Method of producing three-dimensional structures using motor proteins The present invention relates to a method of producing a three-dimensional structure of a nanomaterial on a surface comprising the step of (a) contacting said polymer material affixed to (aa) biological filaments with biologically active motor proteins carried by said surface ; or (ab) biologically active motor proteins with biological filaments carried by said surface in the presence of an energy source in a buffer having a pH value of 5 to 9 and containing monovalent or divalent cations. It is preferred that said nanomaterial is carbon nanotubes or DNA and that said biological filaments are actin filaments or microtubules. It is further preferred that said motor proteins are kinesin or myosin molecules. Also preferred is that said DNA carries metal ions. In particular, the metal ions can be attached to the DNA before or after structure formation. The invention additionally relates to a three-dimensional structure of nanomaterial obtainable by the method of the invention. Said three-dimensional structure preferably is a nanocircuit of metalized DNA.

In the present specification, a number of documents is cited. The disclosure content of these documents, including manufacturers'manuals, is herewith incorporated by reference.

The capability to fabricate structures in the submicron range is essential for the successful development of nanotechnology. Although optimized lithographic processes have reduced the feature sizes on microelectronics chips already below 100 nm, the final resolution of these top-down techniques will be limited. Bottom-up approaches, such as the self-assembly of carbon-nanotubes, quantum dots or biopolymers, are capable of producing structures well below 10 nm. However, the techniques that are currently available to manipulate or arrange such nanostructures in a controlled manner require large, sophisticated and expensive apparatuses (about 10.000 times bigger than the structures to be manipulated). The fact that these machines can usually manipulate only a small number of nanostructures at a time severely limits their applicability for efficient molecular nanoconstruction.

Carbon nanotubes, for example, are of extreme interest for nanoelectronics as they are extremely small but offer a high intrinsic conductivity. The controlled alignment of these nanotubes to given contact points in electronic circuit arrays is one of the major challenges in that field in the moment but no suitable techniques are available (Appell, D. (2002). Wired for success. Nature 419,553-555). A promising approach, where the ends of carbon nanotubes were biofunctionalized in order to achieve the specific binding to predefined contact points was published recently (Williams, K. A., <BR> <BR> Veenhuizen, P. T. M., de la Torre, B. , Eritja, R. , Dekker, C. (2002). Carbon nanotubes with DNA recognition. Nature 420,761).

Another nanomaterial of particular interest is DNA. The size, strength and sequence specificity of DNA molecules make them possible building blocks for the assembly of two and three-dimensional structures on the nanometer scale (N. C. Seeman, Trends in Biotechnology 17, (1999), 437-433). One potential application for such DNA-based structures is in nanoelectronics. Although the intrinsic conductance of DNA is very low (A. J. Storm et al., Appl. Phys. Lett. 79, (2001), 3881-3883), linearly stretched DNA molecules have proven valuable as templates for metallization (E. Braun et al., Nature 391, (1998), 775-778; K. Keren et al., Science 297, (2002), 72-75; M. Mertig et al., NanoLetters 2, (2002), 841-844), for metal-ion insertion (P. Aich et al., Mol Biol <BR> <BR> 294, (1999, ) 477-485), and for the nucleation of metal and semiconductor nanoparticles (A. P. Alivasatos et al., Nature 382, (1996), 609-611; J. J. Storhoff, C.

A. Mirkin, Chem Rev 99, (1999), 1849-1862). Such metalized DNA has sufficient conductance for use as a nanowire (K. Keren et al., Science 297, (2002), 72-75; J.

Richter et al., Appl. Phys. Lett. 78, (2001), 536-538; J. Richter et al., Applied Physics A 74, (2002), 725-728).

In order to use DNA to link the components of a nanocircuit, the molecules must be aligned mechanically, and this poses a major challenge. Recently, a number of physical techniques have been developed to manipulate single DNA molecules (C.

Bustamante et al., Curr Opin struct Biol 10, (2000), 279-285). These techniques include the employment of magnet (S. B. Smith et al., Science 258, (1992), 1122- 1126; C. Gosse, V. Croquette, Biophys J 82, (2002), 3314-3329) or optical tweezers (S. B. Smith et al., Science 271, (1996), 795-799; Y. Arai et al., Nature 399 (6735): (1999), 446-448; J. R. Wenner et al., Biophys J 82, (2002), 3160-3169), microneedles (P. Cluzel et al., Science 271, (1996), 792-794; B. Essevaz-Roulet et al., Proc Natl Acad Sci U S A 94, (1997), 11935-11940) or an atomic force microscope (AFM) (M. Rief et al., Nat Struct Biol 6, (1999), 346-349). However, a major limitation of these techniques is that only one DNA molecule can be manipulated at a time. While this is not a problem for the investigation of the mechanical properties of individual DNA molecules, it severely limits the efficiency of these techniques for DNA-based molecular construction. In addition, these techniques require large, sophisticated and expensive apparatuses, thus hampering parallelization.

One way to circumvent these difficulties and to simultaneously manipulate multiple DNA molecules is to use hydrodynamic flow (T. T. Perkins et al., Science 264, (1994), 822-826; M. Mertig et al., AIP Conference Proceedings 633, (2002), 449- 453), an electric field (R. M. Zimmermann, E. C. Cox, Nucleic Acids Res 22, (1994), 492-497; V. Namasivayam et al., Anal Chem 74, (2002), 3378-3385) or a moving water-air interface (D. Bensimon et al., Physical Review Letters 74, (1995), 4754- 4757; Z. Gueroui et al., Proc NatlAcad Sci U S A 99, (2002), 6005-6010). In this way, many DNA molecules can be simultaneously aligned and stretched. However, to create truly two-or three-dimensional structures, these procedures would have to be applied sequentially in different directions, or additional steps such as the cutting and moving of individual DNA fragments by an AFM (J. Hu et al., Nano Lett. 2, (2002), 55-57) would have to be added. Accordingly, major additional efforts need to be taken in order to achieve the desired goal.

An entirely different technical field covers the detection of motor proteins and the potential in vitro applications such motor proteins may confer. Hess and Vogel, Reviews in Molecular Biotechnology 82 (2001), 67-85 present an overview of available techniques at the time to use motor-protein based technologies for a variety of purposes including nano-electro-mechanical systems (NEMS), self-healing processes, and surface survey including transport of replacement materials in a directed fashion. Loading cargo onto microtubules may be effected by, e. g. antigen- antibody or biotin-avidin interactions. Suggestions for the control of direction and speed include the use of patterned surfaces, flow fields, electrical fields and caged ATP in combination with ATP consuming enzymes. In a more specific application, Hiratsuka and colleagues (Biophys. J. 81 (2001), 1555-1561) have demonstrated that a unidirectional movement of microtubules on kinesin fixed on a glass layer can be achieved by an arrow-headed design of lithographically fabricated tracks. Retainment of microtubules to the tracks was achieved by coating of the glass support with a resist prior to the lithographic process. A different option for keeping motor proteins to predefined movements was explored by Böhm and colleagues (Nanotechnology 12 (2001), 238-244) who immobilized microtubules with glutaraldehyde after isopolar alignment obtained by applying a flow field. Using this approach, the transport of micrometer-sized cargoes such as glass-particles over distances in millimetre ranges by kinesin molecules was observed. One of the documents covered by the review of Hess and Vogel, cited above has been published by Hess and colleagues in Nanoletters 1 (2001), 235-239. Here, the authors have developed a system for the directed movement of microtubules on kinesin-based motor proteins using a patterned polyurethane surface. The movement could be controlled with UV-light activatable caged ATP and an ATP-consuming enzyme, hexokinase. They also showed cargo loading wherein the cargo was coupled to microtubuli via biotin- streptavidin bonds. Suzuki et al. (Biophys. J. 72 (1997), 1997-2001) describe the directed movement of actin filaments along myosin molecules attached to PMMA- coated glass slides. Direct coating of myosin to glass slides resulted in loss of motility.

In summary, there is a need in the art to provide means and methods that allow the convenient and directed construction of fabricated interwoven structures on the nanoscale. The solution to this technical problem is achieved by providing the embodiments characterized in the claims.

Thus, the present invention relates to a method of producing a three-dimensional structure of a nanomaterial on a surface comprising the step of (a) contacting said nanomaterial affixed to (aa) biological filaments with biologically active motor proteins carried by said surface; or (ab) biologically active motor proteins with biological filaments carried by said surface in the presence of an energy source in a buffer having a pH value of 5 to 9 and containing monovalent and/or divalent cations.

The term"three-dimensional structure"refers to the fact that any structure formed by the method of the invention is a spatial structure, irrespective of the size and the dimensions of the nanomaterial employed. It is preferred that the first and second dimensions of the three-dimensional structure correlating to the two dimensions of the surface are large in comparison to the third dimension which is perpendicular to said surface. For example, if the nanomaterial is DNA, then the three-dimensional structure may be a network formed by one or more DNA strands wherein the first and second dimensions mirror the arrangement of DNA strand (s) after the movement of the biological filaments or said motor proteins have completed their movement along said surface and the third dimension essentially corresponds to the diameter of the DNA molecule, or in positions where two DNA stands or two segments of the same DNA strand cross, essentially twice the diameter of a DNA molecule. Alternatively, the two dimensions correlating to the two dimensions of the surface are not large in comparison to the third dimension which is perpendicular to said surface. Also in such cases novel applications such as applications as molecular sieves are envisaged.

The term"nanomaterial"refers to any material which size falls in at least one dimension below 100nm. The nanomaterial may be of organic or inorganic origin. The nanomaterial is in one preferred embodiment a polymer material. It may thus be any material consisting of at least three, preferably at least 10, more preferred at least 100 such as at least 200 or at least 500 and most preferred at least 1000 monomeric units. The monomeric units may be of organic or of inorganic origin. The nanomaterial used in the method of the invention may comprise, at the same time, monomeric units of organic as well as of inorganic origin. It is further envisaged that the monomeric units, either of organic or of inorganic origin, may be heterogeneous within said nanomaterial. In other terms, the invention envisages the use of homopolymers as well as of heteropolymers. A typical and preferred example of such an organic material consisting of heterogeneous monomeric subunits is a nucleic acid such as DNA or RNA being comprised, in its natural state, of four different bases. A further preferred example of such an organic material is a peptide (having up to 30 amino acids arranged usually in linear order) or a polypeptide (having more than 30 amino acids arranged usually in linear order) being comprised, in their natural state, essentially of 20 different amino acids. Examples of inorganic nanomaterial are semiconductor quantum dots (nanocrystals), metallic nanoparticles, and carbon nanotubes.

The term"biological filament"refers to a naturally occurring filamentous structure or a structure derived from such a naturally occurring filamentous structure which retains the function of said filament. The derivatives should return their function to interact with the motor proteins such that the motor proteins can still fulfill their movement and transport functions. Derivatization of such filaments may allow for the binding of additional components to the filaments. The term"biological filament"also includes fragments of naturally occurring biological filaments or muteins thereof that retain the above recited function.

The term"biologically active motor proteins"refers to motor proteins that occur in nature such as kinesin or myosin or biologically active fragments or derivatives of such naturally occurring motor proteins. Naturally occurring motor proteins fulfil the task of transport within a cell. For example, kinesin moves along microtubules carrying, for example, vesicles whereas myosin walks on actin filaments. Motor proteins may be fragmented or derivatized according to procedures known in the art and tested for retaining their movement function. Derivatization of kinesins by mutation has been described in the art; see, for example, Coy et al, Nat. Cell. Biol. 1 (1999), 288-292 ; Hess and Vogel, loc. cit. Biologically active fragments of motor proteins include meromyosin, Insofar, the term"biologically active motor proteins" refers to any type of naturally or not-naturally occurring motor protein that is capable of moving along a substrate and, preferably, at the same time, of carrying a cargo. It is self-explanatory that conditions must be provided that allow the functioning of said biologically active motor proteins as well as of the filamentous interaction partners.

Such conditions include a pH value in the range between 5 and 9 of the buffer in which the movement takes place, more preferred between 6 and 8 and most preferred around 7. Suitable conditions further include the presence of mono-and/or divalent cations. The nature of the cations to be employed and the concentrations thereof required depend, inter alia, on the type of motor protein/filament employed. If the kinesin/microtubule-based system is employed, then, for example, Mg2+ions between 0, uM and 10 mM, preferably 1 mM and an ionic strength between 10 mM and 500 mM, preferably 100-200 mM may be employed.

The"energy source"is advantageously a nucleotide triphosphate such as ATP or GTP.

In accordance with the present invention, an alternative approach is presented in which biological machines, specifically kinesin motor proteins, are used to manipulate, simultaneously, many individual nanomaterial molecules near a surface.

Alternatively, it is possible to manipulate solely one and the same molecule near a surface. These molecules are exemplified by A-phage DNA molecules. While the interaction of motor proteins with filamentous structures in cells has been studied extensively (see (J. Howard, Mechanics of Motor Proteins and the Cytoskeleton, Sinauer Associates, Sunderland, MA (2001) ) for a review), its applicability for nanotechnological tasks has been explored only recently (C. Montemagno, G.

Bachand, Nanotechnology 10, (1999), 225-231; H. Suzuki et al., Biophys J 72, (1997) 1997-2001; D. V. Nicolau et al., Biophys J 77, (1999), 1126-1134; D. V.

Nicolau, R. Cross, Biosens Bioelectron 15, (2000) 85-91; H. Hess et al., Nano Lett. 1, (2001) 235-239; H. Hess, V. Vogel, J. Biotechnol. 82, (2001), 67-85; H. Hess et al., Nano Lett. 2, (2002) 1113-1115; K. J. Böhm et al., Nanotechnology12, (2001), 238- 244) without, however, taking into account the options presented by the present invention. The intriguing feature of motor proteins, such as kinesin, is that they perform very precise nanometer-steps in a highly controlled manner: for example, kinesin takes 8-nm-steps along a microtubule (K. Svobody et al., Nature 362, (1993), 721-727), each step coupled to the hydrolysis of one ATP molecule (D. L. Coy et al., J. Biol. Chem. 274, (1999), 3667-3671). However, because the force produced by one kinesin molecule (about 6 pN (J. Howard, Mechanics of Motor Proteins and the Cytoskeleton, Sinauer Associates, Sunderland, MA (2001) ) is barely sufficient to stretch a A-phage DNA molecule to its contour length of 16.5 um (M. Rief et al., Nat Struct Biol 6, (1999), 346-349), many motors have to be used together. A preferred approach taken in accordance with the present invention is therefore based on a gliding motility assay in which the substrate surface is coated with kinesin motor proteins and microtubules are propelled across the surface by a number of kinesin molecules in the presence of ATP. The binding of DNA to the microtubules was achieved using a biotin-streptavidin linkage (Figure 1).

The general inventive concept is, of course, not confined to the manipulation of DNA molecules. Rather, it is extendable to any nanomaterial that is flexible enough and has the appropriate dimensions to be carried around by motor proteins or by filaments which themselves are being moved by motor proteins. Insofar, the method of the present invention can be put into practice with different biological molecules such as polypeptides or further organic or even inorganic matter. The gist of the invention lies within the fact that cargo is not only carried along a surface as suggested by the prior art but that the movement of the motor proteins/filaments generates a molecular three-dimensional network of the cargo molecule (s) which itself may, depending on the nature of cargo used, find a variety of different applications, preferred embodiments thereof being recited herein below.

In all embodiments of the present invention it is preferred that the nanomaterial is interfaced with the surface in order to generate an anchor point for the production of the three-dimensional structure. Interfacing may be effected directly with the surface or via a molecule attached to the surface. The interfacing may further be effected via a covalent linkage or via a non-covalent linkage such as a van-der-Waals force.

Linkages may, in essence, be the same type of linkages that are used for the loading of cargoes to the motor proteins as described herein and in the art cited in this specification. Additionally, linkages may be of irreversible nature, in particular if covalent linkages are favoured. Other kinds of linkages include oligomere and protein-protein interactions. Further, linkages include those between protein- antibodies, biotin-streptavidin, Ni-NTA, specific motors, filament or DNA binding proteins (or muteins of these proteins). Generation of linkages may also be effected by change of the pH value of the buffer solution, as described, for example, by Allemand et al., Biophys. J. 73 (1997), 2064-2070. In accordance with the above, a further preferred embodiment of the invention relates to a method as above, further comprising, prior to said step (a), the step of (a') fixing a portion of said nanomaterial to said surface. Also, in accordance with the above, the nanomaterial preferably is a polymer material, either of organic or of inorganic origin. As regards the constitution of the polymer material, it refers to any material consisting of at least three, preferably at least 10, more preferred at least 100 such as at least 200 or at least 500 and most preferred at least 1000 monomeric units. The monomeric units may be of organic or of inorganic origin. The polymeric material used in the method of the invention may comprise, at the same time, monomeric units of organic as well as of inorganic origin.

It is further envisaged that the monomeric units, either of organic or of inorganic origin, may be heterogeneous within said polymer material. In other terms, the invention envisages the use of homopolymers as well as of heteropolymers. A typical and preferred example of such an organic polymeric material consisting of heterogeneous monomeric subunits is a nucleic acid such as DNA or RNA being comprised, in its natural state, of four different bases. A further preferred example of an organic polymer material is a peptide (having up to 30 amino acids arranged usually in linear order) or a polypeptide (having more than 30 amino acids arranged usually in linear order) being comprised, in their natural state, essentially of 20 different amino acids.

It is further preferred in accordance with the method of the invention that attached to the surface or contained in the buffered solution is a compound such as taxol or casein that protects the motor proteins or filaments from rapid loss of function or from degradation. These compounds thus fulfil a kind of chaperone function.

The three-dimensional structure may be constructed such that it covers the complete surface. Alternatively, a patchwork or only single spots or areas of three-dimensional structures may be generated on said surface. Whether the whole surface or only (a) portion (s) thereof are covered by the three-dimensional structure may be regulated by the density of motor proteins or of biological filaments attached to the surface. For example, if kinesin is used as a motor protein that is attached to the surface, then a density of at least 1, more preferred at least 10 kinesin molecule per square micron of surface area is required in areas where the generation of said three-dimensional structure is desired. Densities of about 1000 molecules per square micron are also feasible. These density ranges are generally also applicable to other motor proteins.

Patchworks can be produced by precoating of surfaces with materials that allow the attachment of biological filaments or motor proteins or that prevent such an attachment. In the latter case, the surface itself must allow the attachment of biological filaments or motor proteins. An example of a compound useful to attach a motor protein is poly (methylmethacrylate) (PMMA) that binds myosin while allowing the motor protein to support the movement of actin filaments ; see Suzuki et al., loc. cit. In alternative embodiments, patchworks may be generated by structuring of the surface itself, e. g. by introducing tracks into the surface that reduce the movement of motor proteins or filaments outside of said tracks; see, e. g. Hiratsuka et al., loc. cit., Hess and Vogel, loc. cit. It is also possible to combine chemical with mechanical modification of the surfaces in order to secure proper generation of patchworks. The recited steps can, of course, also be employed in the generation of three-dimensional structures that extend over the complete surface or essentially over the complete surface or only a spot thereof.

After or during the generation of the three-dimensional structure, it may be desired to unload the cargo from the filaments or the motor proteins (depending whether option (aa) or (ab) of the method of the invention is employed). Unloading of cargo may be effected by dissolving the linkage between cargo and carrier. For example, if the linking molecules consist of antibody and antigen, an excess of antigen may be provided after completion of the three-dimensional structure. If the linkage is effected by a biotin-streptavidin bridge, modifications of steptavidin may be employed that bind and unbind in response to pH changes or a variety of other parameters; see Stayton et al., Biomol. Eng. 16 (1999), 93-99. The skilled artisan is in a position to devise further methods of unloading pertinent to the teachings of the prior art.

Direction and guidance of carrier molecules may be effected by tracks or coating of the surface with molecules than allow keeping the contact with the carrier as described elsewhere in this specification. Alternatively, hydrodynamic flow, electrical, magnetic or optical fields might be used to effect the direction of the networking.

Combinations of these methods of directing the carrier/cargo complex may be combined with the methodology developed by Hess and colleagues, loc. cit., namely influencing the movement of motors by adding caged ATP and a hexokinase to the system. Alternatively, motion may be directed by simply flowing in and washing out ATP in a controlled faction, e. g. by using a perfusion chamber. In this way, different degrees of freedom for directing the motors/filaments may be obtained.

The advantage of using motor proteins is that the mechanical forces generated by them can be used to transport, collide, direct, stretch and compress nanostructures.

These actions can be controlled locally, they can be turned on and off, mutations of the motor proteins can be developed to better fit the desired actions and protein chemistry can be employed to optimise the biomechanical processes.

The three-dimensional structure obtainable with the method of the invention may be a prescribed, planned structure or it may be a random structure. In other terms, the structure obtainable or obtained with the method of the invention may be an array which is ordered or not ordered.

In a preferred embodiment of the method of the invention, said three dimensional structure is an ordered array that can be used as template for nanoelectrical purposes, for use as a biomolecular data carrier, in the three-dimensional survey of surface structures or for use in the construction of molecular reaction systems. The biomolecular data carrier produced in accordance with the invention is expected to be highly superior in terms of storage as compared to state of the art storage means.

In a further preferred embodiment of the method of the invention, said polymer material is an organic material.

More preferred in accordance with the present invention is that said organic material consists of nucleic acid.

The term"nucleic acid"as used here comprises any type of naturally occurring nucleic acid as well as derivatives thereof. Naturally occurring types of nucleic acid include DNA such as chromosomal DNA (of any type of organism or otherwise naturally occurring, including DNA from prokaryotes, eukaryotes, archaebacteria, prions and viruses including bacteriophages) or plasmid DNA. They further include RNA such as messenger RNA, transfer RNA, ribosomal RNA, small interfering RNA and ribozymes. Derivatives include, but are not limited to, peptide nucleic acids and DNA or RNA molecules incorporating non naturally occurring bases.

Particularly preferred in accordance with the method of the invention is that said nucleic acid is DNA. The DNA may be chromosomal DNA, plasmid DNA etc. as outlined herein above, but also includes cDNA etc.

In a different preferred embodiment of the method of the invention, said method further comprises the step of (b) stretching said nanomaterial.

Stretching of nanomaterial is particularly advantageous with biomolecules such as polypeptides or nucleic acid molecules.

Stretching of nucleic acid molecules, in particular of DNA molecules has the advantage that the sequence specificity of the DNA can be utilized to achieve specific binding of the DNA ends to contact points or of nanomaterial specific sites along the DNA molecule. Other molecules that can be streched include polysaccharides as well as polyethyleneglycol or similar polymeric constructs. As an example of polypeptides, fibrionectin molecules may be stretched.

In a particular preferred embodiment of the method of the invention, it further comprises the step of (c) fixing the nanomaterial, preferably the polypeptides or nucleic acid molecules in a stretched state.

In a further particularly preferred embodiment of the invention, the method further comprises the step of (d) attaching metal ions to said nucleic acid.

This preferred embodiment is particularly useful in the generation of nanocircuits. It envisages the addition of metal ions such as platin, palladium, gold, silver to the nucleic acid molecules before or after the generation of the three-dimensional structure. The metalization procedure is described in DE 101 31 551.1 and DE 196 24 332.7-43. Metal complexes that are loaded to DNA are transferred into conducting connections by chemical reduction after the structure has been generated (M. Mertig, L. Colombi Ciacchi, R. Seidel, W. Pompe, A. De Vita, NanoLetters, 2002,2, 841- 844.).

In an alternative particularly preferred embodiment of the method of the invention, metal ions are attached to said nucleic acid.

In this embodiment which has essentially the same range of applications as outlined above, the metal ions are present in or on the nucleic acid molecules prior to the construction of the three-dimensional network.

It is also preferred in accordance with the method of the invention that said nanomaterial is an inorganic material such as semiconductor and metallic nano- particles.

Particularly preferred is that said inorganic material is carbon nanotubes.

As with three dimensional structures composed of DNA/metal ions, networks of carbon nanotubes will be highly efficient nanocircuits bearing the great advantage that they are inherently conducting and do not necessarily have to be metallized.

A number of biological filaments may be employed in accordance with the present method which are intermediate filaments, collagen filaments, myosin filaments, bacterial flagella, septin filaments, centractin filaments, hemaglobin S filaments and other amyloid or plaque filaments. The method of the invention provides a further preferred embodiment wherein said biological filaments are microtubules or actin filaments. Microtubules and actin filaments have been explored in different applications in the prior art as parts of a cargo-transporting system. Their adaption to the system employed by the present invention therefore appears particularly feasible as has already been demonstrated by the appended examples. Again, actin filaments and microtubules used in accordance with the present invention include biologically active molecules fragmented from naturally occurring molecules or derived therefrom by different means such as by mutation, including substitution, inversion, deletion, duplication etc. If microtubules are employed, then it is also preferred that the number of protofilaments equals 13, since they in this case probably do not rotate.

Similarly, a variety of motor proteins are feasible when it comes to implementing the present invention. These motor proteins include F1-ATPase and others known in the art. The invention envisages in an additional preferred embodiment a method wherein said motor proteins are kinesin or myosin molecules.

It is understood in accordance with the present invention that kinesin (or fragments or derivatives thereof as outlined herein above) is used as a motor protein if microtubules are the biological filaments and myosin molecules (or fragments or derivatives thereof as outlined herein above) are used as motor proteins if actin is employed as the biological filament.

The invention does not exclude, however, that different motor proteins and different filaments are used in the same experiment, e. g. kinesin-gelsolin fusion can be used to move actin filaments along microtubules. Also, the fusion of kinesin heads to myosin tails can be used to move myosin filaments along microtubules.

Depending on the type of motor protein employed, different energy sources and preferably bio-organic energy sources are included into the experimental set-up.

Preferably, said energy source is ATP or GTP. ATP is preferred if kinesin or myosin is used as the motor protein.

It is particularly preferred in accordance with the method of the present invention that the surface is a flat surface. The term"flat surface"means that the surface does not contain irregularities that would prevent the straightforward movement of the carrier molecules (i. e. the filaments or the motor proteins to which the cargo has been attached). Alternatively and particularly suitable for guiding the carrier are surfaces that contain irregularities such as tracks or channels as described, e. g. by Hess et al., loc. cit. or Hiratsuka et al., loc. cit.

Whereas the surface can assume different consistencies such as a semi-solid consistency (such as in a gel) it is further preferred that said surface is a solid surface.

A wide variety of solid surfaces may be applied in connection with the present invention. As regards function or consistency, some of the following examples are particularly preferred: a chip, a glass slide, a metallic surface, a semiconductor surface or a chemically derivatized surface.

Options to carry out the linkage of carrier and cargo have been detailed herein above. Embodiments are preferred wherein said nanomaterial is affixed to said biological filaments or said motor proteins via a non-covalent interaction. It is most preferred that said non-covalent interaction is an interaction of an antibody or a fragment or derivative thereof with a cognate antigen or an interaction between biotin and avidin or streptavidin.

Herein, the term"fragment or derivative"of an antibody includes Fab-fragments, F (ab') 2-fragments, Fv-fragments, scFvs, chimeric or humanized antibodies, bispecific antibodies all of which are known in the art; for some embodiments, see Harlow and Lane, "Antibodies, A laboratory Manual", CSH Press, Cold Spring Harbor 1988.

Via the specific interaction of these molecules, loading of the carrier molecules with their respective cargo molecules may be effected.

In another most preferred embodiment of the present invention, said method comprises, prior to step (a), the step (a') fixing a portion of said nanomaterial to said surface.

Fixing may be effected by a specific or a non-specific mode. Specific modes would include that, for example, the nanomaterial has attached thereto a partner of a specific binding pair as has been outlined above. The other partner of this specific binding pair would be attached to the surface. Attachment to the surface can be at the border of this surface or somewhere within the surface. Attachment to the surface can also be achieved by manipulating the pH-value of the solution comprising the components used in the method of the invention such as lowering the pH-value as has been described in the appended examples.

This embodiment is specifically advantageous since it allows in an easy way the directed construction of a 3-dimensional structure. For example, the attachment of the nanomaterial at a specific position of the surface may be used as a specific starting point for the generation of the 3-dimensional structure.

As has also been shown in the appended examples, fixing of the nanomaterial at a specific position of the surface may be used to direct the movement of the motorprotein or filament attached thereto in a circular fashion. In so far, circular structures may be generated. The densitiy of the filaments or motor proteins attached to the surface may be used to stretch the nanomaterial, preferably the DNA molecules and, accordingly, interwoven circular structures with different diameters may be obtained. Another most advantageous embodiment of the present invention relates to a method further comprising (e) contacting the nanomaterial with a nanomaterial degrading enzyme.

The use of nanomaterial degrading enzymes provides for the option to manipulate the network/three-dimensional structure generated by the action of the motor proteins/filaments.

A number of enzymes degrading nanomaterials such as polymers, in particular of organic nature are known in the art.

The preferred embodiment of the invention, in said method said nanomaterial is a polypeptide and said enzyme is a protease.

Suitable proteases are known in the art, include trypsin and can be applied by the skilled artisan without further ado.

In a further preferred embodiment, said nanomaterial is a nucleic acid molecule and said enzyme is a nucleic acid degrading enzyme.

In the case that a nanocircuit is to be generated, the direction of the current may, for example, be altered. In regard to this embodiment, it is crucial for certain types of enzymes such as DNasel that the reaction conditions are particularly carefully chosen in order to avoid an undesirable degree of digestion which can be either to small or to large. However, controlled digestion with nucleic acid degrading enzymes has been well established in the art and can be adapted by the skilled artisan to the present applications without further ado.

One particularly advantageous means to reduce the risk of uncontrolled digestion pertains to the employment of restriction enzymes and in particular restriction endonucleases. Upon knowledge of the nucleic acid sequence, cuts into the three- dimensional structure may be effected at predetermined positions and only at these positions. Appropriate restriction enzymes are well known in the art and described, for example, in the annually revised catalogue of New England Biolabs, Inc. In accordance with the above, in a further particularly preferred embodimant of the present invention, said nucleic acid degrading enzyme is a restriction enzyme.

As has been also already indicated above, in a further particularly preferred embodiment of the present invention, said contacting with the restriction enzyme results in a partial digest of said nanomaterial and preferably said DNA.

The present invention also relates to a three-dimensional structure of nanomaterial obtainable by the method of the invention.

The three-dimensional structure of the invention is, in a preferred embodiment, a nanocircuit of metalized DNA.

The figures show: Fig. 1 Schematic diagram of the microtubule-DNA transport and manipulation system. Microtubules are driven by kinesin motor proteins over the surface of a casein-coated substrate. The biotinylated end of a DNA molecule is bound to the lightly biotinylated microtubule via a streptavidin link. In the experiments shown in Figures 3 and 4 the second (free) end of the DNA is attached either to the substrate surface or to another microtubule.

Fig. 2 Fluorescent images of a motile microtubule (rhodamine labelled-red) transporting 5 individual DNA molecules (YOYO labelled-green or yellowish when colocalized with the red microtubule ; white arrows) in the direction of the dotted white line.

Fig. 3 Stretching DNA-molecules by motile microtubules. a) Two condensed DNA- molecules attached to the substrate surface by one of their ends are grasped at their second end by motile microtubules (red, moving in the direction of dotted white line) and are consequently stretched (green; white arrows). b) A DNA molecule (initially being transported by one microtubule) is stretched between two motile microtubules.

Fig. 4 A motile microtubule is guided on a"DNA leash"that is attached to its leading edge. In contrast to the other microtubules observed in this experiment (straight dotted lines), the guided microtubule is forced onto a circular path by the pulling force of the stretched DNA molecule.

The examples illustrate the invention.

Example 1: Generation of three-dimensional DNA structures using the kinesin/microtubule system To demonstrate the transport and stretching of the DNA molecules by motile microtubules, a microscope assay was used. First a casein-containing solution (0.5 mg/ml in BRB80) was perfused into a flowcell (A 4 mm wide flow cell was built from a microscope slide (FISHERfinest Premium Plain, 75 x 25 x lmm), a coverslip (Menzel-Glaeser, 18 x 18 mm) and 2 pieces of double sided tape (Scotch 3M, thickness 0.1 mm) ), and the casein allowed to adsorb to the surfaces for 5 minutes in order to reduce the denaturation of kinesin and to prevent the sticking of microtubules. Then the motor-containing solution (5 ug/ml kinesin, 1 mM Mg-ATP, 0.2 mg/ml casein in BRB80) was perfused. After 5 minutes the solution was exchanged for a motility solution containing the microtubules with bound DNA (Rhodamine labelled, biotinylated microtubules were polymerised from 10 pI bovine brain tubulin (4 mg/ml, mixture of 3 thodamine labelled/4 biotinylated/9 unlabeled tubulin units) in BRB80 buffer (80 mM potassium PIPES, pH=6. 9,1 mM EGTA, 1mM MgCiz) with 4 mM MgCl2, 1 mM Mg-GTP and 5% DMSO at 37°C. After 30 minutes the microtubule polymers were stabilized and 100fold diluted into room temperature BRB80 containing 10 uM taxol. To remove any free (unpolymerized) tubulin, 400 p1 of the resulting microtubule solution (0. 32 uM) was centrifuged at 178. 000g in a Beckman airfuge for 5 minutes. The pellet was resuspended in 200 pI BRB10 (10 mM potassium PIPES, pH=6. 9,1 mM EGTA, 1 mM MgCI2) containing 10 pM taxol, yielding a microtubule solution with about 0.64 uM polymerised tubulin (also termed B-MT 50). The ends of A-phage DNA were biotinylated using the Kleenos-fragment of polymerase I and biotinylated dCTP (13), thus yielding fully double-stranded DNA with 4 biotin molecules on one end and 6 biotin molecules on the other. For fluorescent labelling, 5 pl of biotinylated \DNA (100 ug/ml) was mixed with 44 pi nanopure water and 1 ul YOYO-1 iodide stock solution (25x diluted in TBE buffer (45 mM Tris-borate, 1 mM EDTA) ). The solution was incubated for at least 30 minutes at 4°C in the dark. Streptavidin was bound to the biotinylated tubulin sites on the microtubule lattice by mixing B-MT 50 with streptavidin (40 nM in BRB10). In order to achieve a rapid binding, and thus to avoid any crosslinking of microtubules, the mixing was performed while vortexing the solution. The labelled A-DNA (diluted to 2 pg/ml in BRB 10 containing 10 uM taxol) was then added to the microtubule- streptavidin solution. (50 nM biotinylated tubulin, 0. 5 ug/ml DNA, 1mM Mg-ATP, 0.2 mg/ml casein, 10 uM taxol in BRB10). Total internal reflection fluorescence (TIRF) microscopy (through the objective, 100x, NA = 1.45, Zeiss, Germany) was used to image the YOYO-labeled A-phage DNA molecules (The 488 nm line from an Argon/Krypton ion laser (Innova 90, Coherent, UK) was coupled into an optical single mode, polarization maintaining fiber (OZ optics, Canada) and injected offcenter into the epi-illumination port of a Zeiss Axiovert 200M optical microscope. Rhodamine labelled microtubules were imaged by conventional epifluorescence microscopy.

Images were acquired every 5 seconds with an exposure time of 100 ms using an intensified cooled CD camera (I-PentaMax, Princeton Instruments, USA), and the Metamorph imaging system (Universal Imaging, USA)). Individual DNA molecules, which condense into 1 um-diameter random coils due to their high flexibility, were clearly observed as they were transported across the kinesin-coated surface on the gliding microtubules (Figure 2).

The forces generated by the motor proteins can be used to stretch condensed DNA molecules. In one method, the pH of the motility solution was lowered to 6.0 resulting in a number of DNA molecules binding to the surface by one end (J. F. Allemand et al., Biophys J 73, (1997), 2064-2070). Moving microtubules then picked up the other end of the DNA, presumably via the biotin-streptavidin linkage. The DNA was stretched (Figure 3a), sometimes up to its 16. 5-pm contour length, until the DNA detached from the surface or broke (Breakage of the DNA in our experiments was presumably due to the photocleavage of the YOYO-labeled DNA. See also : B.

Akerman, E. tuite, Nucleic Acids Res 24, (1996), 1080-1090). In another method, DNA was stretched between two moving microtubules (Figure 3b). In these sets of experiments, the speeds and the trajectories of the moving microtubules were not dramatically influenced by the binding to the DNA molecule. This is due to the high motor densities of up to 1000 motor molecules per square micron of surface area. At these densities, there were about 10-100 kinesin molecules interacting with a 5 um long microtubule (J. Howard et al., Nature 342, (1989), 154-158; W. O. Hancock, J.

Howard, J. Cell Biol. 140, (1998), 1395-1405), producing forces up to 60-600 pN.

Interestingly, DNA can also be be used to control the movement of the microtubules.

This was achieved by lowering the density of the motors and increasing the stability of the DNA by reducing the YOYO-labeling. Microtubules could then be stopped completely or their trajectories altered so that they were forced to move along circular paths (Figure 4).

We have demonstrated a novel method for the manipulation of individual DNA molecules based on force generation by motor proteins. Thus we have borrowed a biological principle, as it is used in living cells, to fulfil specific tasks in an engineering environment. In contrast to conventional DNA manipulation methods, our approach offers the potential for the simultaneous and yet individual operation on multiple biomolecules. However, to be useful for building nanostructures, the manipulation needs to be more precise in both position and direction. We believe that the further combination of concepts from biology and material science might overcome these challenges. For example, to create nanocircuits, DNA could be bound with one end to specifically prepared contacts in nanofabricated arrays and gliding microtubules, whose movement is guided along predefined tracks (H. Hess et al., nano Lett. 1, (2001) 235-239; H. Hess, V. Vogel, J. Biotechnol. 82, (2001), 67-85; H. Hess et al., Nano Lett. 2, (2002), 1113-1115), could be used to link the appropriate network points. Prior to metallization the sequence specific information within the DNA strands could be used to cut unwanted connections. Because each step in such a procedure would allow the simultaneous operation on many DNA molecules, complex network structures might be produced with high efficiency. Beyond the application of our approach for DNA manipulation, we foresee a potential of this techniques also for the transport and positioning of other nanostructures, such as functionalized carbon nanotubes (K. A. Williams et al., Nature 420, (2002), 761).

Example 2: Sequel of steps observed by applying the method of the invention with kinesin motor proteins and phage X DNA At the beginning of the observation a A-phage DNA molecule was stretched between the substrate surface and a microtubule to its full contour length of about 16. 5 um.

The microtubule was stopped by the pulling force of the DNA molecule and buckled due to the forces generated by the kinesin motors attached to the rear part of the microtubule. After detachment of the surface bound end of the DNA, the microtubule continued its movement. The microtubule then transported five condensed DNA molecules. The binding of the DNA molecules to the microtubules was quite reliable, i. e. there were no DNA molecules lost on the way. The maximum apparent distance of transport was only limited by the field of view and by the total time of the image acquisition.

Grasping of two DNA ends by moving microtubules and simultaneous stretching of DNA between substrate surface and microtubules were subsequently observed. The images shown in Figure 3a were derived from this sequence.

We also observed transport of DNA on a moving microtubule and stretching of that DNA molecule by a second microtubule. The images shown in Figure 3b were derived from this sequence.

In another experiment, guiding of a microtubule on a circular path due to the pulling force of the stretched DNA molecule was observed. Observation started at time zero.

The images shown in Figure 4 were derived from this sequence.

A second example of a moving microtubule was observed which was forced onto a circular path due to the pulling force of a stretched DNA molecule. The microtubule performed about 5 turns with a radius of about 2 um. A similar spiral movement of motor-driven filaments, whose motion is interrupted by surface defects, has been described in L. Bourdieu et al., Phys. Rev. Lett. 75, (1995), 176-179.