on the exact sequence of the leucine zipper domain (Harbury et al., 1993, A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants, Science 262: 1401-07). While only dimers are disclosed herein for simplicity, it would be apparent to one of ordinary skill in the art that trimeric and tetrameric units may also be used for the construction of assembly units for use in staged assembly of nanostructures according to the methods disclosed herein. In certain embodiments, trimeric and tetrameric units could be especially useful for incorporation of functional elements that, e.g., require two or more chemical moieties for proper activity, for example, the incorporation of two cysteine moieties for binding of gold particles. Several non-limiting examples of leucine-zipper domains are provided in Table 3 below.

[0091] Table 3 shows canonical leucine zippers and high stability dimerization sequences. The top line shows register of the repeat unit. Residues in the a and d positions are generally hydrophobic and control the oligomerization. Residues in the e and g positions are generally charged and create salt bridges to stabilize the oligomerization. 6

[0092] Many naturally occurring leucine zippers may be used according to the methods of the invention, including those found in the yeast transcription factor GCN4 and in the mammalian Fos, Jun and Myc oncogenes. Additional proteins containing leucine zippers and other coiled coil-type oligomerization sequences can be identified by searching public protein databases such as SWISS-PROT/TrEMBL (Bairoch and Apweiler, 2000, The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000, Nucl. Acids Res. 28: 45-48). Table 4 shows the results of such a search, using the keywords “coiled coil” and “dimer.”

[0093] In Table 4, the common names of genes are listed, as well as their SWISS-PROT accession numbers, sequence description and sequence. The SWISS-PROT accession number is a unique identifier for a sequence record. An accession number applies to the complete record and is usually a combination of a letter(s) and numbers, such as a single letter followed by five digits (e.g., Q12345) or a combination of six letters and digits (e.g., Q1Z2F3). The coiled coil sequences are underlined. 7