Abstract:
A composition comprising a double-stranded DNA filament having a plurality of single-species of oligonucleotide and chain terminator units, and a method for forming such filaments are disclosed. The oligonucleotide include a first segment, a second segment, pre-determined lengths, and pre-adjusted ratios of A:T/G:C between 1% and 50%. The chain terminator units include nucleotides of the first or second segment.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This Non-Provisional Patent Application is a continuation of, and claims the benefit of, U.S. patent application Ser. No. 12/438,986, entitled “Self-Assembling Oligonucleotides and Methods,” which is the National Stage of International Application No. PCT/US2007/019036, filed on Aug. 29, 2007, which claims the benefit of U.S. Provisional Application No. 60/841,039, filed on Aug. 29, 2006, all of which are hereby incorporated by reference herein in their entireties including all references and appendices cited therein. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to self-assembling oligonucleotides and methods of their use, e.g., in micro- and nano-fabrication. 
       REFERENCES 
       [0003]    Braun, E. et al., Nature (London) 391.775 (1998). 
         [0004]    Richter, J. et al., Appl. Phys. Lett. 78, 536 (2001). 
         [0005]    Hainfeld, J. F. et al. Microsc. Microanal., 7, 1034 (2001). 
         [0006]    Bhalla, V. et al. EMBO Reports, Vol. 4, No.5, 442-445 (2003). 
         [0007]    Fink, H. W. and Shonenberger, C. Nature, Vol. 398,407-410 (1999). 
       BACKGROUND OF THE INVENTION 
       [0008]    Under existing silicon-based computer chip technology, the number of transistors on a chip has doubled about every 18 months according to Moore&#39;s law. This pace is likely to slow considerably because of physical and atomic constraints on silicon chips. For instance, the wire components used in silicon chips cannot be made smaller than 300 nm due to the wavelength of light used in lithography. (Hainfeld, 2001). Enlarging silicon computer chips will accommodate greater processing power. Yet in personal computing applications, silicon chips can only be enlarged so much, especially in the context of laptop computers. Additionally, there is a need to for small powerful-chips to be used in cellular phones, PDAs, and other handheld devices. Transistors can be made smaller if they are made from nanowires having a diameter of less than 10 nm. Use of these smaller transistors could dramatically increase the number of transistors per area of the chip, allowing computer chips to become smaller and more powerful. 
         [0009]    DNA may be used create nanowires because it is sufficiently small and can be made or fashioned as elongate filaments. Double-stranded DNA has a width of approximately 2 nm and each base pair adds approximately 0.34 nm in length to the molecule. Recently, double-stranded DNA has been shown to conduct electricity. (Porath et al. 2000). The conductive ability of DNA may be as good as that of a semiconductor. (Fink and Shonenberger, 1999). 
         [0010]    Further, the conductivity of a DNA strand may be enhanced in a variety of ways. In one approach, the filament is doped with metal ions, such as Zn, Co, Ni. With sufficient doping, the conductivity of the filament may be enhanced several orders of magnitude. Alternatively, nucleic acid filaments may be coated with a conductive film, such as a gold or silver film applied by metal atom deposition of other coating methods (Braun 1998 and Richter 2001). Another exemplary DNA nanowire may be created by binding gold to a DNA filament. (Hainfeld, 2001). This is further described in US Patent Publications 2006/0154380 and 2002/0016306, which are hereby incorporated by reference. DNA filaments may be connected to other circuit elements by hybridization with a single-stranded DNA sequence, using standard DNA splicing and ligation methods. (Hainfeld, 2001). Other circuit elements include other DNA nanowires or solid-state circuit elements that are covalently bound to a single-stranded DNA molecule. 
         [0011]    Each DNA strand that comprises a nanowire may be made using a commercially available polynucleotide synthesizer, yet this approach is suboptimal. The maximum length of a DNA that can be feasibly made using this technique is 100 base pairs. Furthermore, DNA synthesizers will contain shorter DNA strands as impurities. Alternatively, the DNA used to make a nanowire can be assembled from smaller pure oligonucleotides. 
         [0012]    It would therefore be desirable to provide oligonucleotides that can be easily synthesized in bulk and which can be assembled readily into desired lengths and/or filament bundles. 
       SUMMARY OF THE INVENTION 
       [0013]    In one aspect, the invention includes a composition of individual single-stranded oligonucleotides capable of self-assembly. Each oligonucleotide is composed of two adjacent segments that can hybridize to the same segment in another oligonucleotide molecule of the composition. Under hybridization conditions, the oligonucleotides will self-assemble to form a double-stranded polynucleotide in which the first and second segments of an oligonucleotide in one strand of the filament are hybridized with complementary first and second segments, respectively, of two adjacent oligonucleotides in the second strand of the filament, where each strand is composed of a plurality of said oligonucleotides. 
         [0014]    The first and second segments in the single-stranded oligonucleotides may be the same or different lengths and may be palindromic and/or self-complementary. The oligonucleotides may have a phosphate group at their 5′-ends to allow the 5′-end of one oligonucleotide to be ligated to the 3′-end of an adjacent polynucleotide on the same strand, thus to form double-stranded filaments with integral strands. 
         [0015]    The individual molecules may each contain between 1-3 nucleotide bases between the first and second segments, such that each substantially contiguous strand in the double stranded filament contains periodic gaps that are 1-3 bases in length and offset from gaps in the other strand in the filament. 
         [0016]    The first segment of the oligonucleotides may be composed substantially of A and T bases only, and the second segment, of substantially of G and C bases only, such that the double stranded filament has regions composed substantially of paired A and T bases only alternating with regions of paired G and C bases only. 
         [0017]    For use in forming a double-stranded DNA having a selected length, the oligonucleotides may include a plurality of N sets of different-sequence, but same-length oligonucleotides, where the 5′-end sequence in each set of oligonucleotides is complementary to the 5′-end sequence in only one other set of oligonucleotides, and where the 3′-end sequence in each set of oligonucleotides is complementary to 3′-end sequence in only one other set of oligonucleotides. In this embodiment, the pair of complementary, substantially contiguous single strands of a double-stranded nucleic acid filament is composed of only one oligonucleotide from each of the N sets of oligonucleotides. 
         [0018]    Alternatively, for use in forming a double-stranded DNA having a selected length, the oligonucleotides may include a plurality of N sets of different-sequence, different-length oligonucleotides, where the 5′-end sequence in each set of oligonucleotides is the same length as and complementary to the 5′-end sequence in only one other set of oligonucleotides, and where the 3′-end sequence in each set of oligonucleotides is the same length as and complementary to 3′-end sequence in only one other set of oligonucleotides. The pair of complementary, substantially contiguous single strands of a double-stranded nucleic acid filament is composed of only one oligonucleotide from each of the N sets of oligonucleotides. 
         [0019]    In another aspect, the invention includes a method for forming a double-stranded duplex nucleic acid filament, by reacting in solution, under nucleic acid-acid hybridization conditions, the oligonucleotides described above. 
         [0020]    The method may further include ligating adjacent ends of the oligonucleotides forming each strand of the double-stranded filament, where the oligonucleotides each have a 5′-end phosphate group, and the ligating is carried out with a ligase enzyme in the presence of ATP. 
         [0021]    Where the method is employed to produce a double-stranded duplex nucleic acid filament of a defined length, the individual oligonucleotides may include (i) include a plurality of N sets of different-sequence, but same-length oligonucleotides, where the 5-end sequence in each set of oligonucleotides is complementary to the 5′-end sequence in only one other set of oligonucleotides, and where the 3′-end sequence in each set of oligonucleotides is complementary to 3′-end sequence in only one other set of oligonucleotides, or (ii) a plurality of N sets of different-sequence, different-length oligonucleotides, where the 5′-end sequence in each set of oligonucleotides is the same length as and complementary to the 5′-end sequence in only one other set of oligonucleotides, and where the 3′-end sequence in each set of oligonucleotides is the same length as and complementary to 3′-end sequence in only one other set of oligonucleotides. 
         [0022]    For use in forming a bundle of sequence-aligned double-stranded nucleic acid filaments, the oligonucleotide self-assembly step may be carried out in the presence of a selected-length double-stranded template filament having a sequence formed by self-assembly of such oligonucleotides, wherein the template filament directs the assembly of the oligonucleotides to form a bundle of sequence-aligned double-stranded nucleic acid filaments. 
         [0023]    For use in producing a double-stranded duplex nucleic acid filament bridging a pair of selected regions on a substrate, the method may involve the step of (a) anchoring at each region on the substrate. a single-stranded nucleic acid fragment whose sequence is complementary to one segment of the oligonucleotides, (b) exposing the substrate and anchored single-stranded nucleic acid fragments to a solution of the oligonucleotides, under nucleic acid hybridization conditions, and (c) by this exposing, forming between the two regions, and connected therewith, a double-stranded nucleic acid filament composed of overlapping oligonucleotides. 
         [0024]    Each region of the solid support may be a gold film electrode, and the fragments may be attached to the film through a disulfide linkage between a surface-bound thiol reagent and a phosphorothio group at the 3′-end of the fragment. 
         [0025]    For use in creating an electrically conductive filament between the first and second regions, the double-stranded nucleic acid filament formed in step (c) may be coated with an electrically conductive film, or may be treated with a solution of a Zn, Co, or Ni salt, under conditions effective to incorporate Zn, Co, or Ni atoms between the filament&#39;s nucleic acid bases. 
         [0026]    These and other objects and features of the present invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0027]      FIG. 1  shows steps for hybridizing oligonucleotides with 5′ and 3′ palindromic sequences to make a double-stranded polynucleotide. 
           [0028]      FIG. 2  shows addition of phosphate groups at the 5′ end of the oligonucleotides so that ligation can occur without the need for treating the oligonucleotides with a kinase before reaction. 
           [0029]      FIG. 3A  shows a scheme of making a polynucleotide using a single oligonucleotide that has palindromic sequence at the 5′-end of length equal to that of the palindromic sequence at the 3′-end. 
           [0030]      FIGS. 3B -3C shows a scheme for making a polynucleotide using a single oligonucleotide that has sequence at the 5′-end of length different from the sequence at the 3′-end. 
           [0031]      FIG. 4A  shows a scheme for making a polynucleotide of a specific length using multiple oligonucleotides of equal length with sequences at the 5′-end and the 3′-end. The length is specifically determined because the growing double-stranded polynucleotide will hybridize with only one other polynucleotide in each step. 
           [0032]      FIG. 4B  shows a scheme for making a polynucleotide of a specific length using multiple oligonucleotides with sequences at the 5′-end and the 3′-end. In the hybridization, each successive oligonucleotide that hybridizes to the growing double-stranded polynucleotide is one base pair longer than the preceding oligonucleotide. The length is specifically determined because the growing double-stranded polynucleotide will hybridize with only one other polynucleotide in each step. 
           [0033]      FIG. 5  shows the synthesis of a double-stranded polynucleotide connected to a solid support. The oligonucleotide used to make the polynucleotide can hybridize to the single-stranded oligonucleotides that are chemically bound to the solid support. 
           [0034]      FIG. 6  shows the synthesis of bundled double-stranded polynucleotides in which one of the polynucleotides is chemically bound to a solid support. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0035]    I. Definitions 
         [0036]    The terms below have the following definitions unless indicated otherwise. 
         [0037]    “Oligonucleotide” means a natural nucleic acid, typically in single-stranded form, such as DNA or RNA, or a nucleic acid analog, having either nonribose subunits, e.g., morpholino subunits, or charged or uncharged backbone linkages other than natural phosphodiester linkages, e.g., phosphorothioate, phosphoroamidate, phosphorodiamidate, and the like. An “oligonucleotide” has a sequence of bases that allows it to hybridize, under suitable hybridization conditions, with a complementary sequence in another oligonucleotide via Watson-Crick base pairing. Oligonucleotides are typically less than 100 bases in length, typically 8-30 bases. The terms DNA, nucleic acid, including RNA, and nucleic acid analogs are used interchangeably. 
         [0038]    A “palindromic sequence” is a DNA locus whose 5′-to-3′ sequence is identical on each DNA strand. The sequence is the same when one strand is read left to right and the other strand is read right to left. 
         [0039]    A double-strand filament is an oligonucleotide or polynucleotide filament having a length of from less than 100 bases (oligonucleotide strands) to a length of up to several hundred or several thousand bases (polynucleotide strands), and formed of complementary or substantially complementary strands, where each strand is formed of ligated or unligated side-by-side oligonucleotides, and each oligonucleotide in one strand is hybridized with portions or segments of two adjacent oligonucleotides on a second strand. 
         [0040]    An oligonucleotide strand in a double-stranded nucleic acid filament is “substantially contiguous” if the confronting tail-to-head nucleotide bases in adjacent oligonucleotides forming the strand are immediately adjacent, or are spaced at most by 1-3 bases. 
         [0041]    II. Oligonucleotide composition and self-assembly 
         [0042]    The invention includes, in one aspect, individual oligonucleotides that can self-assemble under hybridizing conditions, to form a double-stranded oligonucleotide or polynucleotide filament. Each oligonucleotide includes a 5′-end segment or sequence and a 3′-end segment or sequence. Each segment of an oligonucleotide is capable of hybridizing with the corresponding 5′-end or 3′-end segment of at least one other oligonucleotide in the composition. Although the invention is illustrated with respect to oligonucleotides whose end segments are composed exclusively of pyrimidine bases (A or T or U) or purine bases (G or C), either or both regions may contain mixtures or both base types, as long as the sequences allow for self-assembly by hybridization, as described herein. 
         [0043]    A. Compositions with Single Oligonucleotide Sequences 
         [0044]    This section describes an embodiment in which the individual oligonucleotides in a self-assembly composition are identical, that is, the composition includes a single oligonucleotide species, and each individual oligonucleotide (oligonucleotide molecule) has the same sequence and length. As will be seen, this embodiment has advantages of simplicity and cost, but the self-assembly reaction with a single oligonucleotide species may be more difficult to control, for example, to achieve desired length filaments. 
         [0045]    The oligonucleotides in this embodiment are capable of self-assembling to form “open-ended” double-stranded nucleic acid filaments, according to the self-assembly scheme illustrated in  FIG. 1 . The figure shows an 8-mer oligonucleotide  10  having a 5′-end palindromic segment AATT, and a 3′-end palindromic segment CCGG. As seen in the figure, two such oligonucleotides are capable of forming, under hybridization conditions, a double-stranded complex  12  in which the two 3′-end segments are hybridized by Watson-Crick, and the two adjacent 5′-end segment are single-stranded overhangs. The overhangs, in turn, can now hybridize with the same oligonucleotides in solution, to form increasingly longer double-stranded complexes, such as complex  14  formed after 2 oligonucleotide additions, where each two oligonucleotide additions add one oligonucleotide to each strand of the complex. Thus, after 2n−1 additions, the double-stranded complex shown at  16  is formed that has a pair .of complementary, substantially contiguous single strands, indicated at  16   a ,  16   b , in which the first and second segments of an oligonucleotide in one strand of the filament are hybridized with complementary first and second segments, respectively, of two adjacent oligonucleotides in the second strand of the filament, and each strand is composed of n such oligonucleotides. 
         [0046]    That is, the filament shown in  FIG. 1  has a pair of hybridized double stranded lengths corresponding to n repeats of the sequence in each oligonucleotide, and two overhangs that together make up the sequence of a single oligonucleotide. Although the two overhangs are shown on the same strand, addition of a single oligonucleotide to the filament would of course place one overhang on each strand. 
         [0047]    It will be appreciated that during the self-assembly reaction, many double-stranded filament complexes are being formed, and these may have a range of filament lengths. The self-assembly reaction may be terminated, at a desired end point, e.g., desired average filament length, by controlling the concentration of oligonucleotide in the reaction, and/or changing the reaction conditions, e.g., by cooling, to block further hybridization. More generally, the “length” of the filament is dictated by the (a) concentration of the oligonucleotides in the reaction, (b) the sequence (G/C rich vs. A/T rich), (c) the number of self-associating bases (pairing) and (d) the conditions under which self-associating event occurs. Conditions may include: (1) solvents/buffers, (2) temperature of the self-assembly solution, (3) presence of “ligating” enzymes, (4) “chain terminating” DNA units (see below), (5) presence of additional chemistry at the 3′-5′ gap to favor the passage of electrons/ electricity, and (6) the use of substrates/templates/devices, which act as mold for the alignment and ordered stacking of DNA polymers. 
         [0048]    Chain terminators are DNA sequences designed to stop the self-assembling process of palindromic sequences by competing with the self-assembly process. These units lack the second palindromic unit, therefore can only occur once for each chain elongation. Chain terminators may be useful to stop the self-assembly process to a desirable number of events, so that a more homogeneous family of polymers is generated. The effect of chain termination by these DNA molecules can be monitored by adding them to the above reactions. Concentration of chain terminators and time of addition to the self-annealing reactions will be monitored. Chain terminating DNA units may be useful as anchoring moieties. For example, the presence of a cross linking chemical at the 5′-end of these units will favor the use of DNA polymers in conjunction with activated solid surfaces. Biotin-bearing DNA terminators can be used to link the DNA polymers to avidin coated surfaces, etc. 
         [0049]    Ideally, for bio-circuits applications, homogeneous polymerization will be achieved so that filaments of self-associating DNA are obtained with similar length and folding characteristics. It will be desirable to produce DNA filaments of similar shape and length to construct biotransistors and other “information” devices. Compositions designed to form specified filament lengths are described further below. 
         [0050]    In one embodiment, the individual oligonucleotides each contain between 1-3 nucleotide bases between said first and second segments. As can be appreciated, when the oligonucleotides in this embodiment self-assemble, each of the resulting strands forming the double-stranded filament contains periodic gaps that are 1-3 bases in length and offset from gaps in the other strand in the filament. These gaps may be used, for example, to insert metal ions or atoms or other groups capable of modifying the electronic properties of the strands. Alternatively, filaments with such 1-3 based gaps can be enzymatically modified to fill in and ligate the gaps, forming filaments in which each strand is continuous and integral. 
         [0051]    In another embodiment, and the one illustrated herein, the first segment of the oligonucleotides is composed substantially of A and T bases only, and the second segment of the oligonucleotides is composed of substantially of G and C bases only, yielding a double stranded filament has regions composed substantially of paired A and T bases only alternating with regions of paired G and C bases only. This arrangement of alternating regions of A:T and G:C confers selected electronic properties on the filaments, due to the different donor:acceptor nature of the two regions. 
         [0052]    Following the self-assembly reaction illustrated with respect to  FIG. 1 , the two strands of a filament may be individually ligated to form continuous covalently linked strands without breaks between adjacent oligonucleotides. This may be done, as illustrated in  FIG. 2 , by providing the oligonucleotides with 5′-end phosphate groups, according to standard oligonucleotide synthesis methods. The double-stranded filament formed by self-assembly will then have 5′-end phosphate groups spaced at even intervals along the length of each strand shown at  18  in  FIG. 2 . By reacting the resulting filament with a ligase enzyme, e.g., T4 ligase, in the presence of ATP and under suitable ligation conditions, both strands are internally linked to produce the continuous-strand complex shown at  20  in  FIG. 2 . Following ligation, the double-stranded complexes may be fractionated, e.g., by gel electrophoresis, to yield fragments of desired lengths. 
         [0053]    In the embodiments illustrated above, all of the oligonucleotides have the same 5′-end and 3′-end region sequence and length, as noted, and both regions are palindromic, that is, have the same sequence when read from 5′-to-3′ in one strand and from 3′-to-5′ in the opposite strand. Both of these segments or regions are illustrated as 4-base segments. Typically, however, the oligonucleotide segments will be longer, e.g., 6-15 bases of longer, to provide greater thermal stability at a suitable reaction temperature, e.g., room temperature or greater. Further, the percentage of A:T/G:C may be varied according to known method to achieve a desired duplex stability. 
         [0054]      FIGS. 3A-3C  show various oligonucleotides having different A:T/G:C ratios and/or different palindromic sequences. The oligonucleotide  10  in  FIG. 3A  is the same as that shown in  FIGS. 1 and 2 , and produces the double-stranded filament shown at  16 . Oligonucleotide  22  in  FIG. 3B , representing an oligonucleotide useful in constructing an A:T rich filament, contains an 8-base 5′-end A-A-A-A-T-T-T-T segment and a 4-base C-C-G-G segment, resulting in a double-stranded filament  24  having a 12-base double-stranded repeat sequence and an 8-base and 4-base overhang. Oligonucleotide  26  in  FIG. 3C . representing an oligonucleotide useful in constructing a GUCCI rich filament, contains a four-base 5′-end A-A-T-T segment and an 8-base G-C-G-C-G-C-G-C segment, resulting in a double-stranded filament  28  having a double-stranded 12-base repeat and an 4-base and 8-base overhang. The ratios, lengths and sequences of AT and GUCCI regions may be adjusted to achieve desired electronic, e.g., semiconductor properties in the filaments, as discussed below. 
         [0055]    B. Compositions with Multiple Oligonucleotide Sequences 
         [0056]    This section describes a composition of the invention having N different sequence and/or different length oligonucleotide species. As will be seen, the compositions are designed for self-assembly of filaments having a defined length and sequence, and typically form filaments containing in each strand, one of each of the N different-sequence or different-length oligonucleotides. 
         [0057]    The embodiment illustrated in  FIG. 4A  contains a plurality (N) of different-sequence, but same-length oligonucleotide species, three of which are indicated at  30 ,  34 , and  38  in  FIG. 4A . In this embodiment, one of the 5′-end or 3′-end segments of a given oligonucleotide species is complementary to the corresponding 5′-end or 3′-end segment of one other oligonucleotide species in the composition, and the other of the two segments is complementary to the corresponding segment of a third oligonucleotide species in the composition. In addition, at least one of the species may have a self-complementary sequence that allows for initial oligonucleotide hybridization. 
         [0058]    For example, oligonucleotide 30 in the composition has a palindromic 3′-end G-G-C-C sequence that allows the oligonucleotide to form a duplex dimer  32 . However, since the 5′-end regions of this oligonucleotide are not complementary, self-assembly by that oligonucleotide alone ends. A second oligonucleotide species  34  in the composition has a 5′-end segment that is complementary to the 5′-end segment of oligonucleotide  30 , allowing this species to hybridize to both strands of complex  32 , through the 5′-end segment overhangs, to form complex  36  that is expanded bi-directionally. Similarly, a third species  38  having a 3′-end segment that is complementary to the overhang 3′-end segments of the complex  36 , allowing this species to hybridize to both strands of complex  36 , through the 3′-end segment overhangs to form complex  40 , again by bi-directional expansion. 
         [0059]    After each hybridization step, the pattern described in the second and third hybridization steps continues. The fifth, seventh and additional odd-numbered oligonucleotides must have a 3′ segment that is the reverse compliment of the 3′ segment of the fourth, sixth and previous even-numbered oligonucleotide. Furthermore, the sixth, eighth, and additional even-numbered oligonucleotides must have a 5′ segment that is the reverse compliment of the 5′ segment of the fifth, seventh, and previous odd-numbered oligonucleotides. Bidirectional growth continues in this manner until all N different-sequence species are incorporated into each of the two filament strands, as indicated at  42  in the figure. 
         [0060]    Although only one of the oligonucleotide species described above has a self-complementary segment (oligonucleotide  30 ), it will be appreciated that other species may also have a self-complementary segment, since the stability of any two-oligonucleotide hybrids, where only one of the two segments are hybridized, would be substantially less than the stability of the same species in a growing strand, where both segments would be hybridized, and the species would therefore tend to participate preferentially in filament formation. 
         [0061]    In another embodiment, illustrated in  FIG. 4B , the composition of the invention includes N different-sequence and different length species, but where, as above, even- or odd-numbered species have 5′-end or 3′-end segments that are complementary to the 5′-end or 3′-end segments of odd- or even numbered species, respectively. As illustrated, an “initial” species  44  has a self-complementary 3′-end region G-G-C-C capable of forming duplex hybrids, with non-self-complementary 5′-end T-T-T-A-A segments. A second species  48  has a 5′-end segment A-A-A-T-T that is complementary to the two overhangs in complex  46 , to form complex  50  by bi-directional expansion by one oligo. A third species  52  has a 3′-end segment C-C-G-G-G segment that is complementary to the two overhangs in complex  50 , to form complex  54 , which then expands by addition of species  56 , and so forth, as above, until a duplex filament  58  having N different-sequence, different-length oligonucleotides in each strand is formed. Thus, the final length of the filaments formed is uniform and equal to the combined lengths of each of the N oligonucleotide species. 
         [0062]    III. Construction of a Nucleotide-Based Nanowire 
         [0063]    In one aspect, the filaments formed by self-assembly, as above, may be used in forming patterned-wire nanostructures, such as wires, in a micro- or nano-fabrication method, as illustrated in  FIG. 5 . The figure shows a microfabrication substrate  60  having two spaced regions,  62 ,  64 , such as electrode regions. Each region has attached to its surface, one or more oligonucleotides, such as oligonucleotide  66  on region  62  and oligonucleotide  68  on region  64 . The substrate regions may carry an electrically conductive film formed of gold, silver, or the like. The oligonucleotides may be attached to a gold film through thiol linkages or a disulfide linkage between a surface-bound thiol reagent and a phosphorothio group at the 3′-end of the oligo, as described, for example, in U.S. Pat. Nos. 6,369,206; 5,521,289; 6,369,206; 6,645,721; and 6,733,975, all of which are incorporated herein by reference. The bound oligonucleotides are complementary to the overhangs in a double-stranded filament, such as filament  70  similar to filament shown at  16  in  FIG. 1 . 
         [0064]    To form the filament connection between the two regions, the region is exposed to a solution of the oligonucleotides, under suitable hybridization conditions, allowing both anchorages of the oligonucleotides to the attached oligonucleotides, and self-assembly to form filament connections, such as connection  70 , across the two regions. 
         [0065]    Where it is desired to form the shortest connection between a pair of anchor regions, e.g., substantially straight lines, the self-assembly composition may include N different-length and/or different-sequence oligonucleotides, as described above with respect to  FIG. 4A or 4B , to form filaments whose selected final length is selected to just bridge the distance between the anchoring regions. Following self-assembly, the wire may be ligated to form a continuous filament covalently bridging the two regions. 
         [0066]    Where the filaments between substrate regions are intended to function as semiconductor or conductor wires, the filaments may be further coated with a conductive or other desired film, according to known micro- or nano-fabrication methods, such as low-angle deposition. 
         [0067]    In another embodiment, the double-stranded nucleic acid filament may or may not be treated with a zinc, cobalt, or nickel salt under conditions effective to incorporate Zn, Co, or Ni atoms between the bases of the nucleic acid filament. U.S. Pat. Nos. 5,824,473; 6,432,641; and 6,974,703 are incorporated herein by reference. 
         [0068]    In still another embodiment, the filaments may serve as photomask lines, allowing the substrate surface to be irradiated, to render with linear regions below the filament either susceptible or resistant to surface photoetching, as one step in a micro- or nano-fabrication process. 
         [0069]      FIG. 6  illustrates a method for forming sequence-aligned&#39; bundles of filaments, such as indicated at  82 . In this method, a single, continuous double-stranded filament  78  having a known length and sequence is prepared, e.g., by solid-phase synthesis, and joined to two regions  74 ,  76  on a substrate  72 , as shown at the top in the figure. As seen, filament  78  has the same repeating, offset-oligonucleotide sequence as that formed by self-assembly of the oligonucleotides  80 . When the substrate  72  and attached defined-length filament  78  is exposed to a solution of oligonucleotides  80 , the latter self-assemble into additional filaments, with the self-assembly being guided by the existing filament, so that each new filament tends to adopt a sequence alignment with the existing “template” filament(s), forming a bundle of filaments, like filament  84  that are all in sequence alignment. 
         [0070]    Although the invention has been described with reference to particular embodiments and applications, it will be appreciated that various changes and modifications may be made without departing from the claimed invention.