NUCLEIC ACID MEDIATED ELECTRON TRANSFER

Abstract: The present invention provides for the selective covalent modification of nucleic acids with redox active moieties such as transition metal complexes. Electron donor and electron acceptor moieties are covalently bound to the ribose-phosphate backbone of a nucleic acid at predetermined positions. The resulting complexes represent a series of new derivatives that are bimolecular templates capable of transferring electrons over very large distances at extremely fast rates. These complexes possess unique structural features which enable the use of an entirely new class of bioconductors and photoactive probes.

Description: NUCLEIC ACID MEDIATED ELECTRON TRANSFER This application is a continuation-in-part application of U S S N 08/166,036, filed December 10, 1993, and of U.S S N 08/475,051 , filed June 7 1996 FIELD OF THE INVENTION The present invention is directed to electron transfer via nucleic acids More particularly, the invention is directed to the site-selective modification of nucleic acids with electron transfer moieties such as redox active transition metal complexes to produce a new series of biomaterials and to methods of making and using them The novel biomaterials of the present invention may be used as bioconductors and diagnostic probes BACKGROUND OF THE INVENTION The present invention, in part relates to methods for the site-selective modification of nucleic acids with redox active moieties such as transition metal complexes, the modified nucleic acids themselves, and their uses Such modified nucleic acids are particularly useful as bioconductors and photoactive nucleic acid probes The detection of specific nucleic acid sequences is an important tool for diagnostic medicine and molecular biology research Gene probe assays currently play roles in identifying infectious organisms such as bacteria and viruses in probing the expression of normal genes and identifying mutant genes such as oncogenes, in typing tissue for compatibility preceding tissue transplantation in matching tissue or - blood samples for forensic medicine, and for exploring homology among genes from different species Ideally, a gene probe assay should be sensitive, specific and easily automatable (for a review, see Nickerson, Current Opinion in Biotechnology 4 48-51 (1993)) The requirement for sensitivity (i e low detection limits) has been greatly alleviated by the development of the polymerase chain reaction (PCR) and other amplification technologies which allow researchers to amplify exponentially a specific nucleic acid sequence before analysis (for a review, see Abramson et al , Current Opinion in Biotechnology, 4 41-47 (1993)) Specificity, in contrast, remains a problem in many currently available gene probe assays The extent of molecular complementarity between probe and target defines the specificity of the interaction Variations in the concentrations of probes, of targets and of salts in the hybridization medium, in the reaction temperature, and in the length of the probe may alter or influence the specificity of the probe/target interaction It may be possible under some limited circumstances to distinguish targets with perfect complementarity from targets with mismatches, although this is generally very difficult using traditional technology, since small variations in the reaction conditions will alter the hybridization New experimental techniques for mismatch detection with standard probes include DNA ligation assays where single point mismatches prevent ligation and probe digestion assays in which mismatches create sites for probe cleavage Finally, the automation of gene probe assays remains an area in which current technologies are lacking Such assays generally rely on the hybridization of a labelled probe to a target sequence followed by the separation of the unhybπdized free probe This separation is generally achieved by gel electrophoresis or solid phase capture and washing of the target DNA, and is generally quite difficult to automate easily The time consuming nature of these separation steps has led to two distinct avenues of development One involves the development of high-speed, high-throughput automatable electrophoretic and other separation techniques The other involves the development of non-separation homogeneous gene probe assays For example, Gen-Probe Inc , (San Diego, CA) has developed a homogeneous protection assay in which hybridized probes are protected from base hydrolysis, and thus are capable of subsequent chemiluminescence (Okwumabua et al Res Microbiol 143 183 (1992)) Unfortunately, the reliance of this approach on a chemiluminescent substrate known for high background photon emission suggests this assay will not have high specificity EPO application number 86116652.8 describes an attempt to use non-radiative energy transfer from a donor probe to an acceptor probe as a homogeneous detection scheme However, the fluorescence energy transfer is greatly influenced by both probe topology and topography, and the DNA target itself is capable of significant energy quenching, resulting in considerable variability Therefore there is a need for DNA probes which are specific, capable of detecting target mismatches, and capable of being incorporated into an automated system for sequence identification As outlined above, molecular biology relies quite heavily on modified or labelled oligonucleotides for traditional gene probe assays (Oligonucleotide Synthesis A Practical Approach Gait et al , Ed , IRL Press Oxford, UK, 1984, Oligonucleotides and Analogues A Practical Approach Ed F Eckstein, Oxford University Press, 1991) As a result, several techniques currently exist for the synthesis of tailored nucleic acid molecules Since nucleic acids do not naturally contain functional groups to which molecules of interest may easily be attached covalently methods have been developed which allow chemical modification at either of the terminal phosphates or at the heterocyclic bases (Dreyer et al Proc Natl Acad Sci USA 1985, 82 968) For example, analogues of the common deoxyπbo- and πbonucleosides which contain ammo groups at the 2' or 3' position of the sugar can be made using established chemical techniques (See Imazawa et al , J Org Chem 1979, 44 2039, Imazawa et al , J Org Chem 43(15) 3044 (1978), Verheyden et al , J Org Chem 36(2) 250 (1971), Hobbs et al , J Org Chem 42(4) 714 (1977)) In addition, oligonucleotides may be synthesized with 2'-5' or 3'-5' phosphoamide linkages (Beaucage et al , Tetrahedron 49(10) 1925 (1992), Letsinger, J Org Chem , 35 3800 (1970), Sawai, Chem Lett 805 (1984), Oligonucleotides and Analogues A Practical Approach, F Eckstein, Ed Oxford University Press (1991)) The modification of nucleic acids has been done for two general reasons to create nonradioactive DNA markers to serve as probes, and to use chemically modified DNA to obtain site-specific cleavage To this end, DNA may be labelled to serve as a probe by altering a nucleotide which then serves as a replacement analogue in the nick translational resynthesis of double stranded DNA The chemically altered nucleotides may then provide reactive sites for the attachment of immunological or other labels such as biotm (Gilliam et al , Anal Biochem 157 199 (1986)) Another example uses ruthenium derivatives which intercalate into DNA to produce photoluminescence under defined conditions (Friedman et al , J Am Chem Soc 112 4960 (1990)) In the second category, there are a number of examples of compounds covalently linked to DNA which subsequently cause DNA chain cleavage For example 1 ,10- phenanthrolme has been coupled to single-stranded oligothymidylate via a linker which results in the cleavage of poly-dA oligonucleotides in the presence of Cu 2+ and 3- mercaptopropionic acid (Francois et al , Biochemistry 27 2272 (1988)) Similar experiments have been done for EDTA 1 -Fe(ll) (both for double stranded DNA (Boutoπn et al , FEBS Lett 172 43-46 (1986)) and triplex DNA (Strobel et al , Science 249 73 (1990)), porphyπn-Fe(lll) (Le Doan et al Biochemistry 25 6736-6739 (1986)), and 1 ,10-phenanthronιne-Cu(l) (Chen et al , Proc Natl Acad Sci USA, 83 7147 (1985)), which all result in DNA chain cleavage in the presence of a reducing agent in aerated solutions A similar example using porphynns resulted in DNA strand cleavage and base oxidation or cross-linking of the DNA under very specific conditions (Le Doan et al Nucleic Acids Res 15 8643 (1987)) Other work has focused on chemical modification of heterocyclic bases For example the attachment of an inorganic coordination complex, Fe-EDTA, to a modified internal base resulted in cleavage of the DNA after hybridization in the presence of dioxygen (Dreyer et al , Proc Natl Acad Sci USA 82 968 (1985)) A ruthenium compound has been coupled successfully to an internal base in a DNA octomer, with retention of both the DNA hybridization capabilities as well as the spectroscopic properties of the ruthenium label (Telser et al , J Am Chem Soc 111 7221 (1989)) Other experiments have successfully added two separate spectroscopic labels to a single doubie-stranded DNA molecule (Telser et al , J Am Chem Soc 111 7226 (1989)) The study of electron transfer reactions in proteins and DNA has also been explored in pursuit of systems which are capable of long distance electron transfer To this end, intramolecular electron transfer in protein-protein complexes, such as those found in photosynthetic proteins and proteins in the respiration pathway, has been shown to take place over appreciable distances in protein interiors at biologically significant rates (see Bowler et al , Progress in Inorganic Chemistry Bioinorganic Chemistry, Vol 38, Ed Stephen J Lippard ( 990) In addition, the selective modification of metalloenzymes with transition metals has been accomplished and techniques to monitor electron transfer in these systems developed For example, electron transfer proteins such as cytochrome c have been modified with ruthenium through attachment at several histidines and the rate of electron transfer from the heme Fe 2* to the bound Ru 3* measured The results suggest that electron transfer "tunnel" pathways may exist (Baum, Chemical & Engineering News, February 22, 1993, pages 2023, see also Chang et al , J Am Chem Soc 113 7056 (1991)) In related work, the normal protein insulation, which protects the redox centers of an enzyme or protein from nondiscπminatory reactions with the exterior solvent, was "wired" to transform these systems from electπcal insulators into electrical conductors (Heller, Ace Chem Res 23 128 (1990)) There are a few reports of photoinduced electron transfer in a DNA matrix In these systems, the electron donors and acceptors are not covalently attached to the DNA, but randomly associated with the DNA, thus rendering the explicit elucidation and control of the donor-acceptor system difficult For example the intense fluorescence of certain quaternary diazoaromatic salts is quenched upon intercalation into DNA or upon exposure to individual mononucleotides, thus exhibiting electron donor processes within the DNA itself (Brun et al . J Am Chem Soc 113 8153 (1991)) Another example of the difficulty of determining the electron transfer mechanism is found in work done with some photoexcitable ruthenium compounds Early work suggested that certain ruthenium compounds either randomly intercalate into the nucleotide bases, or bind to the helix surface (Purugganan et al , Science 241 1645 (1988)) A recent reference indicates that certain ruthenium compounds do not intercalate into the DNA (Satyanarayana et al , Biochemistry 31(39) 9319 (1992)), rather, they bind non-covalently to the surface of the DNA helix In these early experiments, various electron acceptor compounds, such as cobalt, chromium or rhodium compounds were added to certain DNA-associated ruthenium electron donor compounds (Puragganan et al., Science 241 1645 (1988), Orellana et al , Photochem Photobiol 499 54 (1991), Brun et al , J Am Chem Soc 113 8153 (1991), Davis, Chem -Biol Interactions 62 45 (1987), Tomaiia et al , Ace Chem. Res , 24 332 (1991)) Upon addition of these various electron acceptor compounds, which randomly bind non-covalently to the helix, quenching of the photoexcited state through electron transfer was detected The rate of quenching was dependent on both the individual electron donor and acceptor as well as their concentrations, thus revealing the process as bimolecular In one set of experiments, the authors postulate that the more mobile surface bound donor promotes electron transfer with greater efficiency than the intercalated species, and suggest that the sugar-phosphate backbone of DNA, and possibly the solvent medium surrounding the DNA, play a significant role in the electron transport (Purugganan et al , Science 241 1645 (1988)) In other work, the authors stress the dependence of the rate on the mobility of the donor and acceptor and their local concentrations, and assign the role of the DNA to be primarily to facilitate an increase in local concentration of the donor and acceptor species on the helix (Orellana et al , supra) In another experiment, an electron donor was reportedly randomly intercalated into the stack of bases of DNA, while the acceptor was randomly associated with the surface of the DNA The rate of electron transfer quenching indicated a close contact of the donor and the acceptor, and the system also exhibits enhancement of the rate of electron transfer with the addition of salt to the medium (Fromherz et al , J Am Chem Soc 108 5361 (1986)) In all of these experiments, the rate of electron transfer for non-covalently bound donors and acceptors is several orders of magnitude less than is seen in free solution An important stimulus for the development of long distance electron transfer systems is the creation of synthetic light harvesting systems Work to date suggests that an artificial light harvesting system contains an energy transfer complex, an energy migration complex, an electron transfer complex and an electron migration complex (for a topical review of this area, see Chemical & Engineering News, March 15, 1993, pages 38-48) Two types of molecules have been tried a) long organic molecules, such as hydrocarbons with covalently attached electron transfer species, or DNA, with intercalated, partially intercalated or helix associated electron transfer species, and b) synthetic polymers The long organic molecules, while quite rigid, are influenced by a number of factors, which makes development difficult. These factors include the polarity and composition of the solvent, the orientation of the donor and acceptor groups, and the chemical character of either the covalent linkage or the association of the electron transfer species to the molecule The creation of acceptable polymer electron transfer systems has been difficult because the available polymers are too flexible, such that several modes of transfer occur Polymers that are sufficiently rigid often significantly interfere with the electron transfer mechanism or are quite difficult to synthesize Thus the development of an electron transfer system which is sufficiently rigid, has covalently attached electron transfer species at defined intervals, is easy to synthesize and does not appreciably interfere with the electron transfer mechanism would be useful in the development of artificial light harvesting systems In conclusion, the random distribution and mobility of the electron donor and acceptor pairs, coupled with potential short distances between the donor and acceptor, the loose and presumably reversible association of the donors and acceptors, the reported dependence on solvent and broad putative electron pathways, and the disruption of the DNA structure of intercalated compounds rendering normal base pairing impossible all serve as pronounced limitations of long range electron transfer in a DNA matrix Therefore, a method for the production of rigid, covalent attachment of electron donors and acceptors to provide minimal perturbations of the nucleic acid structure and retention of its ability to base pair normally, is desirable The present invention serves to provide such a system, which allows the development of novel bioconductors and diagnostic probes. SUMMARY OF THE INVENTION The present invention provides for the selective modification of nucleic acids at specific sites with redox active moieties such as transition metal complexes An electron donor and/or electron acceptor moiety are covalently bound preferably along the ribose- phosphate backbone of the nucleic acid at predetermined positions The resulting complexes represent a seπes of new derivatives that are biomolecular templates capable of transferring electrons over very large distances at extremely fast rates These complexes possess unique structural features which enable the use of an entirely new class of bioconductors and diagnostic probes Accordingly, the invention provides polydentate nucleosides, which are nucleosides containing a polydentate ligand covalently attached at the 2' or 3' position of said nucleoside, in the presence or absence of a transition metal Phosphoramidite polydentate nucleosides are also provided Further provided are oligonucleotides covalently attached to a solid support, with at least one nucleoside of said oligonucleotide is a polydentate nucleoside Additionally provided are single-stranded nucleic acids containing at least one electron donor moiety and at least one electron acceptor moiety The electron donor and acceptor moieties are covalently attached to the nucleic acid, and at least one of the electron transfer moieties attached to a polydentate nucleoside Additionally provided are compositions comprising a first single stranded nucleic acid containing at least one covalently attached electron donor moiety and a second single stranded nucleic acid containing at least one covalently attached electron acceptor moiety At least one of the electron donor and acceptor moieties is attached to a polydentate nucleoside Further provided are methods of detecting a target sequence in a nucleic acid sample comprising hybridizing a single stranded nucleic acid containing at least one covalently attached electron donor moiety and at least one covalently attached electron acceptor moiety to the target sequence to form a hybridization complex At least one of the electron transfer moieties is attached to a polydentate nucleoside Detection of the target sequence may then occur Additionally provided are methods of detecting a target sequence in a nucleic acid comprising a first target domain and a second target domain adjacent to the first target domain. The method comprises hybridizing a first nucleic acid containing at least one covalently attached electron donor moiety to said first target domain and hybridizing a second nucleic acid containing at least one covalently attached electron acceptor moiety to said second target domain. At least one of the electron transfer moieties is attached to a polydentate ribonucleoside. Further provided are methods for making nucleic acids with covalently attached electron transfer moieties comprising incorporating a polydentate nucleoside into a nucleic acid, wherein the polydentate nucleoside may or may not have a bound transition metal. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates all the possible orientations of electron donor (EDM) and electron acceptor (EAM) moieties on a single stranded nucleic acid Figure 2 illustrates the possible orientations of electron transfer moieties EDM and EAM on two adjacent single stranded nucleic acids These orientations also apply when the two probes are separated by an intervening sequence Figure 3 illustrates a series of amino-modified nucleoside precursors prior to incorporation into an oligonucleotide Figure 4A and 4B depict the structure of electron transfer moieties Figure 4A depicts the general formula of a representative class of electron donors and acceptors Figure 4B depicts a specific example of a ruthenium electron transfer moiety using bisbipyridine and imidazole as the ligands. Figure 5 is a schematic showing transition metals bound to the ribose-phosphate backbone in a variety of positions. M is a transition metal. M, is bound via an amine on the 2' carbon of the ribose; an electron must travel through 4 σ bonds to enter the pi-orbitals (the "pi-way") of the stacked bases M 2 and M 3 are bound via a phosphoramide-type linkages, and electrons must travel through 7 σ bonds to enter the pi-way, respectively M A is bound via an amine on the 3' carbon of the nbose, and an an electron traverses through 5 σ bonds Figures 6A, 6B, 6C, and 6D depict the attachment of a modified nucleosides to control pore glass (CPG) and the formation of a single stranded nucleic acid with elongation and attachment of transition metal complexes as the exemplified electron transfer species The experimental conditions are outlined in Example 9 Figure 6A depicts the formation of 2'-amιno-2'-deoxyuπdιne derivatized to control pore glass (CPG) 2'- amino modified undine is depicted, although any base may be used As is known in the art, phosphoramidite nucleosides are added to the derivatized nucleoside, after removal of the DMT protecting group, as generally depicted in Figure 6B, using the UCTCCTACAC sequence as an example The addition of a 5' terminal phosphoramidite 2-amιno-deoxyuπdιne, with a DMT protecting group, results in a single stranded nucleic acid containing a 3' and 5' 2'-amιno modified nucleoside Figure 6C depicts the addition of the electron transfer species, exemplified by two ruthenium transition metal complexes, ιm(bpy) 2 Ru and Ru(ll)(NH 3 )„py Figure 6D depicts the synthesis of a single stranded nucleic acid modified at the 3' terminus with a 2'-amιno modified nucleoside and at the 5' terminus with a polydentate nucleoside Figure 7 depicts the addition of a polydentate ligand to the C-terminus of PNA Figures 8A and 8B depicts attachment of the ammo-modified nucleic acids of the invention to electrodes (A) depicts the attachment to glassy carbon electrodes R is the oligonucleotide, and GCE is a glassy carbon electrode (B) depicts the attachment of the ammo-modified nucleic acids of the invention to oxidized surfaces using silane reactions Figure 9A and 9B depict the synthesis of a polydentate nucleoside While not shown, a preferred synthesis utilizes NaCNBH 4 in place of BH 4 -, included with the aldehyde, as a one step synthesis The reduction occurs slowly, while the reaction of the amine with the aldehyde occurs quickly, thus allowing the synthesis of the final product Figure 9B depicts the protection of the secondary amine as a tπflouroacetamide via reaction with excess S-ethyl-tπfluorothioacetate in ethanol DETAILED DESCRIPTION Unless otherwise stated, the term "nucleic acid" or "oligonucleotide" or grammatical equivalents herein means at least two nucleotides covalently linked together A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, a nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al , Tetrahedron 49(10) 1925 (1993) and references therein, Letsinger, J Org Chem 35 3800 (1970) Spπnzl et al , Eur J Biochem 81 579 (1977), Letsinger et al , Nucl Acids Res 14 3487 (1986), Sawai et al, Chem Lett 805 (1984), Letsinger et al , J Am Chem Soc 110 4470 (1988), and Pauwels et al , Chemica Scnpta 26 141 91986)), phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J Am Chem Soc 114 1895 (1992), Meier et al , Chem Int Ed Engl 31 1008 (1992), Nielsen, Nature, 365 566 (1993), Carlsson et al , Nature 380 207 (1996), all of which are incorporated by reference) These modifications of the ribose-phosphate backbone may be done to facilitate the addition of electron transfer moieties, or to increase the stability and half-life of such molecules in physiological environments Particularly preferred are peptide nucleic acids (PNA) These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids This results in two advantages First, the PNA backbone exhibits improved hybridization kinetics PNAs have larger changes in the melting temperature (Tm) for mismatched versus perfectly matched basepairs DNA and RNA typically exhibit a 2-4°C drop in Tm for an internal mismatch With the non-ionic PNA backbone, the drop is closer to 7-9°C This allows for better detection of mismatches Similarly, due to their non-ionic nature, hybridization of the bases attached to these backbones is relatively insensitive to salt concentration This is particularly advantageous in the systems of the present invention, as a reduced salt hybridization solution has a lower Faradaic current than a physiological salt solution (in the range of 150 mM) The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyπbo- and πbo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine and hypoxathanine, etc In some instances, e g in the case of an "intervening nucleic acid", the term nucleic acid refers to one or more nucleosides As used herein, the term "nucleoside" includes nucleotides The terms "electron donor moiety", "electron acceptor moiety", and "electron transfer moieties" or grammatical equivalents herein refers to molecules capable of electron transfer under certain conditions It is to be understood that electron donor and acceptor capabilities are relative, that is, a molecule which can lose an electron under certain experimental conditions will be able to accept an electron under different experimental conditions It is to be understood that the number of possible electron donor moieties and electron acceptor moieties is very large, and that one skilled in the art of electron transfer compounds will be able to utilize a number of compounds in the present invention Preferred electron transfer moieties include, but are not limited to, transition metal complexes, organic electron transfer moieties and electrodes In a preferred embodiment, the electron transfer moieties are transition metal complexes Transition metals are those whose atoms have an incomplete d shell of electrons Suitable transition metals for use in the invention include, but are not limited to cadmium (Cd) magnesium (Mg), copper (Cu), cobalt (Co) palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru), rhodium (Rh) osmium (Os), rhenium (Re), platmium (Pt), scandium (Sc), titanium (Ti), Vanadium (V) chromium (Cr), manganese (Mn), nickel (Ni), Molybdenum (Mo), technetium (Tc) tungsten (W) and indium (Ir) That is, the first series of transition metal, the platinum metals (Ru Rh, Pd Os, Ir and Pt), along with Re, W, Mo and Tc, are preferred Particularly preferred are ruthenium rhenium, osmium, platinium and iron The transition metals are complexed with a variety of ligands to form suitable transition metal complexes, as is well known in the art Suitable ligands include, but are not limited to, -NH 2 , pyridine, pyrazine, isonicotinamide, imidazole, bipyπdine and substituted derivative of bipyndine, phenanthro nes particularly 1 10-phenanthrolιne (abbreviated phen) and substituted derivatives of phenanthrohnes such as 4,7- dimethylphenanthrohne, dipyndophenazine, 1 ,4,5,8,9,12-hexaazatrιphenylene (abbreviated hat), 9,10-phenanthrenequιnone dnmine, 1 ,4,5,8-tetraazaphenanthrene (abbreviated tap), 1 ,4,8,11-tetra-azacyclotetradecane, diaminopyπdine (abbreviated damp), porphyπns and substituted derivatives of the porphyπn family A general formula that is representative of a class of donors and acceptors that may be employed is shown in figure 4A The groups R 1 , R 2 , R 3 , R 4 , and R 5 may be any coordinating ligand that is capable of covalently binding to the chosen metal and may include any of the above ligands In a preferred embodiment, as is more fully described below, two or more of the coordination atoms are covalently attached to the πbose, forming a covalently attached polydentate ligand, as is generally described below The structure of a ruthenium electron transfer species using bisbipyπdine and imidazole as the ligands is shown in figure 4B Specific examples of useful electron transfer complexes include, but are not limited to, those shown in Table 1 TABLE 1 Donors Acceptors Ru(bpy) 2 ιm-NH 2 -U Ru(NH 3 ) 5 -NH 2 -U Ru(bpy) 2 ιm-NH 2 -U Ru(NH 3 )„py-NH 2 -U Ru(bpy) 2 ιm-NH 2 -U Ru(NH 3 ) 4 ιm-NH 2 -U trans-Ru(cyclam)py Where Ru = ruthenium bpy = bisbipyndine im = imidazole py = pyridine cyclam = 1 ,4,8, 11 -tetra-azacyclotetradecane Other suitable moieties include bιs(phenanthrolιne) (dιpyrιdophenazιne)Ru(ll) (abbreviated [Ru(phen) 2 dppz] *2 ), bιs(9,10-phenthrenequιnone dιιmιne)(phenanthrolιne)Rh(lll), abbreviated [Rh(phι) 2 phen] +3 , trιs(phenanthrolιne)Ru(ll) (abbreviated [Ru(o-phen) 3 ] *2 ), Co(phen) 3 *3 Co(bpy) 3 ' 3 , Rh(phen) 3 *3 , Cr(phen) 3 *3 , Ru(bpy) 2 (dppz) t2 , and Ru(bpy) 2 When the electron transfer moiety is a transition metal complex, a preferred embodiment utilizes two or more coordination atoms for the chelation of the transition metal covalently attached to the nbose By covalently attaching a polydentate ligand to the nbose, two or more coordinating atoms are provided. Thus, in a preferred embodiment, the invention provides polydentate nucleosides and nucleic acids containing them The addition of a further covalently attached coordination atom to the modified nbose significantly increases the stability and binding of the transition metal complex to the nbose This is beneficial in a number of respects First of all, the polydentate ligands attached to the nbose result in tighter binding of the metal to the nbose of the nucleic acid This allows for selective addition of transition metal complexes onto a single stranded nucleic acid For example, incorporating a nucleotide containing a monodentate ligand, such as the 2' and 3' ammo-modified nucleotides described herein, to one end of the nucleic acid and a nucleotide containing a bi- or polydentate ligand to the other end allows for selective addition of an electron donor or acceptor to just one end, followed by the addition of the other electron transfer moiety to the other end, without resulting in all four possible combinations, as is more fully outlined below Furthermore, the increased stability and binding of the transition metal complexes to the nucleotide due to the polydentate ligand allows the attachment of the transition metal complex to a nucleotide prior to incorporation into a nucleic acid Thus, modified nucleotides with attached transition metal complexes may be made and then added to a solid phase oligonucleotide synthesis, thus allowing attachment of the transition metal electron transfer moiety at any position, thus greatly enhancing the ease of synthesis of the nucleic acids of the present invention This for example allows the incorporation of two different transition metal complexes, i e an electron donor and an electron acceptor, to the same oligonucleotide, using traditional solid support syntheses Thus, the present invention provides nucleosides with polydentate ligands attached to the nbose at either the 2' or 3' position, to form polydentate nucleosides A "polydentate ligand" is a ligand for a transition metal that contains at least two coordination atoms The polydentate ligand is covalently attached to the nucleoside as depicted herein to form a polydentate nucleoside of the general structure shown in Structure 1 Structure 1 The R r (X-R 2 ) n group is the polydentate ligand, with n ranging from 1 , to form a bidentate ligand, to the number of coordination atoms that may be bound by any particular transition metal Thus, for example, in the case of metals that bind 6 coordination atoms such Ru, Rh, Os, Fe, Re, etc the upper limit of n is six to form hexadentate ligands Y, and Y 2 may be hydrogen, hydroxy, a phosphoramidite moiety, a protecting group, or a phosphodiester bond or analog linking the polydentate nbose at either the 3' or 5' position to a nucleic acid, such as phosphoramide, phosphorothioate, phosphorodithioate, 0-methylphophoroamιdιte linkages and peptide nucleic acid backbones and linkages Y 2 may also be a solid support such as control pore glass (CPG) or other polymeric supports, as is known in the art The R groups, R, and R 2 in Structure 1 , are coordination atoms for the transition metal, and are generally independently nitrogen or oxygen, depending on the transition metal, as is known in the art For example, Ru, Rh, Re, Os, etc , nitrogen coordination atoms are preferred, for Fe and Co, mixtures of nitrogen and oxygen are preferred The R groups may have additional atoms associated with them, such as hydrogen atoms, such as in the case of secondary amines X is an alkyl or aryl linker moiety that serves to covalently link the R groups in a conformation suitable for binding to the transition metal Generally, X may be an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl moiety The X moieties are chosen to confer to the polydentate ligand the correct coordination geometry for the transition metal chosen As will be appreciated by those in the art, a wide variety of X moieties may be used Thus for example, X moieties are easily chosen for metals that prefer octahedral coordination geometry such as Ru, Fe, Os, Re, Cr, Mn, Rh, Re, etc Similarly, suitable X moieties are chosen for transition metals that prefer square planar geometry (i e Pt and Pd), or tetrahedral geometry (i e Ni) A bidentate ligand attached at the 2' position is depicted in Structure 2, although as will be appreciated by those in the art, polydentate ligands may be attached at the 3' position as well Structure 2 The X moiety joining any two R groups together may fuse with one of the R groups to form ring structures, either mono- or polycychc rings, as is depicted in Structure 3 (described in the Examples) or Structure 4 Structure 3 Similarly, the X and an R group may form morpholine, phenanthrohnes, bipyπdines, catacols, and other heteroaryl groups Preferred X groups include, but are not limited to, ethylene (-CH 2 CH 2 -), benzyl and other polyaromatic rings, such as aryl and heteroaryl groups As will be appreciated by those in the art, additional coordination atoms R may be added via additional X groups to nucleotides of Structure 1 to form tn-, tetra-, penta- and hexadentate ligands A representative tπdentate ligand is shown in Structure 5 The upper limit of coordination atoms will again depend on the transition metal used, for example, many of the transition metals useful herein are hexadentate (Fe, Os, Ru, Rh, Re, etc), and thus can bind up to six coordinating atoms Structure 5 R 3 is an additional coordination atom X 2 is an additional linker moiety, and can be the same or different from X, It should be appreciated that the geometry and angles of the Structure 5 polydentate ligand are not accurate, but are merely representational The structure of the attached polydentate ligand may be linear, i e only one end of the polydentate ligand is attached to the nbose, as is generally depicted in the Structures or may form a ring structure, i e both ends of the polydentate ligand may be attached to the nbose, i e using a Schiff s base, tetraazo or porphyπn structures as the polydentate ligand In one embodiment, the polydentate nucleoside is formed using a nucleoside containing an ammo group at both the 2' and 3' position on the nbose, as is depicted in Structure 6 Structure 6 The structures herein are depicted in the absence of the transition metal and any necessary additional ligands, although similar to Figure 4A and B, the compounds may include the metal and additional ligands as well, as is depicted in Structure 7 (corresponding to Structure 2) Structure 7 L is a co-hgand, that provides at least one coordination atom for the chelation of the metal ion As will be appreciated by those in the art, the number and nature of the co- ligand will depend on the coordination number of the metal ion Mono-, di- or polydentate co- gands may be used Thus, for example, when the metal has a coordination number of six, two coordination atoms are provided by the bidentate ligand covalently attached to the nucleoside and four coordination atoms are provided by the co-hgands Thus, m = four, when all the co-hgands are monodentate, m = 2, when the co-hgands are bidentate, or m = 3, for two monodentate co-hgands and a bidentate co-hgand Suitable ligands are listed above The structures herein depict the polydentate nbose with the polydentate ligands attached to at the 2' position of the nbose As will be appreciated by those in the art, the polydentate ligand may also be attached at the 3' position when the nbose is at the 3' terminus of the nucleic acid In a preferred embodiment, the R, coordination atom is directly attached to the nbose ring via the 2' or 3' carbon In alternate embodiments, there may be additional atoms between the R1 coordination atom and the carbon of the nbose, although, as outlined herein, the linker is preferably short, as to prevent intercalation Thus, methylene groups (-CH2-), ethylene groups (-CH2CH2-), acetene groups (-CHCH-), etc may be used In addition to transition metal complexes, other organic electron donors and acceptors may be covalently attached to the nucleic acid for use in the invention These organic molecules include, but are not limited to, πboflavin, xanthene dyes, azine dyes, acndine orange, /V,/V-dimethyl-2,7-diazapyrenium dichloride (DAP 2 "), methylviologen ethidium bromide, quinones such as N,N'-dιmethylanthra(2,1,9- ef6,5,10- d'e'Odnsoquinoline dichloride (ADIQ 2* ), porphyπns ([meso-tetrakιs(N-methyl-x- pyπdιnιum)porphyπn tetrachloride], varlamine blue B hydrochloride, Bindschedler's green, 2,6-dιchloroιndophenol, 2,6-dιbromophenolιndophenol, Brilliant crest blue (3- amιno-9-dιmethyl-amιno-10-methylphenoxyazιne chloride), methylene blue, Nile blue A (aminoaphthodiethylaminophenoxazine sulfate), ιndιgo-5,5',7,7'-tetrasulfonιc acid, ιndιgo-5,5' 7-trιsulfonιc acid, phenosafranine, ιndιgo-5-monosulfonιc acid, safranine T, bιs(dιmethylglyoxιmato)-ιron(ll) chloride, indulme scarlet, neutral red, and substituted derivatives of these compounds In one embodiment, the electron donors and acceptors are redox proteins as are known in the art However, redox proteins in many embodiments are not preferred In a particularly preferred embodiment, an electron transfer moiety comprises an solid support such as an electrode to which the nucleic acid is attached covalently or otherwise That is, the electrode serves as either the electron donor or acceptor, as is more fully described below The techniques used in this embodiment are analogous to the wiring of proteins to an electrode except that the nucleic acids of the present invention are used rather than a redox protein (see for example Gregg et al , J Phys Chem 95 5970 (1991 ), Heller et al , Sensors and Actuators R , 13-14 180 (1993), and Pishko et al , Anal Chem , 63 2268 (1991)) Electrode attachment is utilized in initiating electron transfer via an applied potential and for electronic methods of electron transfer monitoring In a preferred embodiment, electron transport between the electrode and the nucleic acid can be indirect, utilizing electron transport mediators which are free in solution or imbedded in a gel or polymer to provide a type of electronic coupling between the electrode and the nucleic acids In a preferred embodiment, the electron transfer moiety-modified nucleic acids of the invention are attached via such a matrix Matrix attachment has several advantages for use in a nucleic acid gene sensor Because of the 3-dιmensιonal nature of the polymer, large numbers of modified nucleic acid probes can be attached to a small surface area of electrode Using a highly porous "hydrogel," rates of nucleic acid hybridization can be quite high, nearly matching that of nucleic acid in solution For example, polymers with covalently attached redox molecules behave as highly effective electron transfer mediators Siloxane and ethylene oxide polymers, modified with ferrocene molecules, demonstrated electron transfer between enzymes and an electrode, for example, flexible siloxane and ethylene oxide polymers covalently attached to ferrocene or Os(bpy) 2 have been shown to be highly effective redox polymers for mediating electron transfer from several enzymes to an electrode (see Boguslavsky et al , Solid State Ionics, V 60, p 189 (1993)) Similarly, a redox- conducting epoxy cement has been prepared (see Hellar et al J Phys Chem , 95 5970 (1991 )) Cross linked redox gels for amperometπc biosensors applications have also been prepared with glucose oxidase electrically connected to electrodes so that electrons were shown to flow from the enzyme, through the polymer and to the electrode (see Hellar, A , et al , Anal Chem , 62, 258 (1990)) In this embodiment it is preferred that a redox polymer such as a poly-(vιnylpyrιdιne) complex of Os(bby) 2 CI be cross-linked with an epoxide such as diepoxide to form a redox-conducting epoxide cement which is capable of strongly binding to electrodes made of conductive material such as gold vitreous carbon graphite, and other conductive materials This strong attachment is included in the definition of "covalently attached" for the purposes of this embodiment The epoxide cross-linking polymer is then reacted with, for example, an exposed amine, such as the amine of an amino- modified nucleic acid described above covalently attaching the nucleic acid to the complex, forming a "redox hydrogel" on the surface of the electrode In an analogous fashion chemically modified DNA can be substituted for the redox enzyme or mediator with the result of electron transfer processes being observed from a transition metal-modified DNA moiety through a coupled redox conducting polymer to an electrode Suitable mediators include water soluble ferrocene/ferπcinium hydrσquinones/quinones, reducible and oxidizable components of organic salts, cobaltocenes, the hexa- and octacyanides of molybdenum tungsten and iron In addition, macrocycles and chelating ligands of transition metals such as cobalt, ruthenium and nickel are used, including Co(ethylenedιamιne) 3 and Ru(ethylenedιamιne) 3 and the tπsbypyπdyl and hexamine complexes of transition metals such as Co, Ru, Fe, and Os (see Alyanasundaram, supra) In a preferred embodiment, electron transport between the electrode and the nucleic acid can be direct via a covalent bond One advantage of these systems is that the orientation of the DNA probe can be influenced to reduce any bending back of the probe onto the electrode Also, more precise control of applied potential and measured current is associated with short covalent linkages versus gels and polymers In a preferred embodiment, the covalent bonds must be highly conducting such as in a redox polymer (Hellar, A Ace Chem Res Vol 23, p 128, 1990) Alternatively, if they are poorly conducting, the length of the linkage must be kept short Accordingly, a preferred embodiment has an electron traversing no more than about five σ bonds, with no more than three being especially preferred Carbon paste and glassy carbon rods have proven reliable and effective as electrodes in a variety of chemical sensors, including sensitive glucose oxidase enzyme-based biosensors, and may be used in the present invention In addition, flexible siloxane and ethylene oxide polymers covalently attached to ferrocene or Os(bpy) 2 molecules have been shown to be highly effective redox polymers for mediating electron transfer from several enzymes to an electrode Amino-πbose modified nucleic acids are attached to carbon electrodes by variations of these literature techniques Finally, nucleic acids are more directly attached to oxidized carbon electrodes via guanosine residues, using known carbodiimide and N- hydroxysuccinimide chemistry In a preferred embodiment, glassy carbon electrodes (GCEs) are used In this embodiment, amine groups such as outlined above on the 2' or 3' carbon of the nbose ring are used for attachment The reaction proceeds via the oxidation of an amine group to a cation radical which forms a chemically stable and covalent bond between the amine and the edge plane of the GCE surface (see Deinhammer, R, et al Langmuir 10 1306 (1994)) This synthetic approach has been well characterized using X-ray photo-electron spectroscopy and cyclic voltammetry The yield using this chemistry can be quite high, approximately 1 x 10 10 molecules/cm 2 The amine compound forms a stable bond to the carbon surface, and steric effects influence binding efficiency The reactivity of primary amines is substantially higher than secondary amines, the binding of tertiary amines is not observed at all Employing the amino-modified (primary amine group) oligonucleotides described earlier, the procedure developed by Deinhammer, R, ef al to prepare the GCEs for electrochemical treatment in amine containing solution is depicted in Figure 8A In addition, DNA has been immobilized onto GCEs using a water soluble carodimide (Mikkelsen et al , Electroanalysis 4 929 (1992)) In a preferred embodiment, the nucleic acids of the invention are attached to gold electrodes Several methods are available for the covalent attachment of redox active species to gold surfaces and electron transfer reactions with these materials have been observed Hydroxy thiols (OH(CH 2 ) x SH) of varying lengths are prepared by variation of literature procedures (see Miller, C et al J Phys Chem 95 877 (1991 ) and Chidsey, C E D , Science, V 251 , p 919, (1991)) Example 8 outlines the preparation of hydroxyl thiols which are attached to gold electrodes Alternative procedures for the preparation of hydroxythiols are known in the art Au electrodes or surfaces are prepared by literature procedures and the modified hydroxythiols adsorbed onto the Au In an additional embodiment, the modified nucleic acids of the invention are covalently attached to thin film oxidized surfaces It has been reported that a variety of compounds can be covalently bonded (in the form of monolayers) to thin-film Sn0 2 , Tι0 2 , and Ru0 2 and Pt electrodes (see Lenhard, J and Murray, R J Electroanal Chem 78 195 (1977)) Reversible electrochemistry of surface bound complexes such as 3,5-dιnιtrobenzamιde to electrodes has been observed The reported complexes are attached to the electrode via an amide bond linkage Employing these literature procedures, analogous derivatives using amino-modified oligonucleotides described in this work can be prepared and are schematically represented in Figure 8B. Accordingly, using the above methods, oligonucleotides may be attached to a solid support such that the electrode serves as either the electron donor moiety or the electron acceptor moiety Thus, all combinations of electron donors and acceptors may be made: two transition metal complexes; two organic electron transfer species; one transition metal, one organic moiety, one transition metal and an electrode; and one organic moiety and an electrode. The choice of the electron transfer species will depend in part on the method of initiation and detection required, as is more fully described below. The term "target sequence" or grammatical equivalents herein means a nucleic acid sequence on a single strand of nucleic acid. The target sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, mRNA, or others. It may be any length, with the understanding that longer sequences are more specific. As is outlined more fully below, probes are made to hybridize to target sequences to determine the presence or absence of the target sequence in a sample. Generally speaking, this term will be understood by those skilled in the art. The probes of the present invention are designed to be complementary to the target sequence, such that hybridization of the target sequence and the probes of the present invention occurs. As outlined below, this complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. A variety of hybridization conditions may be used in the present invention As is known in the art, "high" stringency usually refers to conditions such as 0 1XSSC at 65°C, reduced stringency conditions include 2-5XSSC at 25-50 α C The hybridization conditions may also vary when a non-ionic backbone, i.e PNA is used, as is known in the art The terms "first target domain" and "second target domain" or grammatical equivalents herein means two portions of a target sequence within a nucleic acid which is under examination. The first target domain may be directly adjacent to the second target domain, or the first and second target domains may be separated by an intervening target domain. The terms "first" and "second" are not meant to confer an orientation of the sequences with respect to the 5'-3' orientation of the target sequence. For example, assuming a 5'-3' orientation of the complementary target sequence, the first target domain may be located either 5' to the second domain, or 3' to the second domain The present invention is directed, in part, to the site-selective modification of nucleic acids with redox active moieties such as transition metal complexes for the preparation of a new series of biomaterials capable of long distance electron transfer through a nucleic acid matrix The present invention provides for the precise placement of electron transfer donor and acceptor moieties at predetermined sites on a single stranded or double stranded nucleic acid In general, electron transfer between electron donor and acceptor moieties in a double helical nucleic acid does not occur at an appreciable rate unless nucleotide base pairing exists in the sequence between the electron donor and acceptor in the double helical structure This differential in the rate of electron transfer forms the basis of a utility of the present invention for use as probes In the system of the present invention, where electron transfer moieties are covalently bound to the backbone of a nucleic acid, the electrons putatively travel via the π-orbitals of the stacked base pairs of the double stranded nucleic acid The electron transfer rate is dependent on several factors, including the distance between the electron donor-acceptor pair, the free energy (ΔG) of the reaction, the reorganization energy (λ), the contribution of the intervening medium, the orientation and electronic coupling of the donor and acceptor pair, and the hydrogen bonding between the bases The contribution of the intervening medium depends in part, on the number of sigma (o) bonds the electron must traverse from the electron donor to reach the bases stack, or to exit the stack to reach the electron acceptor As is shown in Figure 5, when the metal is bound to the nbose-phosphate backbone via an amine moiety at the 2' carbon of the nbose, an electron must travel through four σ bonds to reach the stack the metal to nitrogen bond, the nitrogen to 2' carbon bond, and from the 2' carbon to the base, or vice versa depending on the direction of the electron flow Since the base of the nucleotide is conjugated in some degree, the base can be considered to be the edge of the "π-way", that is, the conjugated π orbitals of the stacked base pairs When the metal is bound to the ribose-phosphate backbone via the 3' carbon of the nbose, an electron must traverse through 5 σ bonds When the metal is bound via phosphoramide-type linkages an electron must traverse through 7 σ bonds In the preferred embodiments, the compositions of the invention are designed such that the electron transfer moieties are as close to the "pi-way" as possible without significantly disturbing the secondary and tertiary structure of the double helical nucleic acid, particularly the Watson-Crick basepaiπng The effect on the electron transfer rate by the hydrogen bonding between the bases is a dependence on the actual nucleic acid sequence, since A-T pairs contain one less hydrogen bond than C-G pairs However, this sequence dependence is overshadowed by the determination that there is a measurable difference between the rate of electron transfer within a DNA base-pair matrix, and the rate through the ribose- phosphate backbone, the solvent or other electron tunnels This rate differential is thought to be at least several orders of magnitude, and may be as high as four orders of magnitude greater through the stacked nucleotide bases as compared to other electron transfer pathways Thus the presence of double stranded nucleic acids, for example in gene probe assays, can be determined by comparing the rate of electron transfer for the unhybndized probe with the rate for hybridized probes In one embodiment the present invention provides for novel gene probes, which are useful in molecular biology and diagnostic medicine In this embodiment single stranded nucleic acids having a predetermined sequence and covalently attached electron donor and electron acceptor moieties are synthesized The sequence is selected based upon a known target sequence, such that if hybridization to a complementary target sequence occurs in the region between the electron donor and the electron acceptor, electron transfer proceeds at an appreciable and detectable rate Thus, the present invention has broad general use, as a new form of labelled gene probe In addition, since detectable electron transfer in unhybndized probes is not appreciable, the probes of the present invention allow detection of target sequences without the removal of unhybndized probe Thus, the present invention is uniquely suited to automated gene probe assays or field testing In a preferred embodiment, the probes are used in genetic diagnosis For example, probes can be made using the techniques disclosed herein to detect target sequences such as the gene for nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which is a gene associated with a variety of cancers, the Apo E4 gene that indicates a greater risk of Alzheimer's disease, allowing for easy presymptomatic screening of patients, mutations in the cystic fibrosis gene, or any of the others well known in the art In an additional embodiment, viral and bacterial detection is done using the complexes of the invention In this embodiment, probes are designed to detect target sequences from a variety of bacteria and viruses For example, current blood-screening techniques rely on the detection of anti-HIV antibodies The methods disclosed herein allow for direct screening of clinical samples to detect HIV nucleic acid sequences, particularly highly conserved HIV sequences In addition, this allows direct monitoring of circulating virus within a patient as an improved method of assessing the efficacy of anti-viral therapies Similarly, viruses associated with leukemia, HTLV-I and HTLV-II, may be detected in this way Bacterial infections such as tuberculosis may also be detected In a preferred embodiment, the nucleic acids of the invention find use as probes for toxic bacteria in the screening of water and food samples For example, samples may be treated to lyse the bacteria to release its nucleic acid and then probes designed to recognize bacterial strains, including, but not limited to, such pathogenic strains as, Salmonella, Campylobacter, Vibrio cholerae, enterotoxic strains of £ coli, and Legionnaire's disease bacteria Similarly, bioremediation strategies may be evaluated using the compositions of the invention In a further embodiment, the probes are used for forensic "DNA fingerprinting" to match crime-scene DNA against samples taken from victims and suspects The present invention also finds use as a unique methodology for the detection of mutations in target nucleic acid sequences As a result, if a single stranded nucleic acid containing electron transfer moieties is hybridized to a target sequence with a mutation, the resulting perturbation of the base pairing of the nucleosides will measurably affect the electron transfer rate This is the case if the mutation is a substitution, insertion or deletion Alternatively, two single stranded nucleic acids each with a covalently attached electron transfer species that hybπdize adjacently to a target sequence may be used Accordingly, the present invention provides for the detection of mutations in target sequences Thus, the present invention provides for extremely specific and sensitive probes, which may, in some embodiments, detect target sequences without removal of unhybndized probe This will be useful in the generation of automated gene probe assays In an alternate embodiment double stranded nucleic acids have covalently attached electron donor and electron acceptor moieties on opposite strands Such nucleic acids are useful to detect successful gene amplification in polymerase chain reactions (PCR), thus allowing successful PCR reactions to be an indication of the presence or absence of a target sequence PCR may be used in this manner in several ways For example, if one of the two PCR primers contains a 5' terminally attached electron donor, and the other contains a 5' terminally attached electron acceptor, several rounds of PCR will generate doubly labeled double stranded fragments (occasionally referred to as "amplicons") After appropriate photoinduction, the detection of electron transfer provides an indication of the successful amplification of the target sequence as compared to when no amplification occurs A particular advantage of the present invention is that the separation of the single stranded primers from the amplified double stranded DNA is not necessary, as outlined above for probe sequences which contain electron transfer moieties Alternatively, the detection of a target sequence via PCR is done by attaching one electron transfer moiety species to one or both of the primers The other electron transfer moiety species is attached to individual nucleosides of the PCR reaction pool, as is described herein Incorporation of the nucleosides containing the electron transfer moiety into the nucleic acid during the PCR reaction results in both electron transfer species being attached either to the same single strand or to opposite strands, or both Allowing the newly synthesized nucleic acid to remain in a hybridized form allows the detection of successful elongation via electron transfer, and thus the detection of a target sequence In this way, the present invention is used for PCR detection of target sequences In another embodiment the present invention provides for double stranded nucleic acids with covalently attached electron donor and electron acceptor moieties to serve as bioconductors or "molecular wire" The electron transport may occur over distances up to and in excess of 28A per electron donor and acceptor pair In addition, the rate of electron transfer is very fast, even though dependent on the distance between the electron donor and acceptor moieties By modifying the nucleic acid in regular intervals with electron donor and/or electron acceptor moieties, it may be possible to transport electrons over long distances, thus creating bioconductors These bioconductors are useful in a large number of applications, including traditional applications for conductors such as mediators for electrochemical reactions and processes In addition, these bioconductors may be useful as probes for photosynthesis reactions as well as in the construction of synthetic light harvesting systems The current models for the electron transfer component of an artificial light harvesting system have several problems, as outlined above, including a dependence on solvent polarity and composition, and a lack of sufficient rigidity without arduous synthesis Thus the present invention is useful as both a novel form of bioconductor as well as a novel gene probe The present invention provides nucleic acids with covalently attached electron transfer moieties The electron transfer moieties may be attached to the nucleic acid at a variety of positions In one embodiment, the electron donor and acceptor moieties are added to the 3' and/or 5' termini of the nucleic acid on either the sugar-phosphate backbone or a terminal base In alternative embodiments, the electron donor and acceptor moieties are added to the backbone of one or more internal nucleosides, that is, any nucleoside which is not the 3' or 5' terminal nucleoside In a further embodiment, the electron donor and acceptor moieties are added to the backbone of both internal and terminal nucleosides In a preferred embodiment, the electron transfer moieties are attached to the ribose- phosphate backbone in a number of positions As shown in Figure 5 several positions are possible, with attachment to a nbose of the ribose-phosphate backbone being particularly preferred Accordingly, in Figure 5, the most preferred site of attachment of a electron transfer moiety is M 1t followed by M 4 , M 2 and M 3 , in that order In a preferred embodiment, the electron transfer moieties are attached at the 2' or 3' position on the nbose, with 2' being particularly preferred In a preferred embodiment, the electron transfer moieties do not intercalate, and are attached such that do not intercalate Thus, while it is possible to utilize a "linker", such as alternating double bonds to attach the electron transfer moiety to the nucleic acid, the linker is either preferably not longer than the equivalent of one or two nucleosides in length, or is not significantly flexible to allow intercalation Preferably, if linkers are used, they are attached via the nbose of the nucleic acid backbone In one embodiment, the electron transfer moieties are added to the bases of the terminal nucleosides Thus, when the target sequence to be detected is n nucleosides long, a probe can be made which has an extra terminal nucleoside at one or both of the ends of the nucleic acid (n + 1 or n + 2), which are used to covalently attach the electron transfer moieties but which do not participate in basepair hybridization This extra terminal nucleoside is important since attachment of electron transfer moieties to an internal nucleoside base is expected to perturb Watson-Cnck basepainng That is, the base used for covalent attachment should be outside of the region used to identify the target sequence Additionally, it is preferred that upon probe hybridization, the terminal nucleoside containing the electron transfer moiety covalently attached at the base be directly adjacent to Watson-Cnck basepaired nucleosides, that is, the electron transfer moiety should be as close as possible to the stacked π-orbitals of the bases such that an electron travels through a minimum of σ bonds to reach the "n-way", or alternatively can otherwise electronically contact the π-way In one embodiment, a single stranded nucleic acid is labelled with an electron transfer moiety via the terminal bases at both ends Alternate embodiments utilize a terminal base and a 5' or a 3' terminal ribose-phosphate attachment as described above In further embodiments, compositions are provided comprising a first single stranded nucleic acid containing an electron donor covalently attached at a terminal base and a second single stranded nucleic acid containing an electron acceptor covalently attached at a position as described above, that is, at a 5', 3' or internal position, alternatively, the electron donor and acceptor may be switched A particularly preferred embodiment utilizes an electrode as one of the electron transfer moieties with the other electron transfer moiety being attached to a terminal base preferably on the same single strand The present invention further provides methods for the site-specific addition of electron transfer moieties to nucleic acids As outlined above, the electron transfer moieties may be added at the 2' or 3' position of a nbose of the ribose-phosphate backbone, to a 3' or 5' terminal base, or to an internal nucleoside using peptide nucleic acid linkages, phosphoramidate bonds, phosphorothioate bonds, phosphorodithioate bonds, or O-methyl phosphoramidate bonds Molecular mechanics calculations indicate that perturbations due to the modification of at the nbose of the terminal nucleosides of nucleic acids are minimal, and Watson- Cnck base pairing is not disrupted (unpublished data using Biograf from Molecular Simulations Inc , San Diego, CA) For attachment to a nbose, a preferred embodiment utilizes modified nucleosides to attach the electron transfer moieties Preferably amino-modified nucleosides and nucleosides are used In an alternate embodiment, thio-modified nucleosides are used to attach the electron transfer moieties of the invention The modified nucleosides are then used to site-specifically add a transition metal electron transfer moiety, either to the 3' or 5' termini of the nucleic acid, or to any internal nucleoside Either the 2' or 3' position of the nbose may be altered for attachment at the 3' terminus, for attachment to an internal nbose or the 5' terminus, the 2' position is preferred Thus, for example, the 2 position of the nbose of the deoxyπbo- or πbonucleoside is modified prior to the addition of the electron transfer species, leaving the 3' position of the nbose unmodified for subsequent chain attachment if necessary In a preferred embodiment, an ammo group is added to the 2'or 3' carbon of the sugar using established chemical techniques (Imazawa et al , J Org Chem , 44 2039 (1979), Hobbs et al , J Org Chem 42(4) 714 (1977) Verheyden et al J Org Chem 36(2) 250 (1971)) In a preferred embodiment, the 2' or 3' ammo modified nucleosides are converted to polydentate ligands as outlined herein and in Example 9 This may be done in a wide variety of ways, as will be appreciated by those in the art robin start here For polydentate ligands comprising oxygen coordination atoms, the hydroxyl groups at the 2' and 3' position of nbose may be used to form polydentate ligands, as will be appreciated by those in the art The amino-modified nucleosides made as described above are converted to the 2' or 3' modified nucleotide tπphosphate form using standard biochemical methods (Fraser et al , Proc Natl Acad Sci USA, 4 2671 (1973)) In a preferred embodiment, the modified nucleosides comprise polydentate nucleosides as are described herein As exemplified in Figure 9, a variety of polydentate ligands may be made starting from the amino-modified nucleosides Modified nucleosides for the attachment of the electron transfer moieties to the bases, is done as outlined in Telser, supra, both of which are expressly incorporated by reference These modified nucleosides are then incorporated at either the 3' or 5' terminus as outlined below Thus a modified nucleoside includes a 2' or 3'-amιno or thio-modified nucleoside, nucleosides modified at the bases, or polydentate nucleosides Once the modified nucleosides are prepared protected and activated, they may be incorporated into a growing oligonucleotide by standard synthetic techniques (Gait, Oligonucleotide Synthesis A Practical Approach, IRL Press, Oxford, UK 1984, Eckstein) in several ways In one embodiment, one or more modified nucleosides are incorporated into a growing oligonucleotide chain by using standard molecular biology techniques such as with the use of the enzyme DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase, reverse transcriptase, and RNA polymerases For the incorporation of a 3' modified nucleoside to a nucleic acid, terminal deoxynucleotidyltransferase may be used (Rathff, Terminal deoxynucleotidyltransferase In The Enzymes, Vol 14A P D Boyer ed pp 105-118 Academic Press, San Diego, CA 1981) Alternatively, and preferably, the ammo nucleoside is converted to the phosphoramidite or H-phosphonate form, which are then used in solid-phase or solution syntheses of oligonucleotides In this way the modified nucleoside, either for attachment at the nbose (i e ammo- or thiol-modified nucleosides) or the base, is incorporated into the oligonucleotide at either an internal position or the 5' terminus This is generally done by protecting the 5' position of the nbose with 4',4-dιmethoxytrιtyl (DMT) followed by reaction with 2-cyanoethoxy-bιs- dnsopropylaminophosphine in the presence of diisopropylammonium tetrazolide to give the phosphoramidite as is known in the art, although other techniques may be used as will be appreciated by those in the art See Gait, supra, Caruthers, Science 230 281 (1985), both of which are expressly incorporated herein by reference For attachment of an electron transfer moiety to the 3' terminus, a preferred method utilizes the attachment of the modified nucleoside to solid supports such as controlled pore glass (CPG) or other polymeπc supports, as are known in the art for the synthesis of oligonucleotides In this embodiment, the modified nucleoside is protected at the 5' end with DMT, and then reacted with succinic anhydride with activation The resulting succinyl compound is attached to CPG or other polymeric supports as is known in the art Further phosphoramidite nucleosides are added, either modified or not, to the 5' end after deprotection In other embodiments, the electron transfer moiety or moieties are added to the middle of the nucleic acid, i e to an internal nucleoside This may be accomplished in three ways In a preferred embodiment, a modified nucleoside is incorporated at the 5' terminus as described above In this embodiment, oligonucleotide synthesis simply extends the 5' end from the modified nucleoside using standard techniques This results in an internally modified oligonucleotide In one embodiment, the nucleosides are modified to contain an aromatic amine capable of binding an electron transfer moiety at either the 2' or 3' position of the nbose For example, one of the nitrogens of imidazole can be attached at the 2' or 3' position of the nbose and thus used to attach the electron transfer moiety such as a transition metal complex This may effectively reduce the number of σ bonds an electron must travel through to reach the "pi-way" since the imidazole offers substantially less resistance to electron transfer as compared to a σ bond In a preferred embodiment, the imidazole is attached at the 2' position of the nbose In an alternate embodiment, the imidazole is attached at the 3' position The imidazole- modified nucleoside may be incorporated into an oligonucleotide as outlined herein for modified nucleosides In an alternate embodiment, electron transfer moieties are added to the backbone at a site other than nbose, resulting in an internal attachment For example, phosphoramide rather than phosphodiester linkages can be used as the site for transition metal modification These transition metals serve as the donors and acceptors for electron transfer reactions While structural deviations from native phosphodiester linkages do occur and have been studied using CD and NMR (Heller, Ace Chem Res 23 128 (1990), Schuhmann et al J Am Chem Soc 113 1394 (1991)), the phosphoramidite internucleotide link has been reported to bind to complementary polynucleotides and is stable (Beaucage et al , supra, and references therein, Letsinger, supra, Sawai, supra, Jager, Biochemistry 27 7237 (1988)) In this embodiment, dimers of nucleotides are created with phosphoramide linkages at either the 2'-5' or 3'-5' positions A preferred embodiment utilizes the 3'-5' position for the phosphoramide linkage, such that structural disruption of the subsequent Watson-Cnck basepaiπng is minimized These dimer units are incorporated into a growing oligonucleotide chain, as above, at defined intervals, as outlined below robin start here Thus, the present invention provides methods for making a nucleic acid with covalently attached electron transfer moieties In a preferred embodiment, the method is for making a nucleic acid with an electron transfer moiety attached at the 3' terminus of said nucleic acid The method comprises attaching a modified nucleoside to a solid support, and adding phosphoramidite nucleosides to the 5' terminus of the modified nucleoside to form a nucleic acid The nucleic acid is then optionally cleaved from the CPG using known methods The nucleic acid may be then hybridized to its complement, to protect the bases from modification, and the electron transfer moiety is added to the 2'-amιno modified nucleoside ln a preferred embodiment, methods for making a nucleic acid with an electron transfer moiety attached at the 5' terminus are provided The method comprises attaching a nucleoside to control pore glass, and adding phosphoramidite nucleosides to the 5' terminus of the nucleoside to form a nucleic acid A 2' or 3' am o modified nucleoside is added to the 5' terminus, and the nucleic acid is cleaved from the CPG The nucleic acid is hybridized to its complement, and the electron transfer moiety is added to the 2' or 3'-amιno modified nucleoside In a preferred embodiment, a method for making a single stranded nucleic acid with electron transfer moieties attached at both the 3' and 5' terminus The method comprises attaching a modified nucleoside to control pore glass The modified nucleoside may be either amino-modified, for attachment via the nbose as descπbed herein or modified at the base Additional phosphoramidite nucleosides are added to the 5' terminus of the modified nucleoside to form a nucleic acid A modified phosphoramidite nucleoside is further added to the 5' terminus of the nucleic acid, which is then cleaved off the control pore glass and hybridized to its complement An electron donor moiety is added to one modified nucleoside and an electron acceptor moiety is added to the other modified nucleoside It should be understood that it is important that the basepaiπng of the nucleoside bases is not significantly perturbed in order to allow hybridization, good electron transfer rates, and the detection of mismatches Thus for example, the transition metal moieities when attached to the nucleic acids of the invention, do not intercalate, i e insert and stack between the basepairs of the double stranded nucleic acid Intercalation of the transition metals with the accompanying ligands disturbs the basepaiπng, and thus hinders the transfer of electrons and the identification of mismatches Similarly, with the exception of terminal bases, as is outlined below, attaching the transition metal complexes at the nucleoside bases (Telser et al , supra) also disturbs the basepaiπng and impedes the identification of mismatches It should be noted that when using the above techniques for the modification of internal residues it is possible to create a nucleic acid that has an electron transfer species on the next-to-last 3' terminal nucleoside, thus eliminating the need for the extra steps required to produce the 3' terminally labelled nucleoside In a further embodiment for the modification of internal residues, 2' or 3' modified nucleoside tπphosphates are generated using the techniques described above for the 3' nucleoside modification The modified nucleosides are inserted internally into nucleic acid using standard molecular biological techniques for labelling DNA and RNA Enzymes used for said labelling include DNA polymerases such as polymerase I, T4 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase, reverse transcriptase and RNA polymerases such as E coli RNA polymerase or the RNA polymerases from phages SP6, T7 or T3 (Short Protocols in Molecular Biology, 992 Ausubel et al Ed pp 3 11-3 30) As described above, the electron transfer moiety, preferably a transition metal complex, may be attached to any of the five bases (adenine, thymme, uracil, cytosme, guanine and other non-naturally occurring bases such as inosme, xanthme, and hypoxanthine, among others) This is done using well known techniques, see Telser et al , J Am Chem Soc 111 7226-7232 (1989), Telser et al , J Am Chem Soc 111 7221-7226 (1989) As outlined herein, these terminally modified nucleosides may be attached to the nucleic acid enzymatically as is known in the art, using DNA polymerases, alternatively, the modified nucleosides may be incorporated into a growing oligonucleotide chain using traditional phosphoramidite chemistry during oligonucleotide synthesis as is outlined herein The exposed amine or other ligand at the 2' or 3' position of the nbose, the phosphoramide linkages, or the other linkages useful in the present invention, are readily modified with a variety of electron transfer moieties, and particularly transition metal complexes with techniques readily known in the art (see for example Millet et al, in Metals in Biological Systems, Sigel et al Ed Vol 27, pp 223-264, Marcell Dekker Inc New York, 1991 and Durham, et al in ACS Advances in Chemistry Series, Johnson et al Eds , Vol 226, pp 180-193, American Chemical Society, Washington D C , and Meade et al , J Am Chem Soc 111 4353 (1989)) Generally, these techniques involve contacting a partially chelated transition metal complex with the amine group of the modified nucleoside The organic electron transfer species are also added to the functional group of the modified nucleoside such as an amine group, using techniques known in the art When peptide nucleic acids (PNA) are used, attachment of the electron transfer moieties proceeds as follows The am o group at the N-terminus of the PNA will bind a partially chelated transition metal or organic electron transfer moiety similar to the amino-modified nbose (Figure 9A) Addition to the carboxy terminus proceeds as is generally outlined in Figure 9B Additionally, for single stranded PNAs, one electron transfer moiety may be attached to the N-terminus, and the other electron transfer moiety is attached to the terminal base at the carboxy terminus Alternatively, both transfer moieties are attached to terminal bases Similar combinations may be made for two single stranded nucleic acids, each containing an electron transfer moiety in addition, the present invention provides a novel method for the site specific addition to the ribose-phosphate backbone of a nucleic acid of electron donor and electron acceptor moieties to a previously modified nucleoside In one embodiment, the electron donor and acceptor moieties are attached to the modified nucleoside by methods which utilize a unique protective hybridization step In this embodiment, the modified single strand nucleic acid is hybridized to an unmodified complementary sequence This blocks the sites on the heterocyclic bases that are susceptible to attack by the transition metal electron transfer species When the terminal bases are to be labelled with electron transfer species, the complementary sequence does not extend to the base to be labelled That is, a complementary sequence of n nucleosides in length is chosen for hybridization to a probe sequence of n+1 or n+2, such that the terminal base is not protected Thus the unprotected base is exposed to the electron transfer moiety such that the moiety is attached to the base After successful addition of the desired metal complex, the modified duplex nucleic acid is separated into single strands using techniques well known in the art In a preferred embodiment, single stranded nucleic acids are made which contain one electron donor moiety and one electron acceptor moiety The electron donor and electron acceptor moieties may be attached at either the 5' or 3' end of the single stranded nucleic acid Alternatively, the electron transfer moieties may be attached to internal nucleosides, or one to an internal nucleoside and one to a terminal nucleoside It should be understood that the orientation of the electron transfer species with respect to the 5'-3' orientation of the nucleic acid is not determinative Thus, as outlined in Figure 1, any combination of internal and terminal nucleosides may be utilized in this embodiment In an alternate preferred embodiment, single stranded nucleic acids with at least one electron donor moiety and at least one electron acceptor moiety are used to detect mutations in a complementary target sequence A mutation, whether it be a substitution, insertion or deletion of a nucleoside or nucleosides, results in incorrect base pairing in a hybridized double helix of nucleic acid Accordingly, if the path of an electron from an electron donor moiety to an electron acceptor moiety spans the region where the mismatch lies, the electron transfer will be eliminated or reduced such that a change in the relative rate will be seen Therefore, in this embodiment, the electron donor moiety is attached to the nucleic acid at a 5 position from the mutation, and the electron acceptor moiety is attached at a 3' position, or vice versa In this embodiment it is also possible to use an additional label on the modified single stranded nucleic acid to detect hybridization where there is one or more mismatches If the complementary target nucleic acid contains a mutation electron transfer is reduced or eliminated To act as a control, the modified single stranded nucleic acid may be radio- or fluorescently labeled, such that hybridization to the target sequence may be detected, according to traditional molecular biology techniques This allows for the determination that the target sequence exists but contains a substitution, insertion or deletion of one or more nucleosides Alternatively, single stranded nucleic acids with at least one electron donor moiety and one electron acceptor moiety which hybridize to regions with exact matches can be used as a controls for the presence of the target sequence It is to be understood that the rate of electron transfer through a double stranded nucleic acid helix depends on the nucleoside distance between the electron donor and acceptor moieties Longer distances will have slower rates and consideration of the rates will be a parameter in the design of probes and bioconductors Thus, while it is possible to measure rates for distances in excess of 100 nucleosides, a preferred embodiment has the electron donor moiety and the electron acceptor moiety separated by at least 3 and no more than 100 nucleosides More preferably the moieties are separated by 8 to 64 nucleosides, with 15 being the most preferred distance In addition, it should be noted that certain distances may allow the utilization of different detection systems For example, the sensitivity of some detection systems may allow the detection of extremely fast rates, i e the electron transfer moieties may be very close together Other detection systems may require slightly slower rates, and thus allow the electron transfer moieties to be farther apart In an alternate embodiment, a single stranded nucleic acid is modified with more than one electron donor or acceptor moiety For example, to increase the signal obtained from these probes, or decrease the required detector sensitivity, multiple sets of electron donor-acceptor pairs may be used As outlined above, in some embodiments different electron transfer moieties are added to a single stranded nucleic acid For example, when an electron donor moiety and an electron acceptor moiety are to be added, or several different electron donors and electron acceptors, the synthesis of the single stranded nucleic acid proceeds in several steps First partial nucleic acid sequences are made, each containing a single electron transfer species, i e either a single transfer moiety or several of the same transfer moieties using the techniques outlined above Then these partial nucleic acid sequences are hgated together using techniques common in the art, such as hybridization of the individual modified partial nucleic acids to a complementary single strand, followed by ligation with a commercially available ligase Alternatively, single stranded nucleic acid may be made by incorporating an ammo modified nucleoside at two positions using the above techniques As a result of the synthesis, one of the am o modified nucleosides has a temporary protecting group on the amine such as DMT Upon hybridization to the complementary unmodified strand, the unprotected amine is exposed to the first electron transfer moieity, i e either a donor or an acceptor, resulting in covalent attachment The protecting group of the protected amino-modified nucleoside is then removed, and the hybrid is contacted with the second electron transfer species, and the strands separated, resulting in a single strand being labelled with both a donor and acceptor The single strand containing the proper electron transfer moieties is then purified using traditional techniques tn a preferred embodiment, single stranded nucleic acids are made which contain one electron donor moiety or one electron acceptor moiety. The electron donor and electron acceptor moieties are attached at either the 5' or 3' end of the single stranded nucleic acid Alternatively, the electron transfer moiety is attached to an internal nucleoside. It is to be understood that different species of electron donor and acceptor moieties may be attached to a single stranded nucleic acid. Thus, more than one type of electron donor moiety or electron acceptor moiety may be added to any single stranded nucleic acid. In a preferred embodiment, a first single stranded nucleic acid is made with on or more electron donor moieties attached A second single stranded nucleic acid has one or more electron acceptor moieties attached In this embodiment, the single stranded nucleic acids are made for use as probes for a complementary target sequence. In one embodiment, the complementary target sequence is made up of a first target domain and a second target domain, where the first and second sequences are directly adjacent to one another. In this embodiment, the first modified single stranded nucleic acid, which contains only electron donor moieties or electron acceptor moieties but not both, hybridizes to the first target domain, and the second modified single stranded nucleic acid, which contains only the corresponding electron transfer species, binds to the second target domain. The relative orientation of the electron transfer species is not important, as outlined in Figure 2, and the present invention is intended to include all possible orientations. In the design of probes comprised of two single stranded nucleic acids which hybridize to adjacent first and second target sequences, several factors should be considered These factors include the distance between the electron donor moiety and the electron acceptor moiety in the hybridized form, and the length of the individual single stranded probes. For example, it may be desirable to synthesize only 5' terminally labelled probes. In this case, the single stranded nucleic acid which hybridizes to the first sequence may be relatively short, such that the desirable distance between the probes may be accomplished. For example, if the optimal distance between the electron transfer moieties is 15 nucleosides, then the first probe may be 15 nucleosides long ln one aspect of this embodiment, the two single stranded nucleic acids which have hybridized to the adjacent first and second target domains are hgated together prior to the electron transfer reaction This may be done using standard molecular biology techniques utilizing a DNA ligase, such as T4 DNA ligase In an alternative embodiment, the complementary target sequence will have a first target domain, an intervening target domain, and a second target domain In this embodiment, the first modified single stranded nucleic acid, which contains only electron donor moieties or electron acceptor moieties but not both, hybridizes to the first target domain, and the second modified single stranded nucleic acid, which contains only the corresponding electron transfer species, binds to the second target domain When an intervening single stranded nucleic acid hybridizes to the intervening target sequence, electron transfer between the donor and acceptor is possible The intervening sequence may be any length, and may comprise a single nucleoside Its length, however, should take into consideration the desirable distances between the electron donor and acceptor moieties on the first and second modified nucleic acids Intervening sequences of lengths greater than 14 are desirable, since the intervening sequence is more likely to remain hybridized to form a double stranded nucleic acid if longer intervening sequences are used The presence or absence of an intervening sequence can be used to detect insertions and deletions In one aspect of this embodiment, the first single stranded nucleic acid hybridized to the first target domain, the intervening nucleic acid hybridized to the intervening domain, and the second single stranded nucleic acid hybridized to the second target domain, may be hgated together prior to the electron transfer reaction This may be done using standard molecular biology techniques For example, when the nucleic acids are DNA, a DNA ligase, such as T4 DNA ligase can be used The complementary target single stranded nucleic acid of the present invention may take many forms For example, the complementary target single stranded nucleic acid sequence may be contained within a larger nucleic acid sequence, i e all or part of a gene or mRNA, a restriction fragment of a plasmid or genomic DNA, among others One skilled in the art of molecular biology would understand how to construct useful probes for a variety of target sequences using the present invention ln one embodiment, two single stranded nucleic acids with covalently attached electron transfer moieties have complementary sequences, such that they can hybridize together to form a bioconductor In this embodiment, the hybridized duplex is capable of transferring at least one electron from the electron donor moiety to the electron acceptor moiety In a preferred embodiment, the individual single stranded nucleic acids are aligned such that they have blunt ends, in alternative embodiments, the nucleic acids are aligned such that the double helix has cohesive ends In either embodiment, it is preferred that there be uninterrupted double helix base-pairing between the electron donor moiety and the electron acceptor moiety, such that electrons may travel through the stacked base pairs In one bioconductor embodiment, the double stranded nucleic acid has one single strand nucleic acid which carries all of the electron transfer moieties In another embodiment, the electron transfer moieties may be carried on either strand, and in any orientation For example, one strand may carry only electron donors, and the other only electron acceptors or both strands may carry both In one embodiment, the double stranded nucleic acid may have different electron transfer moieties covalently attached in a fixed orientation to facilitate the long range transfer of electrons This type of system takes advantage of the fact that electron transfer species may act as both electron donors and acceptors depending on their oxidative state Thus, an electron donor moiety, after the loss of an electron, may act as an electron acceptor, and vice versa Thus, electron transfer moieties may be sequentially oriented on either strand of the double stranded nucleic acid such that directional transfer of an electron over very long distances may be accomplished For example, a double stranded nucleic acid could contain a single electron donor moiety at one end and electron acceptor moieties, of the same or different composition, throughout the molecule A cascade effect of electron transfer could be accomplished in this manner, which may result in extremely long range transfer of electrons This may be accomplished, for example, by incorporating transition metal complexes that possess a range in oxidation potentials due to ligand substitutions made at the metal center The choice of the specific electron donor and acceptor pairs will be influenced by the type of electron transfer measurement used, for a review see Winkler et al , Chem Rev 92 369-379 (1992) When a long-lived excited state can be prepared on one of the redox sites, direct measurement of the electron transfer rate after photoinduction can be measured, using for example the flash-quench method of Chang et al , J Amer Chem Soc 113 7057 (1991) In this preferred embodiment, the excited redox site, being both a better acceptor and donor than the ground-state species, can transfer electrons to or from the redox partner An advantage of this method is that two electron transfer rates may be measured the photoinduced electron transfer rates and thermal electron-hole recombination reactions Thus differential rates may be measured for hybridized nucleic acids with perfect complementarity and nucleic acids with mismatches In alternative embodiments, neither redox site has a long lived excited state, and electron transfer measurements depend upon bimolecular generation of a kinetic intermediate For a review, see Winkler et al , supra This intermediate then relaxes to the thermodynamic product via intramolecular electron transfer using a quencher, as seen below D-A + hv - D-A ' D-A ' + Q - D-A * + Q D-A + - D * -A D * -A + Q → D-A + Q The upper limit of measurable intramolecular electron transfer rates using this method is about 10 4 per second Alternative embodiments use the pulse-radiolytic generation of reducing or oxidizing radicals, which inject electrons into a donor or remove electrons from a donor, as reviewed in Winkler et al , supra As is appreciated in the art, there are a variety of ways to initiate and detect the electron transfer Electon transfer can be initiated and detected using a wide variety of methods. including, but not limited to. electrical, electrochemical, electromagnetic radiation f optical) and chemical methods It is possible to make a variety of compositions utilizing different electron transfer moieties depending on the desired methods of initiating electron transfer and detection of electron transfer Table 2 depicts a variety of preferred combinations for initiation and detection of electron transfer in the complexes of the invention Table 2 Initiation Detection Description ght hght absorbance, fluorescence, phosphorescence, refractive index, surface plasmon resonance, electron spin resonance ght current amperommetry, voltammetry, capacitance, impedence, opto-electronic detection, photo- amperometry light plus light absorbance, fluorescence, phosphorescence, electronic refractive index, surface plasmon resonance, initiation electron spin resonance light plus current amperommetry, voltammetry, capacitance, electronic impedence, opto-electonic detection, photo- initiation amperommetry, amperommetπc detection, cyclic voltammetry electronic current amperommetry, voltammetry, capacitance, initiation impedence, amperommetπc detection, cyclic voltammetry electronic light chemiluminescence, initiation electrochemiluminescence, electroluminescence By "light" herein is meant electromagnetic radiation, with light in the UV, visible and infrared range being preferred, and UV and visible being the most preferred In a preferred embodiment, initiation of electron transfer is via direct or indirect photoactivation ("light in") Simply, electromagnetic radiation of appropriate wavelength strikes the redox molecule on one end of the DNA causing excitation of a donor moiety electron which either decays immediately or is involved in intramolecular electron transfer The efficiency with which electron transfer is induced depends upon the electronic coupling between the electron donor and acceptor and therefore depends on whether the nucleic acid is single or double stranded In addition, the efficiency of electron transfer depends upon the extinction coefficient of the electron donor at the wavelength of light used (higher is better) and upon the lifetime of the donor electron excited state (longer is better) Preferred donor complexes therefore include acπdine orange, Λ/,Λ/'-dιmethyl-2,7-dιazapyrenιum dichloride (DAP 2* ), methylviologen, ethidium bromide, qu ones such as N,N'-dιmethylanthra(2,1 ,9- def 6,5,10-d 'e 1 dιιsoquιnolιne dichloride (ADIQ 2* ), porphyπns ([meso-tetrakιs(N- methyl-x-pyrιdιnιum)porphyπn tetrachloride] Transition metal donors and acceptors include complexes of ruthenium, rhenium and osmium (most preferred) where at least one of the ligands is a chromophore Photoactivation can also be used to excite "mediators" that transfer energy to the electron donor moiety on the DNA via an inter-molecular process Such mediators include water soluble and stable complexes of the transition metals, including molybdenum and tungsten halides, tnsbipyπdyl complexes of rhenium, osmium and ruthenium In addition, other examples include bipyπdyl and pyπdyl complexes such as Re(bpy)(CO) 3 X where X is a halide and Re(py) 4 0 2 Other examples include transition metal dimers such as [Re 2 CI 8 ] 2 and [Pt 2 (P 2 0 5 H 2 ) 4 ] 4 Ruthenium tπsbypyπdine (Ru 2* (bpy) 3 ) is most preferred In the preferred embodiment, electron transfer occurs after photomduction with a laser In this embodiment, electron donor moieties may, after donating an electron, serve as electron acceptors under certain circumstances Similarly electron acceptor moieties may serve as electron donors under certain circumstances A preferred embodiment utilizes electronic activation, with voltage being preferred A potential is applied to a sample containing modified nucleic acid probes either via a direct linkage of the modified nucleic acid to an electrode or using electron transport mediators Direct linkage can involve a redox active polymer to shuttle electrons from (and to, if the electrode is also used for detection) the electrode Such polymers are outlined below Alternatively, the direct connection can involve a relatively poorly conducting linkage provided the linkage is kept reasonably short (less than six sigma bonds) Preferred linkages will be three or fewer sigma bonds in length to allow efficient transfer of electrons from the electrode, as is outlined below Indirect electron transfer initiation involves electron transfer mediators or effective diffusional electron donors and acceptors such as water soluble ferrocene/ferπcinium, hydroquinones/qumones, reducible and oxidizable components of organic salts, cobaltocenes, the hexa- and octacyanides of molybdenum, tungsten and iron In addition, other examples include macrocycles and chelating ligands of transition metals such as cobalt, ruthenium and nickel, including Co(ethylenedιamιne) 3 and Ru(ethylenedιamιne) 3 and the tπsbypyridyl and hexamine complexes of transition metals such as Co, Ru, Fe, and Os See K Alyanasundaram, Coord Chem Rev V 46, p 159, 1982 Finally, organic molecules such as 4,4'-bιpyrιdιne and 4- mercaptopyπdme are examples where ferrocene is most preferred Precise control and variations in the applied potential can be via a potentiostat and a three electrode system (one reference, one sample and one counter electrode) This allows matching of applied potential to peak electron transfer potential of the system which depends in part on the choice of electron acceptors attached to the nucleic acid High driving forces are achieved using bisbipyπdyl complexes of transition metals, for example, ruthenium and rhenium bisbipyπdyl complexes such as (Ru(bpy) 2 ιm-) as electron acceptors Alternatively, electrochemical initiation of electron transfer may be used The redox states of the electron donating and accepting moieties attached to nucleic acid can be electrochemicaly changed using water soluble chemical oxidants and reductants, either with or without photo- or electrical activation Such compounds include numerous derivatives known in the art (T Kuwana, Electrochemical Studies of Biological Systems, (D T Sawyer Ed ) ACS Sy p Series #38, (1977)) and include hexacyano iron complexes, zinc-mercury amalgam, and tπsphenanthrohne complexes of ruthenium and iron Electron transfer through nucleic acid can be detected in a variety of ways A variety of detection methods may be used, including, but not limited to, optical detection, which includes fluorescence, phosphorescence, and refractive index, and electronic detection, including, but not limited to, amperommetry, voltammetry, capacitance and impedence These methods include time or frequency dependent methods based on AC or DC currents, pulsed methods, lock-in techniques, filtering (high pass, low pass, band pass), and time-resolved techniques including time-resolved fluoroscence In some embodiments, all that is required is electron transfer detection, in others, the rate of electron transfer may be determined In one embodiment, the efficient transfer of electrons from one end of a nucleic acid double helix to the other results in stereotyped changes in the redox state of both the electron donor and acceptor With many electron transfer moieties including the complexes of ruthenium containing bipyndine, pyridine and imidazole rings, these changes in redox state are associated with changes in spectral properties ("light out") Significant differences in absorbance are observed between reduced and oxidized states for these molecules These differences can be monitored using a spectrophotometer or simple photomultiplier tube device In this embodiment possible electron donors and acceptors include all the derivatives listed above for photoactivation or initiation Preferred electron donors and acceptors have characteristically large spectral changes upon oxidation and reduction (large extinction coefficient "deltas") resulting in highly sensitive monitoring of electron transfer Such examples include Ru(NH 3 ) 4 py and Ru(bpy) 2 ιm as preferred examples It should be understood that only the donor or acceptor that is being monitored by absorbance need have ideal spectral characteristics That is, the electron acceptor can be optically invisible if only the electron donor is monitored for absorbance changes In a preferred embodiment, the electron transfer is detected fluorometπcally Numerous transition metal complexes, including those of ruthenium, have distinct fluorescence properties Therefore, the change in redox state of the electron donors and electron acceptors attached to the nucleic acid can be monitored very sensitively using fluorescence Highly efficient electron transfer through double stranded nucleic acid can, for example, result in the production of fluorescent Ru(4,7-bιphenyl 2 - phenanthrohne) 3 2+ at one end of a nucleic acid probe when the electron transfer moiety on the other end is excited The production of this compound can be easily measured using standard fluorescence assay techniques For example, laser induced fluorescence can be recorded in a standard single cell fluoπmeter, a flow through "on¬ line" fluoπmeter (such as those attached to a chromatography system) or a multi- sample "plate-reader" similar to those marketed for 96-well immuπo assays Alternatively, fluorescence can be measured using fiber optic sensors with nucleic acid probes in solution or attached to the fiber optic Fluorescence is monitored using a photomultiplier tube or other light detection instrument attached to the fiber optic The advantage of this system is the extremely small volumes of sample that can be assayed In addition, scanning fluorescence detectors such as the Fluorlmager sold by Molecular Dynamics are ideally suited to monitoring the fluorescence of modified nucleic acid molecules arrayed on solid surfaces The advantage of this system is the large number of electron transfer probes that can be scanned at once using chips covered with thousands of distinct nucleic acid probes Many transition metal complexes display fluorescence with large Stokes shifts Suitable examples include bis- and trisphenanthrohne complexes and bis- and tπsbipyndyl complexes of transition metals such as ruthenium (see Juris, A , Balzani, V , et al Coord Chem Rev , V 84, p 85-277, 1988) Preferred examples display efficient fluorescence (reasonably high quantum yields) as well as low reorganization energies These include Ru(4,7-bιphenyl 2 -phenanthrohne) 3 2+ and Ru(4,4'-dιphenyl- 2,2'-bιpyπdιne) 3 2* Alternatively, a reduction in fluorescence associated with hybridization can be measured using these systems An electron transfer "donor' molecule that fluoresces readily when on single stranded nucleic acid (with an "acceptor" on the other end) will undergo a reduction in fluorescent intensity when complementary nucleic acid binds the probe allowing efficient transfer of the excited state electron This drop in fluorescence can be easily monitored as an indicator of the presence of a target sequence using the same methods as those above In a further embodiment, electrochemilummescence is used as the basis of the electron transfer detection With some electron transfer moieties such as Ru 2t (bpy) 3 , direct luminescence accompanies excited state decay Changes in this property are associated with nucleic acid hybridization and can be monitored with a simple photomultiplier tube arrangement (see Blackburn, G F Clin Chem 37 1534-1539 (1991 ), and Juris et al , supra In a preferred embodiment, electronic detection is used, including amperommetry, voltammetry, capacitance, and impedence Suitable techniques include, but are not limited to, electrogravimetry, coulometry (including controlled potential coulometry and constant current coulometry), voltametry (cyclic voltametry, pulse voltametry (normal pulse voltametry, square wave voltametry, differential pulse voltametry, Osteryoung square wave voltametry, and coulostatic pulse techniques), stripping analysis (aniodic stripping analysis, cathiodic stripping analysis, square wave stripping voltammetry), conductance measurements (electrolytic conductance, direct analysis), time- dependent electrochemical analyses (chronoamperometry, chronopotentiometry, cyclic chronopotentiometry and amperometry, AC polography, chronogalvametry, and chronocoulometry), AC impedance measurement, capacitance measurement, and photoelectrochemistry In a preferred embodiment, monitoring electron transfer through nucleic acid is via amperometπc detection, either directly using a covalently attached electrode, or indirectly using electron transport "mediators" to shuttle electrons from the nucleic acid to an electrode Modes of attaching nucleic acids to electrodes and possible mediators are described below An amperometnc detector would resemble the numerous enzyme-based biosensors currently used to monitor blood glucose, for example This method of detection involves applying a potential (as compared to a separate reference electrode) between the nucleic acid-conjugated electrode and an auxiliary (counter) electrode in the sample containing target genes of interest Electron transfer of differing efficiencies is induced in samples in the presence or absence of target nucleic acid, that is, the single stranded probe exhibits a different rate than the probe hybridized to the target sequence The differing efficiencies of electron transfer result in differing currents being generated in the electrode The device for measuring electron transfer amperometπcally involves sensitive (nanoamp to picoamp) current detection and includes a means of controlling the voltage potential, usually a potentiostat This voltage is optimized with reference to the potential of the electron donating complex on the nucleic acid Possible electron donating complexes include those previously mentioned with complexes of ruthenium being preferred and complexes of rhenium being most preferred ln a preferred embodiment, alternative electron detection modes are utilizes For example, potentiometπc (or voltammetπc) measurements involve non-faradaic (no net current flow) processes and are utilized traditionally in pH and other ion detectors Similar sensors are used to monitor electron transfer through nucleic acid In addition, other properties of insulators (such as resistance) and of conductors (such as conductivity, impedance and capicitance) could be used to monitor electron transfer through nucleic acid Finally, any system that generates a current (such as electron transfer) also generates a small magnetic field, which may be monitored in some embodiments It should be understood that one benefit of the fast rates of electron transfer observed in the compositions of the invention is that time resolution can greatly enhance the signal-to-noise results of monitors based on absorbance, fluorescence and electronic current The fast rates of electron transfer of the present invention result both in high signals and stereotyped delays between electron transfer initiation and completion By amplifying signals of particular delays, such as through the use of pulsed initiation of electron transfer and "lock-in" amplifiers of detection, between two and four orders of magnitude improvements in signal-to-noise may be achieved In a preferred embodiment, DNA is modified by the addition of electron donor and electron acceptor moieties In an alternative embodiment, RNA is modified In a further embodiment, a double stranded nucleic acid for use as a bioconductor will contain some deoxyπbose nucleosides, some nbose nucleosides, and a mixture of adenosine, thymidine, cytos e, guanine and uraαl bases In accordance with a further aspect of the invention, the preferred formulations for donors and acceptors will possess a transition metal covalently attached to a series of ligands and further covalently attached to an amine group as part of the nbose ring (2' or 3' position) or to a nitrogen or sulfur atom as part of a nucleoside dimer linked by a peptide bond, phosphoramidate bond, phosphorothioate bond, phosphorodithioate bond or O-methyl phosphoramidate bond In a preferred embodiment, an oligonucleotide containing at least one electron transfer moiety is attached to an electrode, which also serves as an electron transfer moiety, thus forming a single stranded nucleic acid with both an electron donor moiety and an electron acceptor moiety attached in the manner outlined above Preferably, the single stranded nucleic acid containing an electron transfer moiety is attached covalently or in such a way that allows the transfer of electrons from the electrode to the single stranded nucleic acid in order to allow electron transfer between the electron donor and acceptor Preferably, the non-electrode electron transfer moiety is attached at or near the terminus of the oligonucleotide, such that the probe sequence to be hybridized to the target sequence is between the donor and acceptor The electrode may be immersed in a sample containing the target sequence such that the target sequence hybridizes to the probe and electron transfer may be detected using the techniques outlined above In an additional embodiment, two nucleic acids are utilized as probes as described previously For example, one nucleic acid is covalently attached to a solid electrode which serves as an electron transfer moiety, and the other, with a covalently attached electron transfer moiety, is free in solution Upon hybridization of a target sequence, the two nucleic acids are aligned such that electron transfer between the electron transfer moiety of the hybridized nucleic acid and the electrode occurs The electron transfer is detected as outlined above, using techniques well known in the art The following examples serve to more fully describe the manner of using the above- described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes The references cited herein are expressly incorporated by reference EXAMPLES The amino-modified monomer units are prepared by variation of published procedures and are incorporated into a growing oligonucleotide by standard synthetic techniques The procedure is applicable to both DNA and RNA derivatives Example 1 Synthesis of an Oligonucleotide Duplex with Electron Transfer Moieties at the 5' Termini In this example an eight nucleotide double stranded nucleic acid was produced, with each single strand having a single electron transfer moiety covalently attached to the 5' terminal undine nucleotide at the 2' carbon of the nbose sugar Step 1 Synthesis of 5'-di(p-methoxyphenyl)methyl ether-2'-(tπfluoroacetamιdo)-2'- deoxyuπdine 2'-(tπfluoroacetamιdo)-2'-deoxyuπdιne (2 0 g, 5 9 mmoles) prepared by minor modification of published procedures (Imazawa, supra) was repeatedly dissolved in a minimum of very dry CH 3 CN and rotary evaporated to dryness and then transferred to inert atmosphere vacuum line and further dried for a period of 1 hour The following procedure for the synthesis of the material was adapted from Gait (supra) Under positive pressure argon, the material was dissolved in freshly dried and distilled pyridine and with stirring, 0 05 equivalents (wt ) of 4-dιmethylamιnopyπdιne (DMAP), 1 5 equivalents of triethylamine (TEA) and 1 2 equivalents of 4,4'-dιmethoxytπtyl chloride (DMTr-CI) were added to the reaction mixture The progress of the reaction was monitored by silica gel TLC (98 2 methylene chloride methanol, mobile phase) After 30 minutes, an additional 0 5 equivalents each of DMTr-CI and TEA were added and the reaction allowed to proceed for an additional three hours To this reaction mixture was added an equal volume of water and the solution extracted several times with diethyl ether The ether layers were rotary evaporated to dryness, redissolved in a minimum amount of methylene chloride and purified by flash chromatography (99 1 methylene chloride methanol, mobile phase) to obtain the 5'-dι(p- methoxyphenyl)methyl ether-2'-(trιfluoroacetamιdo)-2'-deoxyuπdιne product Step 2 5'-2'-amιnouπdιne-GCTACGA and 5'-2'-amιnouπdιne-CGTAGCA 5'-dι(p-methoxyphenyl)methyl ether-2'-(tπfluoroacetamιdo)-2'-deoxyuπdιne was dπed under reduced pressure (glass) and dissolved in freshly dried and distilled CH 3 CN and placed in a specially made conical vial and placed on an ABI DNA synthesizer The program for the preparation of standard (i e unmodified) oligonucleotides was altered during the final base (amino-modified) addition to a 15-30 minute coupling time The oligonucleotide was cleaved from the column by standard procedures and purified by C-18 reverse phase HPLC In this manner 5'-2'-amιnouπdιne-GCTACGA and 5'-2'- aminouπdine-CGTAGCA were prepared In addition, unmodified complementary strands to both products were made for use in the electron transfer moiety synthesis below Step 3 5'-2 '-ruthenium bisbipyridineimidazole-aminouπdine-GCTACGA 5'-2'-amιnouπdιne GCTACGA produced in the previous step was annealed to the complementary unmodified strand using standard techniques All manipulations of the annealed duplex, prior to the addition of the transition metal complex were handled at 4°C In order to insure that the DNA remained annealed during modification, the reactions were performed in 1M salt The 5'-amιno modified duplex DNA was dissolved in 0 2 M HEPES, 0 8 M NaCl, pH 6 8 and repeatedly evacuated on a Schlenk line Previously prepared ruthenium bisbipyπdine carbonate was dissolved in the above buffer and oxygen was removed by repeated evacuation and purging with argon via a Schlenk line The ruthenium complex was transferred to the DNA solution via cannulation (argon/vacuum) and the reaction allowed to proceed under positive pressure argon with stirring for 24 hours To this reaction, 50 equivalents of imidazole was added to the flask and the reaction allowed to proceed for an additional 24 hours The reaction mixture was removed from the vacuum line and applied to a PD-10 gel filtration column and eluted with water to remove excess ruthenium complex The volume of the collected fractions was reduced to dryness via a speed vac and the solid taken up in 0 1 M triethylammonium acetate (TEAC) pH 6 0 The duplex DNA was heated to 60°C for 15 minutes with 50% formamide to denature the duplex The single stranded DNA was purified using a C-18 reverse phase HPLC column equiped with a diode array detector and employing a gradient from 3% to 35% acetonitrile in 0 1 M TEAC, pH 6 0 Step 4 5'-2 -ruthenium tetraminepyπdine-aminouπdine-CGTAGCA 5'-amιnouπdιne-CGTAGCA (0 3μm) was dissolved in 0 2 M HEPES, 0 8 M NaCl buffer, pH 6 8 and degassed on the vacuum line To a 10 ml conical shaped flask equipped with a stirring bar and septum was slurried Ru(lll) tetraaminepyndme chloride (10 μm), in the same buffer In a separate flask, Zn/Hg amalgam was prepared and dried under reduced pressure and the ruthenιum(lll) solution transferred (via cannulation) to the Zn/Hg amalgam The immediate formation of a clear yellow solution (λ max = 406 nm) indicated that the reduced form of the ruthenium had been achieved and the reaction allowed to proceed for 30 minutes This solution was transferred to the flask containing the amino-modified DNA and the reaction allowed to proceed at room temperature for 24 hours under argon The reaction mixture was removed from the vacuum line and a 50 fold excess of cobalt EDTA (Kirschner, Inorganic Synthesis (1957), pp 186) added to the solution The solution was applied to Sephadex G-25 gel filtration column to remove excess ruthenium complex and further purified by reverse phase HPLC as descπbed above The two ruthenium modified nucleotides were annealed by standard techniques and characterized (see Example 5) Example 2 Synthesis of Long DNA Duplexes with Electron Transfer Moieties at the 5' Termini In this example an in vitro DNA amplification technique, PCR (reviewed in Abramson et al , Curr Op in Biotech 4 41-47 (1993)) is used to generate modified duplex DNA by polymerization of nucleotides off modified primer strands (Saiki et al , Science 239487 (1988)) Two oligonucleotides 18 bases in length and not complementary to each other are synthesized with ammo-modification to the 2 -nbose position of the 5 nucleotides, as in example 1 A series of oligonucleotides of increasing lengths starting at 40 bases are chemically synthesized using standard chemistry Each of the PCR templates shares a 5' sequence identical to one modified 18mer The 3' end of the template oligonucleotide shares a sequence complementary to the other 18mer PCR rapidly generates modified duplex DNA by the catalysis of 5'-3' DNA synthesis off of each of the modified 18mers using the unmodified strand as a template One hundred nanomoles of each of the two modified 18mers are mixed in 1 ml of an aqueous solution containing 2 000 units of Taq polymerase, deoxyπbonucleoside tnphosphates at 0 2 M each, 50 mM KCl, 10 mM Tπs-Cl, pH 8 8, 1 5 mM MgCI 2 , 3 mM dithiothreitol and 0 1 mg/ml bovine serum albumin One femtomole of the template strand 40 bases in length is added to the mixture The sample is heated at 94°C for one minute for denaturation, two minutes at 55°C for annealing and three minutes at 72°C for extension This cycle is repeated 30 times using an automated thermal cycler The amplified template sequences with transition metal complexes on both 5' termini are purified by agarose gel electrophoresis and used directly in electron transfer applications Example 3 Synthesis of Covalently Bound Electron Transfer Moieties at Internucleotide Linkages of Duplex DNA In this example, alternative backbones to phophodiester linkages of oligonucleotides are employed Functional groups incorporated into these internucleotide linkages serve as the site for covalent attachment of the electron transfer moieties These alternate internucleotide linkages include, but are not limited to, peptide bonds, phosphoramidate bonds, phosphorothioate bonds, phosphorodithioate bonds and O- methylphosphoramidate bonds The preparation of peptide nucleic acid (PNA) follows literature procedures (See Engholm, supra), with the synthesis of Boc-protected pentaflurophenyl ester of the chosen base (thymidine) The resulting PNA may be prepared employing Merπfield's solid-phase approach (Merπfield, Science 232 341 (1986)), using a single coupling protocol with 0 1 M of the thiminyl monomer in 30% (v/v) DMF in CH 2 CI 2 The progress of the reaction is followed by quantiative ninhydπn analysis (Saπn Anal Biochem , 117 147 (1981)) The resulting PNA may be modified with an appropriate transition metal complex as outlined in example 1 The synthesis of phosphoramidate (Beaucage, supra, Letsinger supra, Sawai, supra) and N-alkylphosphoramidates (Jager, supra) internucleotide linkages follows standard literature procedures with only slight modification (the procedures are halted after the addition of a single base to the solid support and then cleaved to obtain a dmucleotide phosphoramidate) A typical example is the preparation of the phenyl ester of 5'0- ιsobutyloxycarbonylthymιdyl-(3'-5')-5'-amιno-5'-deoxythymιdιne (Letsinger, J Org Chem , supra) The dimer units are substituted for standard oligonucleotides at chosen intervals during the preparation of DNA using established automated techniques Transition metal modification of the modified linkages takes place as described in Example 1 The synthesis of phosphorothioate and phosphorodithioate (Eckstein, supra, and references within) internucleotide linkages is well documented A published protocol utilizes an Applied Biosystems DNA synthesizer using a modified β- cyanoethylphosphoramidite cycle that caps after sulphurization with tetraethylthiuram disulfide (TETD) (Iyer, J Org Chem 55 4693 (1990)) The phosphorothioate and phosphorodithioate analogs are prepared as dimers and cleaved from the solid support and purified by HPLC (acetonitπle/triethylammonium acetate mobile phase) Example 4 Synthesis of Two Oligonucleotides each with an electron transfer oietv at the 5' terminus In this example, two oligonucleotides are made which hybridize to a single target sequence, without intervening sequences One oligonucleotide has an electron donor moiety covalently attached to the 5' terminus, and the other has an electron acceptor moiety covalently attached to the 5' terminus In this example, the electron transfer species are attached via a uradme nucleotide, but one skilled in the art will understand the present methods can be used to modify any of the nucleotides In addition, one skilled in the art will recognize that the procedure is not limited to the generation of 8- mers, but is useful in the generation of oligonucleotide probes of varying lengths The procedure is exactly as in Example 1, except that the 8-mers generated are not complementary to each other, and instead are complementary to a target sequence of 16 nucleotides Thus the final annealing step of step 4 of Example 1 is not done Instead, the two modified oligonucleotides are annealed to the target sequence, and the resulting complex is characterized as in Example 5 Example 5 Characterization of Modified Nucleic Acids Enzymatic digestion The modified oligonucleotides of example 1 were subjected to enzymatic digestion using established protocols and converted to their constituent nucleosides by sequential reaction with phosphodiesterase and alkaline phosphatase By comparison of the experimentally obtained integrated HPLC profiles and UV-vis spectra of the digested oligonucleotides to standards (including 2'-amιnourιdιne and 2'- aminoadenine), the presence of the amino-modified base at the predicted retention time and characteristic UV-vis spectra was confirmed An identical procedure was carried out on the transition metal modified duplex DNA and assignments of constituent nucleosides demonstrated single-site modification at the predicted site Fluorescent labeled amino-modified oligonucleotides It has been demonstrated that the fluorochrome, fluorescein isothiocyanate (FITC) is specific for labeling primary amines on modified oligonucleotides while not bonding to amines or amides present on nucleotide bases (Haugland, Handbood of Fluorescent Probes and Research Chemicals, 5th Edition, (1992)) This reaction was carried out using the amino-ohgonucleotide synthesized as described in example 1 and on an identical bases sequence without the 2'-amιno-πbose group present Fluorescence spectroscopic measurements were acquired on both these oligonucleotides and the results confirm the presence of the amine on the 5'-termmal nbose ring Thermodvnamic Melting Curves of Modified Duplex DNA A well established technique for measuring thermodynamic parameters of duplex DNA is the acquisition of DNA melting curves A series of melting curves as a function of concentration of the modified duplex DNA was measured via temperature controlled UV-vis (Hewlett-Packard), using techniques well known in the art These results confirm that hybridization of the amino-modified and transition metal modified DNA had taken place In addition, the results indicate that the modified DNA form a stable duplex comparable to the stability of unmodified oligonucleotide standards Two Dimensional Nuclear Magnetic Resonance (NMR) Spectroscopy The amino-modified oligonucleotides synthesized as a part of this work were prepared in sufficient quantities (6 micromoles) to permit the assignment of the proton NMR spectra using a 600MHz Vaπan NMR spectrometer Measurement of the rate of electron transfer An excellent review of the measurement techniques is found in Winkler et al , Chem Rev 92 369-379 (1992) The donor is Ru(bpy) 2 (NHuπdιne)ιm, E°~1 V, and the acceptor is Ru(NH 3 ) 4 py(NHurιdιne)ιm, E°~330 mV The purified transition metal modified oligonucleotides (U NHRu(bpy)2im GCATCGA and U NH u < NH3)4(py) , m CGATGCA were annealed by heating an equal molar mixture of the oligonucleotides (30 μmolar 60 nmoles of DNA in 2 ml buffer) in pH 6 8 (100 mM NaPi, 900 mM NaCl) to 60°C for 10 minutes and slowly cooling to room temperature over a period of 4 hours The solution was transferred to an inert atmosphere cuvette equipped with adapters for attachment to a vacuum line and a magnetic stirring bar The solution was degassed several times and the sealed apparatus refilled repeatedly with Ar gas The entire apparatus was inserted into a cuvette holder as part of the set-up using the XeCI excimer-pumped dye laser and data acquired at several wavelengths including 360, 410, 460 and 480 nm The photoinduced electron transfer rate is 1 6 X 10 6 s 1 over a distance of 28 A Example 6 Synthesis of a single stranded nucleic acid labeled with two electron transfer moieties This example uses the basic procedures described earlier to generate two modified oligonucleotides each with an electron transfer moiety attached Ligation of the two modified strands to each other produces a doubly labeled nucleic acid with any of four configurations 5' and 3' labeled termini, 5' labeled terminus and internal nucleotide label, 3' labeled terminus and internal nucleotide label, and double internal nucleotide labels Specifically, the synthesis of an oligonucleotide 24 bases in length with an electron transfer donor moiety on the 5' end and an internal electron transfer moiety is described Five hundred nanomoles of each of two 5'-labeled oligonucleotides 12 bases in length are synthesized as detailed above with ruthenium (II) bisbipyπdine imidazole on one oligonucleotide, "D" and ruthenium (III) tetraamine pyridine on a second oligonucleotide, "A" An unmodified oligonucleotide 24 bases in length and complementary to the juxtaposition of oligonucleotide "D" followed in the 5' to 3' direction by oligonucleotide "A" is produced by standard synthetic techniques Five hundred nanomoles of this hybridization template is added to a mixture of oligonucleotides "A" and "D" in 5 ml of an aqueous solution containing 500 mM Tns-CI, pH 7 5, 50 mM MgCI 2 , 50 mM dithiothreitol and 5 mg/ml gelatin To promote maximal hybridization of labeled oligonucleotides to the complementary strand, the mixture is incubated at 60°C for 10 minutes then cooled slowly at a rate of approximately 10°C per hour to a final temperature of 12°C The enzymatic ligation of the two labeled strands is achieved with T4 DNA ligase at 12°C to prevent the ligation and ohgomeπzation of the duplexed DNA to other duplexes (blunt end ligation) Alternatively, E coli DNA ligase can be used as it does not catalyze blunt end ligation One hundred Weiss units of T4 DNA ligase is added to the annealed DNA and adenosine tπphosphate is added to a final concentration of 0 5 mM The reaction which catalyzes the formation of a phosphodiester linkage between the 5' terminal phosphate of oligonucleotide "A" and the 3' terminal hydroxyl group of oligonucleotide "D" is allowed to proceed for 18 hours at 12°C The reaction is terminated by heat inactivation of the enzyme at 75°C for 10 minutes The doubly labeled oligonucleotide is separated from the singly labeled oligonucleotides and the complementary unlabeled oligonucleotide by HPLC in the presence of urea as in the previous examples The doubly labeled oligonucleotide of this example is ideally suited for use as a photoactive gene probe as detailed beiow Example 7 Use of a doubly modified oligonucleotide with electron transfer moieties as a photoactive probe for homologous nucleic acid sequence detection This example utilizes the oligonucleotide 24mer of example 6 in a unique type of gene- probe assay in which removal of unhybndized probe prior to signal detection is not required In the assay procedure, a region of the gag gene of human immunodeficiency virus type I (HIV-l) is amplified by the polymerase chain reaction (Saiki et al , Science 239 487-491 (1988)) This region of HIV-l is highly conserved among different clinical isolates The amplified target DNA versus controls lacking in HIV-l DNA are added to a hybridization solution of 6XSSC (0 9 M NaCl, 0 09 M Na citrate, pH 7 2) containing 50 nanomoles of doubly labeled 24mer probe of example 6 Hybridization is allowed to proceed at 60°C for 10 minutes with gentle agitation Detection of electron transfer following laser excitation is carried out as in example 5 Control samples which lack the hybridized probe show negligible electron transfer rates Probes hybridized to the gag sequence show efficient and rapid electron transfer through the DNA double helix, providing a highly specific, homogeneous and automatable HIV-l detection assay A similar homogeneous gene probe assay involves the use of two probes, one an electron donor and the other an electron acceptor, which hybridize with the gag region of HIV-l in a tandem configuration, one probe abutting the other In this assay, electronic coupling between the two electron transfer moieties depends entirely on hybridization with the target DNA If appropriate, the electron transfer from one probe to the other is enhanced by the ligation of the juxtaposed ends using T4 DNA ligase as in example 6 Example 8 Preparation of a Hydroxythiol for Attachment to a Gold Electrode OH(CH 2 ) 16 OH was purchased from Aldrich and the monoacetate form prepared by slurring the material in dry CH 2 CI 2 0 5 equiv of dimethylaminopyrtdine was added along with 1 4 equivalents of triethylamine and 1 equivalent of acetic anhydride The reactton was allowed to proceed for 2 hours and purified by flash chromatography (80 20 hexane diethyl ether The monoacetate compound was converted to the monotosylate-monoacetate using p- TSOCI by literature procedures and then treated with tπphenyl methylmercaptan To remove the monoacetate, the product was dissolved in MeOH (1 mmol, 9 ml), cooled to 0°C, and aqueous solution of NaOH (1 mmol, in 2 ml water) added The temperature was allowed to rise to room temperature slowly, and the reaction followed by TLC (5% MeOH/CH 2 CI 2 ) When the ester was gone the mixture was recooled to 0°C, and acidified with KHS0 4 to pH 5-6 using pH paper The MeOH was evaporated and the residue was extracted with CH 2 CI 2 (200 ml), dried (Na 2 S0 4 ), evaporated and checked via TLC The material was phosphoroamidited by standard procedures This material was inserted into the DNA synthesizer and an modified oligonucleotide produced The phosphoramidited oligonucleotide was modified with a ruthenium complex by adding Ru(bpy) 2 C0 3 followed by imidazole to yield a Ru(bpy) 2 ιm oligonucleotide The tπtyl protecting group was removed by dissolving the nucleotide in 200 μl of 0 1 M tπethylammonium acetate (TEAA) buffer, pH 7 5 30 μl of 1 M silver nitrate solution was added and the mixture vortexed and incubated at room temperature for 30 minutes 50 μ of 1 M dithiothntol (DTT) was added, the mixture vortexed and incubated for 15 minutes, at which point it was microcentπfuged for 15 minutes to remove precipitated Ag+DTT The supernatant was collected and the pellet was washed with 100 μl of TEAA buffer and the solutions pooled The resulting oligonucleotide was then attached to the gold surface by standard techniques Example 9 Synthesis of a single stranded nucleic acid containing both an electron acceptor and an electron donor moiety In order to evaluate the path dependent nature of the electron transfer process through duplex DNA, an oligonucleotide was prepared with an electron donor at the 3' end and an electron acceptor at the 5' end This multiply-modified oligonucleotide was prepared by synthesizing a derivative with an amine at the 2'-posιtιon of the terminal nbose of both ends Synthesis of Bis-3',5'-2'-deoxyuridine Oligonucleotides A DMT-2'-N-tπfluoroacetyl-protected phosphoroamidite of 2'-amιno-2'-deoxyuπdιne (U NH2 ) was prepared as described earlier and reacted with succinic anhydride This material was reacted with p-nitrophenol to produce the precursor for the attachment to the controller pore glass (GPG) resin as in Figure 6A The modified oligonucleotide were assembled by standard solid phase automated DNA synthesis techniques and the bιs-3',5' 1 -2'-amιno-2'-deoxyurιdιne oligonucleotide isolated and characterized by mass spectrometry and HPLC digestion analysis In addition, the aminoπbose oligomers and their complements were reacted with FITC under conditions that favor labeling of primary amines As expected, only the 2'-amιno-2'deoxyrιbose site was labeled verifying the presence of a primary amine on the DNA As an example, a 11 base pair sequence was prepared (calc for U NH2 CTCCTACACU NH2 -3229, found 3229 1) and the subsequent digestion map was consistent with the proposed structure The metal modification of the bis-ammo modified oligonucleotide was performed in a similar manner The new metal-modified oligonucleotides were characterized by fluorescent labelling, enzymatic digestion, and duplex-melting temperature studies Thermal denaturing and annealing experiments display similar melting temperatures for both ruthenium and aminoπbose oligomers In addition, the amino-modified duplex DNA has been characterized by 2D NMR These data confirm that the donors and acceptors are covalently attached to the 2'-amιno-2'deoxyπbose position and indicate that the DNA structure is unperturbed by the presence of the ruthenium complexes Example 10 Synthesis of a polydentate nucleoside 2'-amιnouπdιne (10 mmol) and pyπdιne-2-carboxyaldehyde (11 mmol) were heated to reflux in absolute ethanol until TLC (silica gel, 30 70 MeOH CHCI 3 ) showed complete conversion of aminoundme to the less-polar product The solvent was evaporated, the residue dissolved in methanol, and 11 mmol NaBH 4 added with vigorous stirring When hydrogen evolution subsided, the mixture was heated to reflux for 2h and the solvent was evaporated The residue was dissolved in water and purified by cation- exchange chromatography on Dowex AG-50 using 2M NH 3 as eluent As noted herein, the use of NaCNBH 4 allowed the one-pot synthesis of the product The product was then protected as shown in Figure 9

Claims: CLAIMS We claim 1. A nucleoside containing a covalently attached polydentate ligand, said ligand attached at the 2' or 3' position of said nucleoside 2 A nucleoside according to claim 1 wherein said attachment is at the 2' position 3 A nucleoside according to claim 1 wherein said attachment is at the 3" position 4 A phosphoramidite nucleoside containing a covalently attached polydentate ligand, said ligand attached at the 2' position of said nucleoside 5 A composition comprising a first nucleoside containing a covalently attached polydentate ligand, said ligand attached at the 2' position of said nucleoside, said nucleoside covalently attached to control pore glass (CPG) 6 A composition comprising an oligonucleotide covalently attached to control pore glass (CPG), wherein at least one nucleoside of said oligonucleotide is a polydentate modified nucleoside. 7 A composition according to claim 6, wherein said polydentate modified nucleoside is at the 5' terminus of said oligonucleotide 8 A composition according to claim 6, wherein said polydentate modified nucleoside is on an internal nucleoside of said oligonucleotide 9 A composition according to claim 6, further comprising a second 2'-amιno modified nucleotide covalently attached to said oligonucleotide 10 A composition according to claim 1 , 4, 5, or 6, further comprising a transition metal chelated to said polydentate nucleoside 11 A single-stranded nucleic acid containing at least one electron donor moiety and at least one electron acceptor moiety, wherein said electron donor moiety and said electron acceptor moiety are covalently attached to said nucleic acid, and wherein at least one of said electron donor and said electron acceptor moieties is attached to a polydentate nucleoside 12 A nucleic acid according to claim 11 , wherein both of said electron donor and acceptor moieties are attached via polydentate nucleoside 13 A nucleic acid according to claim 11 , wherein the other of said electron donor and acceptor moieties is attached via a terminal base 14 A nucleic acid according to claim 11 , wherein the other of said electron donor and acceptor moieties is attached via the 2' or 3' position of a nbose of the nbose- phosphate backbone 15 A nucleic acid according to claims 11 , 12, 13, or 14 wherein at least one of said electron donor moiety and said electron acceptor moiety is a transition metal complex 16 A composition according to claim 11 , 13, 14, or 15 wherein at least one of said electron donor moiety and said electron acceptor moiety is an electrode 17 A composition according to claim 11, 12, 13, 14, 15, or 16 wherein at least one of said electron donor moiety and said electron acceptor moiety is an organic electron donor or acceptor 18 A composition comprising a first single stranded nucleic acid containing at least one covalently attached electron donor moiety and a second single stranded nucleic acid containing at least one covalently attached electron acceptor moiety, wherein at least one of said electron donor and acceptor moieties is attached to a polydentate nucleoside 19 A method of detecting a target sequence in a nucleic acid sample comprising a) hybridizing a single stranded nucleic acid containing at least one covalently attached electron donor moiety and at least one covalently attached electron acceptor moiety to said target sequence to form a hybridization complex, wherein at least one of said electron donor and acceptor moieties is attached to a polydentate nucleoside, and b) detecting electron transfer between said electron donor and acceptor moieties in the hybridization complex as an indicator of the presence or absence of said target sequence 20 A method of detecting a target sequence in a nucleic acid wherein said target sequence comprises a first target domain and a second target domain adjacent to said first target domain, said method comprising a) hybridizing a first nucleic acid containing at least one covalently attached electron donor moiety to said first target domain, b) hybridizing a second nucleic acid containing at least one covalently attached electron acceptor moiety to said second target domain, wherein at least one of said electron donor and electron acceptor moieties is attached to a polydentate πbonucleoside, and c) detecting electron transfer between said electron donor and acceptor moieties while said first and second nucleic acids are hybridized to said first and second target domains as an indicator of the presence or absence of said target sequence in said nucleic acid sample 21 A method according to claim 19 or 20, wherein both of said electron donor and acceptor moieties are attached to polydentate nucleosides 22 A method according to claim 19 or 20, wherein the other of said electron donor and acceptor moieties is attached via a terminal base 23 A method according to claim 19 or 20, wherein the other of said electron donor and acceptor moieties is attached at the 2' or 3' position of a nbose of the nbose- phosphate backbone 24 A method according to claims 19 and 20 wherein at least one of said electron donor moiety and said electron acceptor moiety is a transition metal complex 25 A method according to claim 19 and 20 wherein at least one of said electron donor moiety and said electron acceptor moiety is an electrode 26 A method according to claim 19 and 20 wherein at least one of said electron donor moiety and said electron acceptor moiety is an organic electron donor or acceptor 27 A method for making a nucleic acid with an electron transfer moiety attached at the 3' terminus of said nucleic acid, said method comprising a) attaching a polydentate nucleoside to control pore glass, and b) adding phosphoramidite nucleosides to the 5' terminus of said modified nucleoside to form a nucleic acid 28 A method for making a nucleic acid with an electron transfer moiety attached at the 5' terminus of said nucleic acid said method comprising a) attaching a nucleoside to control pore glass, b) adding phosphoramidite nucleosides to the 5' terminus of said nucleoside to form a nucleic acid, and c) adding a polydentate nucleoside to the 5' terminus of said nucleic acid 29 A method for making a nucleic acid with an electron transfer moiety attached at an internal nucleoside, said method comprising a) attaching a nucleoside to control pore glass, b) optionally adding phosphoramidite nucleosides to the 5' terminus of said nucleoside to form a nucleic acid, c) adding a polydentate nucleoside to the 5' terminus of said nucleic acid, and d) adding phosphoramidite nucleosides to the 5' terminus of said polydentate nucleoside 30 A method for making a nucleic acid with electron transfer moieties attaohed at the 3' and 5' terminus of said nucleic acid, said method comprising a) attaching a first polydentate nucleoside to control pore glass, b) adding phosphoramidite nucleosides to the 5' terminus of said polydentate nucleoside to form a nucleic acid, and c) adding a second polydentate nucleoside to the 5' terminus of said nucleic 31 A method according to claim 27, 28, 29 or 30, wherein said polydentate nucleoside further comprises a bound transition metal 32. A method according to claim 27, 28, 29, or 30, further comprising the step of cleaving said nucleic acid from said support.

Inventors: MEADE THOMAS J, WELCH THOMAS W, MEADE, THOMAS, J., WELCH, THOMAS, W.
Applicants: CALIFORNIA INST OF TECHN, CALIFORNIA INSTITUTE OF TECHNOLOGY
Application year: 1997
Main classification: C07H19
IPC codes: C07H19, C07H21, C07H23, C12Q1