CA2178618A1 - Nucleic acid mediated electron transfer - Google Patents

Abstract

The present invention provider 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. There complexes possess unique structural features which enable the use of an entirely new class of bioconductors and photoactive probes.

Description

Wo 95~15971 ~ 21 7 ~ ~ 1 8 PCT/US94/13893 .
NUCLEIC ACID M~nTA~r~n ELECTRON TRANSFER
FIELD OF THE INVENTION
The present invention is directed to electron transfer via nucleic acids. Nore particularly, the invention is 5directed 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 .
BA~K~cOuNL) 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 modif ied nucleic acids themselves, and their uses. Such modified nucleic acids are particularly useful as biocnn~l~r~ors and photoactive nucleic acid probes.
The detection of specific nucleic acid seS~uences is an important tool for diagnostic medicine and molecular biology research. Gene probe assays currently play roles wo9S/15971 ' ! ~ i- 2 1 786 1 8 PCr/~US94/138s3 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 5 transplantation, in matching tissue or blood samples for forensic medicine, and for exploring homology ~mong genes from different species.
Ideally, a gene probe assay should be sensitive, 6pecific and easily automatable (for ~ review, see l0 Nickerson, Current Opinion in Biotechnology 4: 48-Sl (1993~ ) . The requirement for sen5itivity (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 15 to amplify exponentially a specific nucleic acid ~;equence before analysis (for a review, see Abramson et al., Current Opinion in Biotechnology, 4:41-47 tl993)) .
specificity, in contrast, remains a problem in many currently available gene probe assays. The extent of 20 molecular complementarity between probe and target def ines the specif icity of the interaction . Variations ln 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 25 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 30 difficult using traditional technology, 6ince small variations in the reaction conditions will alter the hybridization. New experimental techniques for mismatch detection with 6tandard probes include DNA ligation assays where single point mismatches prevent ligation WO 95/15971 ` '; i (' 2 ~ 7 8 6 1 8 PCT/US9~/13893 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 lackiny. Such assays generally rely on the hybridization of a 1 Al~ d probe to a target c~ e followed by the ~;eparation of the unhybridized free probe. This 5eparation is generally achieved by gel electrophoresis or solid phase capture and washing of the target DNA, and is generally guite 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-thluu ~
automatable electrophoretic and other separation technigues. The other involves the development of non-~eparation 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 ~-h~mi l~min~scence.
(Okwumabua et al. Res. Nicrobiol. 143:183 (1992)).
Unfortunately, the reliance of this approach on a chemill~minesc~nt substrate known for high background photon emission ~uggests this assay will not have high pecificity. EPO ~pplication number 86116652.8 describes an attempt to use non-radiative energy transfer from a donor probe to an acceptor probe as a e - detection 5cheme. However, the fluorescence energy transfer i5 greatly influenced by both probe topology and topography, and the DNA target itself is capable of significant energy qll~nching, resulting in considerable variability. Therefore there is a need for DNA probes which are specific, capab~e of detecting Wo 9~/15971 ~ 2 1 7 8 6 1 8 PCT/US94/13893 target mismatches, and capable of being incorporated into an automated sy5tem for 8equence identification.
As outlined above, molecular biology relies guite heavily on modified or labelled oligonucleotides for 5 traditional gene probe assays (Oligonucleotide Synthesis: A Practical Approach. Gait et al., Ed., IRL
Press: Oxford, UX, 1984; oligo~t~clPotides and Analogues:
A Practical Approach. Ed. F. Eckstein, Oxford University Press, 1991). As a result, 5everal techniques currently 10 exist for the synthe5i5 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 modif ication at either 15 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 deoxyribo- ~nd ribonucleosides which contain amino groups at the 2' or 20 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)).
Z5 In addition, oligonucleotides may be synthesized with

2'-5' or 3'-5' phosrhoAmide 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, 30 F. Eckstein, Ed. Oxford University Pres5 (1991) ) .
The modification o~ 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-specif ic cleavage .

~ Wo9S/1597~ 2 ~ 78 6 1 8 PCI/rJS94/13893 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 sltered nucleotides may 5 then provide reactive sites for the ~tta~ t of immunological or other labels such as biotin. tGilliam et al., Anal . Biochem. 157: l99 ~1986) ~ . Another example uses ruthenium derivatives which intercalate into DNA
to produce photol~lmin~ nre under defined conditions.
(Friedman et al., J. Am. Chem. Soc. 112: 4960 (1990) ) .
In the second category, there are a number of examples of ~ _ul.ds covalently linked to DNA which subsequently cause DNA chain cleavage. For example 1,10-phenanthroline has been coupled to single-stranded oligothymidylate via a linker which results in the cleavage of poly-dA oligonucleotides in the presence of Cu2+ and 3-mercaptopropionic acid (Francois et al., Biochemistry 27:2272 (1988) ) . Similar experiments have been done for EDTAI-Fe(II) (both for double stranded DNA
(80utorin et al., FE85 Lett. 172:43-46 (1986) ) and triplex DNA (Strobel et al., Science 249:73 (1990)), porphyrin-Fe(III) (Le Doan et al., Biochemistry 25:6736-6739 (1986) ), and 1,10-phenanthronine-Cu(I) (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 porphyrins resulted in DNA E;trand cleavage, and base oxidation or cross-linking of the DNA under very ~pecific conditions (Le Doan et al., Nucleic Acids Res.
15:8643 (1987) ) .
Other work has focused on chemical modification of heterocyclic bases. For example, the atf~ o~ an inorganic coordination complex, Fe-EDTA, to a modified internal base resulted in cleavage of the DNA after hybridi~ation in the presence of dioxygen (Dreyer et WO951159?1 j ~ 2 1 78 6 1 8 PCTNsg4/l38s3 .

al., Proc. Natl. Acad. sci. T~SA 82:968 t1985) ) . A
ruthenium ~1 has been coupled successfully to an internal base in a DNA octomer, with retention of both the DNA hybridization capabilities as well as the 5 :~e. L- ~,scopic properties of the ruthenium label ~Telser et al., J. Am. Chem. Soc. 111:7221 tl989) ) . Other experiments have successfully added two separate spe- L osco~ic labels to a single double-stranded DNA
molecule (Telser et al., J. Am. Chem. Soc. 111:7226 10 (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 trans~er.
To this end, intramolecular electron transfer in 15 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 ~ignificant rates (see Bowler et al., Progress in 20 Inorganic Chemistry: Bioinorganic Chemistry, Vol. 38, Ed. Stephen J. Lippard (1990). In addition, the selective modification of metalloenzymes with transition metals has been accomplished and technigues to monitor electron transfer in these systems developed. For 25 example, electron transfer proteins such as cytochrome c have been modified with ruthenium through att~~ ~
at several histidines and the rate of electron trans~er from the heme Fe2~ to the bound Ru1i measured. The results ~uggest that electron transfer "tunnel" pathways may 30 exist. (Baum, Chemical & Engineering News, February 22, 1933, 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 nondiscriminatory reactions 35 with the exterior solvent, was "wired" to transform ~ Wo 95/15971 ~ ~ 2 t 7 8 6 1 8 Pc r~uSs~3893 these systems from electrical insulators into electrical conductors tHeller, Acc. Chem. Res. 23:128 (1990)).
There are ~ few reports of photoinduced electron transfer in a DNA matrix. In these systems, the 5 electron donors ~nd 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 f 1UOI escel~ce of certain quaternary 10 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 dif f iculty of determining the electron transfer ~ n~cm is found in work done with some photoexcitable ruthenium compounds. Early work suygested that certain ruthenium _ _u--ds either r~ndomly 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 _ul ds 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 : '-, such AS cobalt, chromium or rhodium . '~
were added to certain DNA-associated ruthenium electron donor -~ '~. (Pur~gganan et al., Science 241:1645 (1988); Orellana et al., Photochem. Photobiol. 499:54 (1991); 8run et ~1., J. Am. Chem. Soc. 113:8153 (1991);
Davis, Chem.-Biol. Interactions 62:45 (1987); Tomalia et al., Acc. Chem. Res., 24:332 (1991)). Upon addition of these variou5 electron acceptor ~ /o~nds, which .. _ _ _ _ . .. .. _ _ _ _ _ _ _ _ _ W095/159?1 2 ~ 786 1 8 Pcr/USs4/l3893 randomly bind non-covalently to the helix, quPnrhin~ of the photoexcited state through electron transfer was detected. The rate of q~Pnrh;n l was dependent on both the individual electron donor and acceptor as well as 5 their cu,.cenL ~.tions, 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 10 species, and suggest that the sugar-phosphate backbone of DNA, and possibly the solvent medium ~7uLLuullding the DNA, play a significant role in the electron transport.
(Purugganan et al., Science 241:1645 tl988)). In other work, the authors stress the dPrPn~lPnc e of the rate on 15 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).
20 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. ~he rate of electron trans~er q~lPnrhing indicated a close contact of the donor and the 25 acceptor, and the system also exhibits Pnh~- -nt 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 30 transfer for r.u., cuv~lently bound donors and acceptors is several orders of magnitude less than is seen in free ~;olution .

~ Wo 9S/15971 ~ 2 1 7 8 6 1 8 PCT/US94/13893 An important stimulus for the deYelopment of long distance electron transf er systems is the creation of synthetic light harvesting systems. Work to date suggests that an artificial light harvesting system 5 contains an energy transfer complex, an energy migration complex, an electron transfer complex ~nd an electron migration complex (for a topical review of this area, see Ch~miC~l & Engineering News, ~arch 15, 1993, pages 38-48). Two types of molecules have been tried: a) long lo 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 15 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 20 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 25 transfer occur. Polymers that are sufficiently rigid often significantly interfere with the electron transfer --- Ani~m or are quite difficult to synthesize.
Thus the devel~, L of an electron transfer system which is ~ufficiently rigid, has covalently attached 30 electron transfer species At defined intervals, is easy to synthesize and does not appreciably interfere with the electron transfer -echAniqm would be useful in the development of artificial light harvesting systems.

wo 9~/15971 ' ` ` 2 1 7 8 6 ~ 8 PCr/USs4/138s3 In conclusion, the random distribution and mobility of the electron donor and acceptor pair5, coupled with potential short distances between the donor and acceptor, the loose and presumably reversible 5 a~sociation of the donors and acceptors, the reported dep~n~n- e on solvent and broad putative electron pathways, and the disruption of the DNA bLLU~ ~UL~ of intercalated ~u.,ds rendering normal base pairinq impossible all serve as pronounred limitations of long l0 range electron transfer in a DNA matrix. Therefore, a method for the production of rigid, covalent atta ' rt of electron donors and acceptors to provide minimal perturbations of the nucleic acid structure and retention of its ability to base pair normally, is 15 desirable. The present invention serves to provide such a system, which allows the dev~ t of novel bioconductors and diagnostic probes.
SUMMARY OF THE INVENTION
The present invention provides for the selective 20 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 25 positions. The resulting complexes rep~esenL a series of new derivatives that are biomolecular templates capable of transferring electrons over very large distances at ~LLr. ly fast rates. These complexes possess uniS~ue bLLU~ LUL~l features which enable the use 30 of an entirely new class of biocondu. Lors and diagnostic probes .
Accordingly, it is an object of the invention to provide a single stranded nucleic acid which has both an WO 95/15971 ' PCT/IIS94/13893 ~78618 electron donor moiety and an electron acceptor moiety covalently attached thereto. These moieties are attached through the ribose phosphate or analogous barl~hon~ o~
the nucleic acid. The single stranded nucleic acid is 5 capable of hybridizing to a complementary target 6e~u~llce in a single stranded nucleic acid, and transferring electrons between the donor and acceptor.
It is a further object of the present invention to provide for a nucleic acid probe which can detect base-o pair mismatches. In this Dmho~ir-r-, the single stranded nucleic acid with a covalently attached electron donor ~nd electron acceptor moiety is hybridized to a complementary tarqet sequence in a single stranded nucleic acid. When the region of 15 hybridization contains at least one base pair mismatch, the rate of electron transfer between the donor moiety and the ~cceptor moiety is decreased or eliminated, as compared to when there is perfect complementarity between the probe and target sequence.
20 It is an additional object o~ the present invention to provide a complex which contains a first single stranded nucleic acid with at least one electron donor moiety and a second single stranded nucleic acid with at least one electron acceptor moiety. As with the other Dmhor3i~ r~s 25 of the present invention, the moieties are covalently linked to the ribose-phosphate backbone of the nucleic acids .
In one aspect of the present invention, the first and Lecond single stranded nucleic acids are capable o~
30 hybridizing to each other to form a double stranded nucleic acid, and of trans~erriny electrons between the electron donor moiety and the electron acceptor moiety.

WO 95/15971 ~ ~ 2 1 7 8 6 1 8 PCTIUS94/13893 In another aspect of the present invention, a target sequence in a single stranded nucleic acid comprises at least first and second target domains, which are directly adjacent to one another. The first single 5 stranded nucleic acid hybridizes to the first target domain and the second single stranded nucleic acid hybridizes to the second target domain, such that the first and second single &tranded nucleic acids are adjacent to each other. This resulting hybridization 10 complex is capable of tran5ferring electrons between the electron donor moiety and the electron acceptor moiety on the f irst and second nucleic acids .
In another aspect of the present invention, a target sequence in a single stranded nucleic acid comprises a 15 first target domain, an intervening target domain, and a second target domain. The intervening target domain compri~es one or more nucleotides. The first and second single stranded nucleic acids hybridize to the first and second target domains. An intervening nucleic acid 20 comprising one or more nucleotides hybridizes to the target intervening domain such that electrons are capable of being transferred between the electron donor moiety and the electron acceptor moiety on the first and ~iecond nucleic acids.
25 The invention also provides for a method of making a single stranded nucleic acid containing an electron transfer ~oiety covalently attached to the 5' terminus of the nucleic acid. The method comprises incorporating a ~odified nucleotide into a growing nucleic acid at the 30 5' position to form a modified single stranded nucleic acid. The ~odified single stranded nucleic acid is then hybridized with a complementary single stranded nucleic acid to form a double stranded nucleic acid. The double stranded nucleic acid is reacted with an electron 35 transfer moiety such that the moiety is covalently WO95/15971 ~ 2 1 786 t 8 Pcr/uss4ll38s3 attached to the modif ied single stranded nucleic acid .
The ~odified single stranded nucleic acid containing the electron transfer moiety is separated from the complementary unmodified single stranded nucleic acid.
S The present invention also provides ~ method for making a single ~tranded nucleic acid containing an electron transfer moiety covalently attached to an internal nucleotide. The method comprises creating a nucleotide dimer joined by a rhocrhoramide bond and incu-~u-e~ting l0 said nucleotide dimer into a growing nucleic acid to form a modified single stranded nucleic acid. The modified single stranded nucleic acid is then hybridized with a complementary single stranded nucleic acid to form a double stranded nucleic acid. The double 15 stranded nucleic acid is reacted with an electron transfer moiety such that the moiety is covalently attached to the modified single stranded nucleic scid.
The modified single 5tranded nucleic acid containing the electron transfer moiety is separated from the 20 complementary unmodified single stranded nucleic acid.
Another aspect of the present inYention provides a method of detecting a target sequence. The method comprises cre2ting a single stranded nucleic acid with an electron donor moiety and an electron acceptor moiety 25 covalently attached. The single stranded nucleic acid containing the electron transfer moieties is then hybridized to the target sequence, and an electron transfer rate determined between the electron donor and the electron scceptor.
BRIEF DESC~I~.lON OF THE ~)RAWINGS
Figure l illustrates possible orientations of electron donor tEDM) and electron acceptor (EAM) moieties on a single stranded nucleic acid.

WO 95/15971 ~ 2 1 7 8 6 1 8 PCTIUS94/13893 Figure 2 illustrates orientatiOnS of electron transfer moieties EDI~ and EA~ on two adjacent single stranded nucleic acids. These orientations also apply when the two probes are separated by an intervening seguence.
5 Figure 3 illustrates a series of amino-modified nucleoside precursors prior to incol~o~ation into an oligonucleotide .
Figures 4A and 4B depict the structure of electron transfer moieties. Figure 4A depicts the gener21 10 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.
DETAILED DESCRIPTION
lS 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 20 cases, as outlined below, a nucleic acid may have an analogous backbone, comprising, for example, rhosphoramide tBeaucage et al., Tetrahedron 49 tl0~ :1925 tl993~ and references therein; Letsinger, J. Org. Chem.
35:3800 tl970~ ~, phosphorothioate, rhosph~rodithioate, 25 O-methylrhophc~roamidite linkages tsee Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press~, or peptide nucleic acid linkages tsee Egholm, J. Am. Chem. Soc. 114:1895 tl992~;
Meier et al., Chem. Int. Ed. Engl. 31:1008 tl992~;
30 Nielsen, Nature, 365:566 tl993~ ~ . The nucleic acids may be single stranded or double stranded, as specified.
The nucleic acid may be DNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ~ wo 9S/15971 2 1 7 8 6 1 8 PCTnJSg4/13893 ribo-nucleotideS, and any combination of uracil, adenine, thymine, cytosine and guanine. In some instances, e.g. in the case of an "interveniny nucleic acid", the term nucleic acid refer6 to one or more S 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 lO 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. Generally, electron transfer lS moieties contain transition metals as ~nel,Ls, but not always.
The term "target sequence" or grammatical eguivalents herein means a nucleic acid sequence on a single strand 20 of nucleic acid. The target sequence may be a portion of a gene, a regulatory sequence, genomic DNA, mRNA, or others. It may be any length, with the understanding that longer sequences are more specific. Generally 8pPAkinq~ this term will be understood by those skilled 25 in the art.
The probes of the present invention are designed to be complementary to the t~rget sequence, such that hybridization of the target sequence and the probes of the present invention occurs. As outlined below, 'chis 30 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 35 hybridization can occur under even the least stringent Wo 95/15971 ~ ? ~ 7 ~ 6 ~ ~ PCT/US94/13893 o~ hybridization conditions, the sequence is not a complementary target sequence.
The terms "~irst target domain" and "second target domain" or grammatical equivalent5 herein means two 5 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 ~irst and second target domains may be separated by an intervening target domain. The terms "f irst" and lO "second" are not meant to confer an orientation o~ the sequences with respect to the 5'-3' orientation of the target sequence. For example, Aqsu~ning a 5'-3' orientation of the complementary target sequence, the first target domain may be located either 5' to the 15 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 20 of long distance electron transfer through a nucleic acid matrix. The present invention provides ~or the precise placement o~ electron trans~er donor and acceptor moieties at predetermined sites on a single stranded or double stranded nucleic acid. In general, 25 electron transfer between electron donor and acceptor moieties in a double helical nucleic acid does not occur nt ~n appreciable rate unless nucleotide base pairing exists in the sequence between the electron donor and acceptor in the double helical structure.
30 This differential in the rate o~ 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 trans~er moieties are covalently bound to the bal-kh~ne of a nucleic acid, the electrons ~ WO 95/15971 ~ ! ' 2 1 7 8 ~ ~ 8 PCrJV594/13893 putatively travel via the ~r-orbitals of the stacked base pairs of the double stranded nucleic acid. The electron transfer rate is dPrpn~lpnt on sever21 factors, including the distance between the electron donor-acceptor pair, 5 the free energy (~G~ of the reaction, the reorganization energy (~), the contribution of the intervening medium, the orientation and electronic coupling of the donor and acceptor pair, and the l.ydLo~-n bonding between the ba~es. The latter confers ~ dPrPn~Pnce on the ~ctual o nucleic acid sequence, since A-T pairs contain one less IIYdLOg~:II bond than C-G pairs. However, this ~u,2~.- e dPrPn~lPnre is overshadowed by the determination that there is a measurable difference between the rate of electron transfer within a DNA ba5e-pair matrix, and the 15 rate through the ribose-phosphate backbone, the ~olvent or other electron tunnels. Thi5 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 20 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 unhybridized probe with the rate for hybridized probes.
25 In one Pmho~ nt, the present invention provides for novel gene probes, which are useful in molecular biology and diagnostic medicine. In this Pmho~lt- ~, single stranded nucleic acids having a predetermined sequence and covalently attached electron donor and electron 30 acceptor moieties are synthP~i7ed. The sequence is selected based upon a known target ~equence, ~uch 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 35 appreciable and detectable rate. Thus, the present invention has broad general use, as a new form of .

Wo 95/15971 ~ ~ 2 ~ 7 ~ 6 ~ 8 PCT/USs4~13893 labelled gene probe. In addition, since detectable electron transfer in unhybridized probes is not appreciable, the probes of the present invention allow detection of target sequences without the removal of 5 unhybridized probe. Thus, the present invention is uniquely suited to automated gene probe assays.
The present invention also finds use ~s a unique methodology for the detection of mutations in target nucleic acid seyue~.ces. As a result, if a single lO stranded nucleic ~cid containing electron transfer moieties is hybridized to a target sequence with a mutation, the resulting perturbation of the base pairing of the nucleotides will measurably affect the electron transfer rate. This is the case if the mutation is a 15 substitution, insertion or deletion. Accordingly, the present invention provides for the detection of mutations in target se~uences.
Thus, the present invention provides for extremely specific and sensitive probes, which may, in some 20 embodiments, detect target sequences without removal o~
unhybridized probe. This will be useful in the generation of automated gene probe assays.
In an alternate ~ho~ t double stranded nucleic acids have covalently attached electron donor and electron 25 acceptor moieties on opposite strands. Such nucleic acids are useful to detect successful gene amplification in polymerase chain reactions (PCR). For example, if one of the two PCR primers contains a 5' terminally attached electron donor, and the other contains a 5 ' 30 t~rm;n:~l ly 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 WO9S/15971 ~c 2 1 786 1 ~3 PCT/Usg4~138s3 s~lccessful 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 5 amplified double stranded DNA is not nec~s~ry, as outlined above for probe ~y~ C~S which contain electron transfer moieties.
In another DmhoAi~ t the present invention provides for double stranded nucleic acids with covalently attached lO electron donor and electron acceptor moieties to serve as bioconductors or "molecular wire". The electron tr~nsport may occur over distances up to and in excess of 2~A per electron donor and acceptor pair. In addition, the rate of electron transfer is very fast, 15 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 20 creating bioconductors. These bioconductors are useful in a large number of applications, including traditional applications for conductors such as mediatprs for electrochemical reactions and processes.
In adaition, these bioconductors may be useful as probes 25 for photosynthesis reactions as well as in the construction of synthetic light harvesting systems. The current models for the electron transfer _ _rent of /m artificial light harvesting system have several problems, ~s outlined above, including a ~l~pDn~Dnre on 30 ~iolvent polArity and composition, and a lack of ~iufficient rigidity without arduous synthesis. Thus the present invention is useful ~s both a novel form of bioconductor as well as a novel gene probe.

Wo 95/15971 ~ 2 ~ 7 8 ~ ~ 8 Pcrlus94ll3893 In addition, the present invention provides a novel method for the site specific addition to the ribose-phosphate b~r~hone of a nucleic acid of electron donor and electron ncceptor moieties to a previously modified 5 nucleotide.
In one embodiment, the electron donor and acceptor moieties are added to the 3' and/or 5' termini o~ the nucleic acid. In alternative Pmho~ s, the electron donor and acceptor moieties are added to the bar~ honP
lO of one or more internal nucleotides, that is, any nucleotide which is not the 3 ' or 5 ' terminal nucleotide. In a further Pmhorlir~nt, the electron donor and acceptor moieties are added to the backbone of both internal and terminal nucleotides.
15 In a preferred PmhoAi- L, the transition metal electron transfer moieties are added through a ~,uce~uLe which utilizes modified nucleotides, preferably amino-modified nucleotides. In this PrhoAit-nt, the electron trans~er moieties are added to the sugar phosphate barl~honP
20 through the nitrogen group in phosphoramide linkages.
The modified nucleotides 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 nucleotide.
25 Molecular r- ' ~nics calculations indicate that ~LLuL~ations due to the modification of the terminal nucleotides of nucleic acids are minimal and ~latson-Crick base pairing is not disrupted (unpublished data using Biograf from Molecular Simulations Inc., San 30 Diego, CA). Accordingly, in one P~nhodi~ L, modified nucleotides are used to add an electron transfer moiety to the 5 ' terminug of a nucleic acid . In this embodiment, the 2 ' position of the ribose of the deoxyribo- or ribonucleoside is modif ied prior to the ~ wo 95/15971 -~ - ~ 2 1 7 8 6 1 8 PCT/US94/13893 addition of the electron transfer species, leaving the

3 ' position of the ribose unmodified for subseguent chain at~Arh~-r-. In a preferred ~mhs~l;r-nt, an amino group is added to the 2 ' carbon of the sugar using S estAhl~hPd rhPmir~l techniques. (Imazawa et al., J.
org. Chem., 44:2039 (1979); Hobbs et ~1., J. Org. Chem.
42(4) :714 (1977); Verheyden et al, J. Org. Chem.
36 (2) :250 (1971) ) .
Once the modified nucleotides are prepared, protected 10 ~nd activated, they may be incorporated into a growing oligonucleotide by standard synthetic technigues (Gait, Oligonucleotide Synthesis: A Practical Approach, IRI, Press, Oxford, UK 1984; Eckstein) as the 5' terminal nucleotide. This method therefore allows the addition 15 of a transition metal electron transfer moiety to the 5 ' terminus of a nucleic acid .
In an alternative ~mho~lir-nt, the 3' terminal nucleoside is modified in order to add a transition metal electron transfer moiety. In this embodiment, the 3' nucleoside 20 is modified at either the 2' or 3' carbon of the ribose sugar. In a preferred embodiment, an amino group is added to the 2 ' or 3 ' carbon of the sugar using est~hl i~hPr9 chemical techniques (Imazawa et al., J. Org.
Chem., 44:2039 (1979); Hobbs et al., J. Org. Chem.
25 42(4) :714 (1977); Verheyden et al. J. org. Chem.
36(2) :250 (1971) ) .
The above procedures are applicable to both DNA and RNA
derivatives as ~hown in f igure 3 .
The amino r-'ified nucleotides made as described above 30 are converted to the 2' or 3' modified nucleotide tr;rhss~h~te form using standard biochemical methods (Fraser et al., Proc. Natl. Acad. Sci. USA, 4:2671 (1973) ) . one or more modified nucleosides are then wo 95115971 2 t 7 8 6 1 8 PCTIUS94113~93 attached at the 3' end using standard molecular biology techniques such as with the use of the enzyme DNA
polymerase I or terminal deoxynucleotidyltr~nsferase (Ratliff, Terminal deoxynucleotidyltransferase. In The Enzy;nes, Vol 14A. P.D. Boyer ed. pp 105-118. Academic E'ress, San Diego, CA. 1981).
In other ~mho~i- ts, the transition metal electron transfer moiety or moieties are added to the middle of the nucleic acid, i.e. to ~n internal nucleotide. This may be accomplished in three ways.
In a preferred ~mhor7i nt, an oligonucleotide is amino-_odified at the 5' terminus as described above. In this t, oligonucleotide synthesis simply extends the 5' end from the amino-modified nucleotide using standard techniques. This results in an internally amino modified oligonucleotide.
In an alternate embodiment, electron transfer moieties are added to the backbone at a site other than ribose.
For example, phosphoramide rather than phosphodiester linkages can be used ~s 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, Acc. Chem. Res. 23:128 tl990~; Schuhmann et al.
J. Am. Chem. Soc. 113:1394 (1991)), the rhosphoramidite internucleotide link has been reported to bind to complementary polynucleotides and is stable (Beaucage etal., supra, andreferencestherein; Letsinger, supra;
Sawai, supra; Jager, Biochemistry 27:7237 (1988) ) . In this ~r~ ;r~nt, dimers of nucleotides are created with rhosrhnramide linkages at either the 2 ' -5 ' or 3 ' -5 ' positions. A preferred embodiment utilizes the 3'-5' position for the phosphoramide linkage, such that ~ WO95115971 , 2 1 78 6 1 8 PCT~lJS94/13893 structural disruption of the subsequent Watson-Crick h.~ r~;ring is minimized. These dimer units are in~ o~oL~Ited into a growing oligonucleotide chain, as above, at defined intervals, as outlined below.
5 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~ termin21 nucleotide, thus eliminating the need for the extra ~teps reguired to 10 produce the 3 ~ terminally labelled nucleotide.
In a further r~mhorli- t for the modification of internal residues, 2' or 3~ modified nucleoside triphosphates are generated using the techniques described above for the 3 ' nucleotide modif ication . ~he modif ied nucleosides 15 are inserted internally into nucleic acid using standard molecular biological techniques for labelling DNA and RNA. Enzymes used for said lAhr~l 1 i n5 include DNA
polymerases such as polymerase I, T4 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase, reverse 20 transcriptase and RNA polymerases such as E. coli RNA
polymerase or the RNA polymerases from phages SP6, T7 or T3 tShort Protocols in Molecular 8iology, 1992.
Ausubel et al. Ed. pp 3.11-3.30).
In a preferred embodiment, the electron donor and 25 acceptor moieties are attached to the modif ied nucleotide by methods which utilize a unique protective hybridization step. In this F ' ~ , the modified ~;ingle strand nucleic acid is hybridized to an unmodified complementary sequence. This blocks the sites 30 on the heterocyclic bases that are susceptible to attack by the transition metal electron transfer species. The exposed amine or other ligand at the 2 ' or 3 ' position o~ the ribose, the phosphoramide linkages, or the other linkages useful in the present invention, are readily _ _ . . , . _ . . , _ .

WO9S/15971 ` 2 1 7~6 ~ 8 PcrluS94/13893 modified with a variety of transition metal complexes with techniques readily known in the art (see for example Millet et al, in Metals in 8iological Systems, Sigel et al. Ed. Vol. 27, pp 223-264, Marcell Dekker 5 Inc. New York, 1991 and Durham, et al. in ACS Advances in Chemistry Series, Johnson et al. Eds., Vol. 226, pp 180-i93, American Chemica1 Society, Washington D.C.; and 25eade et al., J. Am. Chem. Soc. 111:4353 (1989) ) . After ~ ces~ful addition of the desired metal complex, the 10 modified duplex nucleic acid is separated into single strands using techniques well known in the art.
In a preferred I mho~ L, single stranded nucleic acids are made which contain one electron donor moiety and one electron acceptor moiety. The electron donor and 15 electron acceptor moieties may be attached at either the S ' or 3 ' end of the single stranded nucleic acid .
Alternatively, the electron transfer moieties may be attached to internal nucleotides, or one to an internal nucleotide and one to a terminal nucleotide. It should 20 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 tDrmin~1 nucleotides may be utilized in this emhodiment.
25 In an alternate preferred rmhorli- ~ single str~nded 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 30 deletion of a nucleotide or nucleotides, results in inC~L~e ~ base pairing in a hybridized double helix of nucleic ~cid. 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 WO 9SIIS971 ` " ' ' 2 1 7 8 6 1 8 PCT/US94113893 electron transfer will be eliminated or reduced such that a change in the relative rate will be seen.
Therefore, in this Pml~orl;-~~t~ the electron donor moiety i8 attached to the nucleic acid at a 5 ' position from 5 the mutation, and the electron acceptor moiety i8 attached at a 3' position, or vice versa.
In this Pmhorl;- I 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 10 mismatches. If the complementary target nucleic acid contains a mutation, electron transfer is reduced or eliminated . To act as a control, the modif ied single stranded nucleic acid may be radio- or f luorescently labeled, such that hybridization to the target sequence 15 may be detected, according to traditional molecular biology techniques. This allows for the determination that the target seguence exists but contains a substitution, insertion or deletion of one or more nucleotides. Alternatively, single stranded nucleic 20 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 25 transfer through a dou~le stranded nucleic acid helix depends on the nucleotide 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 ~nd bio- ,..d~ ors.
30 Thus, while it is possible to measure rates for distances in excess of 100 nucleotides, a preferred o~ has the electron donor moiety and the electron acceptor moiety separated by at least 3 and no more than 100 nucleotides. More preferably the moieties Wo 95/15971 2 ~ 7 8 6 1 8 PCT/US94/13893 are separated by 8 to 64 nucleotides, with 15 being the most preferred distance.
In addition, it should be noted that certain distances may allow the utilization of different detection 5 systems. For example, the sensitivity of 50me detection systems may allow the detection of e~ ly 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 lO moieties to be farther apart.
In an alternate embodiment, a single stranded nucleic ncid is modified with more than one electron donor or acceptor moiety. For ~xample, to increase the signal obtained from these probes, or decrease the required 15 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 20 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 25 electron transfer species, i.e. either a single transfer moiety or several Or the same trans~er moieties, using the techniques outlined above. Then these partial nucleic acid sequences are ligated together using techniques common in the art, such as hybridization of 30 the individual modified partial nucleic acids to a complementary single strand, followed by ligation with a commercially available ligase.

r WO 9511S971 2 1 7 8 ~i 1 8 PCT/US94/13893 In a preferred ~ l~odir~nt, single 6tranded 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 i5 attached to an internal nucleotide.
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 ~ preferred PmhQdi--~t, a fir5t single stranded nucleic acid is made with on or more electron donor moieties attached. ~ 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 Pmho~i-- t, the complementary target sequence is made up of a f irst target domain and a second target domain, where the first and second sequences are directly adjacent to one another. In this PmhQ~lir-nt, 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 C-~L~S~Yr~ ;n5 electron transfer species, binds to the second target domain. The - 30 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 .

wog5/lss7l ; 21 786~ ~ PCr~Ss4/l3893 In the desiyn of probes comprised of two single stranded nucleic acids which hybridize to adjacent first and second target seguences, several factors should be considered. These factors include the distance between 5 the electron donor moiety and the electron acceptor moiety in the hybridized form, and the length of the individual 6ingle stranded probes. For example, it may be desirable to synthesize only 5' terminally l;-hPlle~
probes. In this case, the single stranded nucleic acid lO 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 nucleotides, then the first probe may be 15 nucleotides 15 long.
In one aspect of this omh~ r-, the two single stranded nucleic acids which have hybridized to the adjacent first and second target domains are ligated together prior to the electron transfer reaction. This 20 may be done using standard molecular biology technigues utilizing a DNA ligase, such as T4 DNA ligase.
In an alternative PmhQ~ t, the complementary target seguence will have a first target domain, an intervening target domain, and a second target domain. In this 25 omho~i~~ L, 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 30 cv~ o1 tling electron transfer species, binds to the eecond target domain. When an intervening single stranded nucleic acid hybridizes to the intervening target seguence, electron transfer between the donor and acceptor is possible. The intervening seguence may be 35 any length, and may comprise a single nucleotide. Its _ _ _ . _ _ _ _ _ _ _ . _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ WO 95/15971 .. . 2 1 7 ~ 6 ~ 8 PCT/US94/13893 length, however, should take into consideration the desirable distances between the electron donor and acceptor moietie5 on the first and second modified nucleic acids. Intervening seguences of lengths greater S than 14 are desirable, 5ince the intervening se4uence is more likely to remain hybridized to form a double stranded nucleic acid if longer intervening 5equences Are used. The presence or absence of an intervening ~equence can be used to detect insertions and deletions.
lO In one aspect of this Pmho~i~ L, the first single stranded nucleic acid hybridized to the f irst 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 15 be ligated together prior to the electron transfer reaction. This may be done using 5tandard molecular biology techniques. For example, when the nucleic acids are DNA, a DNA ligase, such as T4 DNA ligase can be used .
20 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 25 mRNA, a restriction fragment of a plasmid or genomic DNA, among others. One 5killed in the art of molecular biology would understand how to construct useful probes for a variety of target seyuenc~s using the present invention .
30 In one e~o~i~~ t, 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 Pmhot?;--nt, the hybridized duplex is capable of transferring at , . _ _ _ _ _ _ _ _ _ _ , . . _ _ . _ . . _ , _ _ _ _ _ _ _ _ , W0 95/15971 ~ , 2 ~ 7 8 6 1 8 PCT/US94/13893 least one electron from the electron donor moiety to the electron acceptor moiety. In a preferred amhQrl;r-r t, the individual single stranded nucleio acids are aligned such that they have blunt ends; in alternative 5 ~-mhQr~i--nts/ the nucleic acids are aligned such that the double helix has cohesive ends. In either ~mhodir~rL, it i8 preferred that there be uninterrupted double helix base-pairing between the electron donor moiety and the electron acceptor moiety, such that electrons may travel lO through the stacked base pairs.
In one bioconductor ~mho~ t, 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 15 be carried on either strand, and in any orientation.
For example, one strand m~y carry only electron donors, and the other only electron acceptors or both strands may carry both.
In one ~rho~ , the double stranded nucleic acid may 20 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 ~ct as both electron donors and acceptors depending on 25 their oxidative state. Thus, an electron donor moiety, after the loss of an electron, may act ~s 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 30 directional transfer of an electron over very long distances may be accomplished. ~or example, a double stranded nucleic acid could contain a single electron donor moiety ~t one end and electron acceptor moieties, of the same or different composition, throughout the 35 molecule. A cascade effect of electron transfer could _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ .. ... . . .. . .

WO95/159~ 786 1 8 p~ sg41l3893 be accomplished in thi5 manner, which may result in exLL. -ly long range transfer of electrons.
The choice of the specif ic electron donor and acceptOr pairs will be influenced by the type of electron 5 transfer mea.uL~ t used; rOr a review, see WinXler et ~Il., Chem. Rev. 92:369-379 (1992). When a long-lived excited state can be prepared on one of the redox site6, direct measuLO ~r~ of the electron transfer rate after photo;n~ ct;~ can be measured, using for example the 10 flash-guench 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 15 of this method is that two electron transfer rates may be measured: the photoinduced electron transfer rates and thermal ele.LL~ hole recombination reactions .
Thus differential rates may be measured for hybridized nucleic acids with perfect complementarity and nucleic 2 0 acids with mismatches .
In alternative ~rho~;r-nts, neither redox site has a long lived excited state, and electron transfer ~easurements depend upon bimolecular generation of a Xinetic intermediate. For a review, see Winkler et al., 25 supra. This intermediate then relaxes to the thermodynamic product via intramolecular electron transfer using a guencher, 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 uslng this method is about 104 per second.

WO 95/15971 ~ , r 2 1 7 8 6 1 8 PCT/US94/13893 Alternative Pmhorlir~nts use the pul5e-radiolytic generation of reducing or oxidizing radicals, which inject electrons into 2 donor or remove electrons from a donor, as reviewed in Winkler et al., supra.
5 Electron transfer will be initiated using electrical, electrochemical, photon (including laser) or chemical activation of the electron transf er moieties . ~hese events are detected by changes in transient absorption or by fluorescence or rhns~h~rescence or lO chemill~m~nPccPnre of the electron transfer moieties.
In the preferred embodiment, electron transfer occurs after photoinduction with a laser. In this Pmhofli- t, electron donor moieties may, after donating an electron, Eierve as electron acceptors under certain circumstances.
15 Similarly, electron acceptor moieties may serve ~s electron donors under certain circumstances.
In a preferred Pmho~i-^nt, DNA is modified by the addition of electron donor and electron acceptor moieties. In an alternative Pmho~;r^nt, RNA is 20 modified. In a further Pmho~ rt, a double stranded nucleic acid for use as a bioconductor will contain some deoxyribose nucleotides, some ribose nucleotides, and a mixture of adenosine, thymidine, cytosine, guanine and uracil bases.
25 In accordance with a $urther 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 ribose ring (2 ' or 3 ' 30 position) or to a nitrogen or sulfur atom as part of a nucleotide dimer linked by a peptide bond, phosphoramidate bond, phosphorothioate bond, ~ Wo 95/15971 2 ~ 7 8 6 1 8 PCr/uss4/l3893 rhosphorodithioate bond or O-methyl rhosrhoramidate bond .
A general formula is representative of a class of donors and acceptors that may be employed i5 shown in figure 5 4A. In this figure, N may be Cd, Mg, Cu, Co, Pd, Zn, Fe, Ru with the most preferred being ruthenium. The groups R1, R2, R3, R4, and R5 may be any coordinating ligand that is capable of covalently binding to the chosen metal and may include ligands such as NH3, 10 pyridine, isonicofinA~;d~, imidazole, bipyridine, 21nd substituted derivative of bipyridine, phenanthrolines and substituted derivatives of phenanthrolines, porphyrins and substituted derivatives o~ the porphyrin family. The structure of ~ ruthenium electron transfer 15 species using bisbipyridine and imidazole as the ligands is shown in figure 4B. Specific examples of useful electron transfer complexes include, but are not limited to, those ~hown in Table l.
TABLE l 2 0 ponors Acce~tors Ru ( bpy ) 2im-NH2-U Ru ( NH3 ) 5-NH2-U
Ru (bpy ) 2im-NH2-U Ru ( NH3 ) 4py-NH2-U
Ru ( bpy) 2im-NH2-U Ru ( NH3) 4im-NH2-U
Where:
25 Ru - ruthenium bpy - bisbipyridine im ~ imidazole py ~ pyridine It i5 to be understood that the number of possible 30 electron donor moieties and electron acceptor moieties is very large, ~nd that one skilled in the art of WO95/15971 ~ 2 ~ ~86 ~ ~ PCTIUS94/13893 ~
electron transfer cG-ro~n~c will be able to utilize a number of - ~ounr7C in the present invention.
In an alternate embodiment, one of the electron transfer moieties may be in the form of a solid support 6uch as 5 zn electrode. When the other electron transfer moiety ~s in solution the 6ystem is referred to as a heterogenous system as compared to a ~ nuus 6ystem where both electron donor and electron trans~er moities are in the same phase.
10 The techniques used in this P~ho~ nL are analogos 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 15 Actuators R., 13-14:180 (1993); and Pishko et al., Anal.
Chem., 63:2268 (1991) ) . In this e~ho~ir ~, it is preferred that a redox polymer such as a poly-(vinylpyridine) complex of Os (bpy) 2Cl be cross-linked with ~n epoxide such as diepoxide to form a redox-20 conducting epoxide cement which is capable o~ stronglybinding 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 25 this P"~ho~i- t. The epoxide cross-linking polymer is then reacted with, for example, an exposed ~mine, 6uch 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 30 the electrode.
In this P~ho~i- L, a single stranded nucleic acid probe containing at least one electron transfer moiety is attached via this redox hydrogel to the surface of an electrode. Hybridization of a target sequence can then 2 1 7 8 6 1 8 PCTNS9~13893 be measured as a function of conductivity between the electron transfer moiety covalently attached to one end of the nucleic acid and the electrode at the other end.
This may be done using eguipment and techniques well 5 known in the art, such as those described in the references cited above.
In similar G"~ho~ nts, two nucleic acids are utilized as probes as described previously. For example, one nucleic acid is attached to a solid electrode, and the 10 other, with a covalently attached electron transfer moiety, is free in solution. Upon hybridization of a target seguence, the two nucleic acids are aligned such that electron transfer between the electron transfer moiety of the hybridized nucleic acid and the electrode 15 occurs. The electron transfer is detected as outlined above, or by use of amperometric, potentiometric or conductometric electrochemical sensors using techniques well known in the art.
The following examples serve to more fully describe the 20 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 25 lllustrative purposes.
EXANPLES
The amino v ';fied monomer units are prepared by Yariation of published ~uc~-lules and are inCoL~UL~ted lnto a growing oligonucleotide by standard synthetic 30 techniques. The procedure i5 applicable to both DNA and P.NA derivatives.

WO 95/15971 2 1 7 8 6 ~ 8 PCT/US94/13893 Examp l e Synthesis of an Oligonucleotide Duplex with ElectrDn Transfer Moieties at the 5' Termini In this example an eight nucleotide double stranded 5 nucleic acid was ~l o~llced, with each single strand having a single electron transfer moiety covalently attached to the 5 ' terminal uridine nucleotide at the 2 ' carbon of the ribose sugar.
Step l: Synthesis of 5'-di(p-methoxyphenyl)methyl ether-2 ' - (trif luoroacetamido) -2 ' -deoxyuridine 2 ' - (trifluoroacetamido) -2 ' -deoxyuridine (2 . 0 g, 5 . 9 mmoles) prepared by minor modification of published ~ocedu~s (Imazawa, supra) was repeatedly dissolved in a minimum of very dry CR3CN and rotary evaporated to 15 dryness and then transferred to inert atmosphere vacuum line and further dried for a period of l 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 20 distilled pyridine and with stirring, 0. 05 equivalents (wt.) of 4-dimethylaminopyridine (DMAP), 1.5 equivalents of triethylamine (TEA) and l . 2 equivalents of 4, 4 ' -dimethoxytrityl chloride (DMTr-Cl) were added to the reaction mixture. The ~Gy.ess of the reaction was 25 monitored by silica gel TLC (98: 2 methylene chloride:methanol, mobile phase). After 30 minutes, an additional 0.5 equivalents each of DMTr-Cl and TEA were added ~md the reaction allowed to proceed for an additional three hours. To this reaction mixture was 30 added ~n equal vo~ume 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 purif ied by flash chromatogra~hy (99: l methylene 2t ~86 1 8 Wo 95/15971 PCT/US94/13893 chloride:methanol, mobile phase), to obtain the 5'-di (p-methoxyphenyl) methyl ether-2 ' - (trif luoroacetamido) -2 ' -deoxyuridine product.
Step 2: 5 ' -2 ' -aminouridine-GCTACGA and 5 ' -2 ' -aminouridine-CGTAGCA
5 '-di (p-methoxyphenyl) methyl ether-2 ' -(trifluoroacetamido)-2'-deoxyuridine was dried under reduced ~es~.uL~ (glass) and dissolved in freshly dried and distilled CH3CN and placed in a 5pecially made lO 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 (amil-o r-t'ified) addition to a 15-30 minute coupling time. The oligonucleotide was cleaved from the 15 column by standard ~Loc~duLl s and purified by C-18 reverse phase HPLC. In this manner 5'-2'-aminouridine-GCTACGA and 5 '-2 ' -aminouridine-CGTAGCA were prepared .
In addition, unmodified complementary strands to both products were made for use in the electron transfer 20 moiety synthesis below.
Step 3: 5'-2'-ruthenium bisbipyridineimidazole-aminouridine-GCTACGA
5 ' -2 ' -aminouridine GCTACGA produced in the previous step was annealed to the complementary unmodified strand 25 using ~tandard techni~lues. All manipulations of the ~ n~Al~l duplex, prior to the addition of the transition metal complex were handled at 4C. In order to insure that the DNA r. ir~Pd Arlr~Al~ during modi~ication, the reactions were performed in lM salt. The 5'-amino 30 modified duplex DNA was dissolved in 0.2 M HEPES, 0.8 M NaCl, pH 6.8 and repeatedly eYacuated on a Schlenk line. Previously prepared ruthenium bisbipyridine carbonate was dissolved in the above buf f er and oxygen was removed by repeated evacuation and purging with Wo 95/15971 ~ 1 7 ~ ~ 1 8 PCrlUS94113893 2rgon 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 ~LesauLe 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-lO 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. l M triethylammonium acetate (TEAC) pH 6Ø The duplex DNA was heated to 60-C for l5 minutes with 50% fnrr-m;tlP 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. l M TEAC, pH 6. 0.
Step 4: 5 ' -2 ' -ruthenium tetraminepyridine-aminouridine-CGTAGCA
~ ' -aminouridine-CGTAGCA ( O . 3~Lm) was dissolved in o . 2 M
HEPES, 0.8 M NaCl buffer, pH 6.8 and ~egA~sPcl on the vacuum line. To a lO ml conical shaped flask Pq-1irpPcl with a stirring bar and septum was slurried Ru(III) tetraaminepyridine chloride (lO ~m), in the same buffer.
In a separate flask, Zn/Hg amalgam was prepared and dried under reduced pressure and the ruthenium(III) solution transferred (via cannulation) to the Zn/~g amalgam. The immediate formation of a clear yellow solution (A,,,,~x = 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 ~ WO 95~15971 r ~ 2 t 7 8 6 1 8 PCT/U594/13893 ~ixture was removed from the vacuum line and a 50 fold excess of cobalt EDTA (~irschner, Inorganic Synthesis (1957J, pp 186) added to the 501ution. The solution was applied to Sephadex G-25 gel filtration column to remove 5 excess ruthenium complex and further purified by reverse ph~se HPLC as described above. The two ruthenium modified nucleotides were annealed by standard techniques and characterized (see Example S).
Example 2 Synthesis of Long DNA Duplexes with Electron Transfer Moleties 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 15 duplex DNA by polymeri2ation of nucleotides off modified primer strands (Saiki et al., Science 239:487 (1988) ) .
Two oligonucleotides 18 bases in length and not complementary to each other are synthesized with amino-modification to the 2'-ribose position o~ the 5' 20 nucleotides, as in example 1.
A series of oligonucleotides of increasing lengths ~;tarting at 40 bases are chemically synthesized using standard chemistry. Each of the PCR templates shares a 5' seguence identical to one modified 18mer. The 3'5 end of the template oligonucleotide 5hares a sequence ry to the other 18mer.
pCR rapidly generates modif ied duplex DNA by the catalysis of 5'-3' DNA synthesis off of each of the nlodified 18mers using the unmodified strand as a 30 template. One hundred nanomoles of each of the two modi~ied 18mers are mixed in 1 ml of an aqueous solution wo 95/15971 2 1 7 8 6 1 8 PCT/US94/13893 containing 2,000 units of Taq polymerase, deoxyribonucleoside triphosphates at 0 . 2 M each, 50 mM
KCl, l0 mM Tris-Cl, p~ 8.8, 1.5 mM ~gCl2, 3 mM
dithiothreitol and 0.l mg/ml bovine serum albumin. One 5 femtomole of the template strand 40 bases in length is added to the mixture. The sample is heated at 94C for one minute for denaturation, two minutes at 55C for AnnnAl ;n~ and three minutes at ~2C for extension. This cycle ic repeated 30 times using an automated thermal l0 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 ~ound Electron Transfer Moieties at Internucleotide Linkaaes of Du~lex DNA
In this example, alternative backbones to phophodiester linkages of oligonucleotides are employed. Functional 20 groups incuL~u~ated into these internucleotide linkages serve as the site for covalent attA~ of the electron transfer moieties. These alternate internucleotide linkages include, but are not limited to, peptide bonds, rhocrhnramidate bonds, 25 rhosphorothioate bonds, phosphorodithioate bonds and O-methylphosphoramidate bonds.
The preparation of peptide nucleic acid (PNA) follows literature ~oct:-luL~s (See Engholm, supra), with the synthesis of Boc-protected pentaflurophenyl ester of the 30 chosen base (thymidine). The resulting PNA may be prepared employing ~!errifield's solid-phase approach WO 95/15971 ' .! ~" ~_ ~ C 2 1 7 8 6 1 8 PCT/US94/13893 .

(~Serrifield, Science, 232:341 t1986) ), using a sinyle coupling protocol with 0.1 M of the thiminyl monomer in 30% (v/v) D~F in CH2Cl~. The ~LOyL~ss of the reaction i5 followed by guantiative ninhydrin analysis (Sarin, 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, Cupra, 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 6upport and then cleaved to obtain a dinucleotide rh~srh~ramidate). A typical example is the preparation of the phenyl ester of 5'0-isobutyloxycarbonylthymidyl-(3'-5')-5'-amino-5'-deoxythymidine (Letsinger, J. Org.
Chem., supra). The dimer units are substituted for ~tandard oligonucleotides at chosen intervals during the preparation of DNA using established automated technigues. Transition metal modif ication of the modified linkages takes place as described in Example 1.
The synthesis of phosphorothioate and phoayl,c,l~ithioate (Eckstein, supra, and references within) internucleotide l;nlr~ s is well ~- ted. A pl~hl;Ch~d protocol utilizes an Applied Biosystems DNA synthesizer using a modified B-cyanoethylrhosphoramidite cycle that caps after sulphurization with tetraethylthiuram disulfide (TETD) (Iyer, J. Org. Chem. 55:4693 (1990) ) . The rho~phorothioate and rhosphorodithioate analoys are prepared as dimers and cleaved from the solid cupport and purified by HPLC (acetonitrile/triethylammonium acetate mobile phase).

WO 95/15971 ~ ~ ;, 2 1 7 8 6 1 8 PCT/US94/13893 Examp l e 4 Synthesis of Two Oligonucleotides Each with ~n Ele~tron Transfer . oietY at the 5 ' Terminus In this example, two oligonucleotides are made which 5 hybridize to a single target sequence, without intervening ~eguences. One oligonucleotide ~as an electron donor moiety covalently attached to the S ' terminus, and the other has an electron acceptor moiety covalently nttached to the 5 ' terminus . In this 10 example, the electron transfer species ~re attached via a uradine nucleotide, but one skilled in the art will understand the present methods can be u6ed to modify any of the nucleotides. In addition, one skilled in the art will recognize that the p..,.Ldu.e is not limited to the 15 generation of 8-mers, but is useful in the generation of oligonucleotide probes of varying lengths.
The pL oceduL e is exactly as in Example 1, except that the 8-mers generated are not complementary to each other, and instead ~re complementary to a target 20 seguence of 16 nucleotides. Thus the final annealing step of step 4 of Example l i6 not done. Instead, the two modified oligonucleotides are annealed to the target seguence, ~nd the resulting complex is characterized as in Example 5.
Example 5 Char~cterization of Modif ied Nucleic Acids ~n7vmatic diqestion The modif ied oligonucleotides of example 1 were subjected to enzymatic digestion using established 30 protocols and converted to their constituent nucleosides ~ WO95/15971 ` `~ 2178618 PCTIUS94/;3893 by seguential reaction with phosphodiesterase and alkaline phosphatase. By compzrison of the experimentally obtained integrated HPLC profiles and W-vis spectra of the digested oligonucleotides to 5 standards ( ~ nclv~ i n~ 2 ' -aminourid ine and 2 ' -Am~noa/l~nine~ ~ the presence of the amino-modified base at the predicted retention time and characteristic W-vis spectra was confirmed. An identical p~oceluLe was carried out on the transition metal modified duplex DNA
10 and assignments of constituent nucleosides d~ ~cted 6ingle-site modification at the predicted site.
Fluorescent labeled amino-modified oliqonucleotides It has been d LL ated that the f 1U~.~L ~ ~C~ C, 15 fluorescein isothiocyanate (FITC) is specific for li~hDl in5 primary amines on modified oligonucleotides while not bonding to amines or amides present on nucleotide bases (Haugland, HAn-3hood of Fluorescent Probes and Research Chemicals, 5th Edition, (1992~ ) .
20 This reaction was carried out using the amino-oligonucleotide synthesized as described in example 1 and on an identical bases sequence without the 2'-amino-ribose group present. Fluorescence spectroscopic mea,u.~ ~s were acquired on both these 25 oligonucleotides and the results conf irm the presence of the amine on the 5'-terminal ribose ring.
ThermodYnamic Meltin~ Curves of Modified Du~lex DNA
A well established technique for measuring thermodynamic parameters of duplex DNA is the acquisition of DNA
30 melting curves. A series of melting curves as a function of concentration of the modified duplex DNA was measured via temperature controlled W-vis (Hewlett-WO95/15971 2 1 7 8 6 1 g PCrNS94113893 Packard), using techniques well known in the art. Theseresults 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 D;~ nAl Nuclear Macrnetic RPco~l~Ance (I~MR) SDe~;L- vs~
The amino ~ i fied oligonucleotides synthPs:i7Pcl as a part of this work were prepared in fiufficient quantities (6 micromoles) to permit the assignment of the 1H proton NMR spectra using a 600MHz Varian NMR spectrometer.
Meal,uL~ L of the rate of electrgn trAncfer An excellent review of the meax,,~ L terhniqupc is found in Winkler et al., Chem. Rev. 92:369-379 (1992).
The donor is Ru(bpy)2(NHuridine)im, E~l V, and the acceptor is Ru(NH3) jpy(NHuridine) im, E-330 mV. The purified transition metal modified oligonucleotides (U~ bpy)~ GCATCGA and U~u(l~83~ ~py)1~CGATGCA were AnnpAlpd by heating an equal molar mixture of the oligonucleotides (30 ~molar: 60 nmoles of DNA in 2 ml buffer) in pH 6.8 (lO0 mM NaPi, 900 mM NaCl) to 60-C for lO minutes and slowly cooling to room temperature over a period of 4 hours. The 501ution was transferred to an inert at - ,'^re cuvette ~ ;rpPcl with adapters for at~A( L to a vacuum line and a magnetic stirring bar.
The solution was ~l~qAccPrl 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 XeCl excimer-~, -d dye WO 95~15971 ~ f 7 8 6 l 8 PCTIUS94/13893 laser and data acquired at several wavelengths including 360, 410, 460 and 480 nm. The photoinduced electron transfer rate is 1.6 X lO6 s-~ over a distance of 28 A.
.
Examp l e 6 Synthesi6 of a Single Stranded Nucleic Acid Labeled with Two Electron Transfer Moieties This example uses the basic plcce.luL~:s 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.
~5 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 i5 described.
Five hundred nanomoles of each of two 5 ' -labeled oligonucleotides 12 bases in length are synthesized as detailed above with ruthenium (II) bisbipyridine imidazole on one oligonucleotide, "D" and ruthenium (III) tetraamine pyridine on a second oligonucleotide, nA~ .
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 pl ~.luced 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 ~ris-Cl, pH 7.5, 50 mM MgCl2, Wo 95115971 2 1 7 8 6 1 8 Pcr/uss4~l3893 50 mN dithiothreitol and 5 mg/ml gelatin, To promote maximal hybridization of labeled oligonucleotides to the complementary strand, the mixture is incubated at 60OC
for 10 minutes then cooled slowly at a rate of 5 approximately 10C per hour to a final temperature of 12C. The enzymatic ligation of the two labeled strands is achieved with T4 DNA ligase at 12C to prevent the ligation and oligomerization of the duplexed DNA to other duplexes (blunt end ligation). Alternatively, E.
10 ~Qli 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 ~nne~ 1 ecl DNA and adenosine triphosphate is added to a rinal concentration of 0. 5 mM. The reaction which 15 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 12C. The re~ction is terminated by heat inactivation of the 20 enzyme at 75C 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 25 oligonucleotide of this example is ideally suited for use as a photoactive gene probe as detailed below.
Example 7 Use of a Doubly Modified Oligonucleotide with Electron Transfer Noieties as a Photoactive Probe 30 for Detection of ComPlementarv Nucleic Acid Seauçnce This example utilizes the oligonucleotide 24mer of example 6 in a unique type of gene-probe assay in which ~ wo 95/15971 ~ ~ 2 1 7 8 6 1 8 PCI~/US94/13893 removal of unhybridized probe prior to signal detection is not required. In the assay ploce~L~, a region of the gag gene of human ;-~ ~noclPficiency virus type I
(HIV-I) is amplified by the polymerase chain reaction (Saiki et al., science 239:487-491 (1988)). This region o~ HIV-I is highly conserved among different clinical isolates .
The amplified target DNA versus controls lacking in HIV-I 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 60C for 10 ~inutes with gentle agitation. Detection of electron transfer following laser excitation is carried out ~s in example 5. Control samples which lack the hybridized probe show negligible electron transfer rates. Probes hybridized to the qag ~eyuence show efficient and rapid electron transfer through the DNA double helix, providing a highly specific, homogeneous and automatable HIV-I detection assay.
A similar homogeneous gene probe ~ssay involves the use of two probes, one an electron donor and the other an electron acceptor, which hybridize with the ga~ reyion of HIV-I 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 ~ppropriate, the electron transfer from one probe to the other is ~nh~nrDrl by the ligation of the juxtaposed ends using T4 DNA ligase as in example 6.

Claims (46)

Please add the following new claims:

1. A single-stranded nucleic acid containing one or multiple electron donor moieties and one or multiple electron acceptor moieties. wherein said electron donor moieties and said electron acceptor moieties are covalently attached to the ribose-phosphate backbone of said nucleic acid, wherein said electron donor and acceptor moieties are transition metal complexes, wherein electron transfer occurs between said electron donor and acceptor moieties when said single stranded nucleic acid is hybridized to a target sequence.

2 A single-stranded nucleic acid containing one or multiple electron donor moieties and one or multiple electron acceptor moieties, wherein one of the electron donor or acceptor moieties is an electrode and the other is a transition metal complex covalently attached to the ribose-phosphate backbone of said nucleic acid, wherein electron transfer occurs between said electron donor and acceptor moieties when said single stranded nucleic acid hybridizes to a target sequence.

3. A single-stranded nucleic acid according to claim 1 or 2 wherein said transition metal complex is covalently attached to a ribose of the ribose-phosphate backbone of said nucleic acid.

4. A single-stranded nucleic acid according to claim 3 wherein said attachment is at the 2' or 3' position of said ribose.

5. A single-stranded nucleic acid according to claim 4 wherein said attachment is at the 2' position of said ribose.

6. A single single-stranded nucleic acid according to claim 3 wherein there are no more than 5 unconjugated sigma bonds between said transition metal and the base attached to said ribose.

7. A single-stranded nucleic acid according to claim 6 wherein there are no morethan 4 unconjugated sigma bonds between said transition metal and the base attached to said ribose.

8. A composition comprising a first single stranded nucleic acid containing one or multiple electron donor moieties and a second single stranded nucleic acid containing one or multiple electron acceptor moieties, wherein each of said electron donor moieties and electron acceptor moieties are transition metal complexes covalently attached to a ribose of the ribose-phosphate backbone of said first and second single stranded nucleic acids, wherein electron transfer occurs between said electron donor and acceptor moieties when said first single stranded nucleic acid is hybridized to said second single stranded nucleic acid.

9. A composition according to claim 8 wherein said attachment is at the 2' or 3' position of said ribose.

10. A composition according a first single stranded nucleic acid containing one or multiple electron donor moieties and a second single stranded nucleic acid containing one or multiple electron acceptor moieties, wherein one of said electron acceptor or donor moieties is an electrode and the other is a transition metal complex covalently attached to the ribose-phosphate backbone of said nucleic acid, and wherein electron transfer occurs between said electron donor and acceptor moieties when said first single stranded nucleic acid is hybridized to said second single stranded nucleic acid.

11. A composition according to claim 10 wherein said transition metal complex is covalently attached to a ribose of the ribose-phosphate backbone of said nucleic acids.

12. A composition according to claim 8 or 11 wherein there are no more than 5 unconjugated sigma bonds between said transition metal and the base attached to said ribose.

13. A composition according to claim 12 wherein there are no more than 4 unconjugated sigma bonds between said transition metal and the base attached to said ribose.

14. A composition according to claim 11 wherein said attachment is at the 2' or 3 ' position of said ribose.

15 . A composition according a first single stranded nucleic acid containing oneor multiple electron donor moieties and a second single stranded nucleic acid containing one or multiple electron acceptor moieties, wherein said electron donor and acceptor moieties are transition metal complexes covalently attached to a ribose of the ribose-phosphate backbone of said first and second single stranded nucleic acids, and wherein electron transfer occurs between said electron donor and saidelectron acceptor moieties when said first and second single stranded nucleic acids are adjacently hybridized to a target domain.

16. A composition according to claim 15 wherein said attachment is at the 2' or 3' position of said ribose.

17 . A composition according a first single stranded nucleic acid containing oneor multiple electron donor moieties and a second single stranded nucleic acid containing one or multiple electron acceptor moieties, wherein one of the electron transfer moieties is an electrode and the other is a transition metal complex covalently attached to the ribose-phosphate backbone, wherein electron transfer occurs between said electron donor and said electron acceptor moieties when saidsingle stranded nucleic acids are adjacently hybridized to a target sequence.

18 . A composition according to claim 17 wherein said transition metal complex is covalently linked to a ribose of the ribose-phosphate backbone of said nucleic acids.

19. A composition according to claim 18 wherein said linkage is at the 2' or 3' position of said ribose.

20. A composition according to claim 15 or 18 wherein there are no more than 5 unconjugated sigma bonds between said transition metal and the base attached to said ribose.

21. A composition according to claim 20 wherein there are no more than 4 unconjugated sigma bonds between said transition metal and the base attached to said ribose.

22. A composition comprising a first single stranded nucleic acid containing oneor multiple electron donor moieties and a second single stranded nucleic acid containing one or multiple electron acceptor moieties, wherein said electron donor acceptor moieties are transition metal complexes covalently linked to a ribose of the ribose-phosphate backbone of said first and second single stranded nucleic acids, and wherein electron transfer occurs between said electron donor and acceptor moieties when said first single stranded nucleic acid hybridizes to a first domain of a target sequence, said second single stranded nucleic acid hybridizesto a second domain of said target sequence, and an intervening nucleic acid hybridizes to an intervening target domain of said target sequence.

23. A composition according to claim 22 wherein said linkage is at the 2' or 3' position of said ribose.

24. A composition comprising a first single stranded nucleic acid containing oneor multiple electron donor moieties and a second single stranded nucleic acid containing one or multiple electron acceptor moieties, wherein one of said electron donor or acceptor moieties is an electrode and the other is a transition metal complex covalently attached to the ribose-phosphate backbone of said nucleic acid, wherein electron transfer occurs between said electron donor and electron acceptor moieties when said first single stranded nucleic acid hybridizes to a first domain of a target sequence, said second single stranded nucleic acid hybridizes to a second domain of said target sequence, and an intervening nucleic acid hybridizes to anintervening target domain of said target sequence.

25. A composition according to claim 24 wherein said transition metal complex is covalently linked to a ribose of the ribose-phosphate backbone of said nucleic acids.

26. A composition according to claim 25 wherein said linkage is at the 2' or 3' position of said ribose.

27. A composition according to claim 22 or 25 wherein there are no more than 5 unconjugated sigma bonds between said transition metal and the base attached to said ribose.

28. A composition according to claim 27 wherein there are no more than 4 unconjugated sigma bonds between said transition metal and the base attached to said ribose.

29. A method of detecting a target sequence in a nucleic acid sample comprising:

a) hybridizing a single stranded nucleic acid containing one or multiple electron donor moieties and one or multiple electron acceptor moieties to said target sequence to form a hybridization complex, wherein said electron donor and electron acceptor moieties are transition metal complexes covalently attached to the ribose-phosphate backbone of said nucleic acids; and b) detecting electron transfer between said electron donor and acceptor moieties in the hybridization complex as an indicator of the presence or absenceof said target sequence.

30. A method of detecting a target sequence in a nucleic acid sample comprising a) hybridizing a single stranded nucleic acid containing one or multiple electron donor moieties and one or multiple electron acceptor moieties to said target sequence to form a hybridization complex, wherein one of said electron donor or acceptor moieties is an electrode and the other is a transition metal complex covalently attached to the ribose-phosphate backbone of said nucleic acid;
and b) detecting electron transfer between said electron donor moieties and said electron acceptor moieties in the hybridization complex as an indicator of the presence or absence of said target sequence.

31. A method according to claim 29 or 30 wherein said covalent attachment is to a ribose of the ribose-phosphate backbone of said nucleic acid.

32. A method according to claim 31 wherein said linkage is at the 2' or 3' positio of said ribose.

33 A method according to claim 31 wherein there are no more than 5 unconjugated sigma bonds between said transition metal and the base attached to said ribose.

34. A method according to claim 33 wherein there are no more than 4 unconjugated sigma bonds between said transition metal and the base attached to said ribose.

35 . 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 one or multiple electron donor moieties to said first target domain;
b) hybridizing a second nucleic acid containing one or multiple electron acceptor moieties to said second target domain, wherein said electron donor and electron acceptor moieties are transition metal complexes covalently attached tothe ribose-phosphate backbone of said nucleic acids; 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.

36 . 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 one or multiple electron donor moieties to said first target domain;

b) hybridizing a second nucleic acid containing one or multiple electron acceptor moieties to said second target domain, wherein one of said electron donor and acceptor moieties is an electrode and the other is a transition metal complex covalently attached to the ribose-phosphate backbone of said nucleic acid; 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.

37. A method according to claim 35 or 36 wherein said transition metal complex is attached to a ribose of the ribose-phosphate backbone of said nucleic acid.

38. A method according to claim 37 wherein said transition metal complex is attached at the 2' or 3' position of said ribose.

39. A method according to claim 37 wherein there are no more than 5 unconjugated sigma bonds between said transition metal and the base attached to said ribose.

40. A method according to claim 39 wherein there are no more than 4 unconjugated sigma bonds between said transition metal and the base attached to said ribose.

41. A method of detecting a target sequence in a nucleic acid wherein said target sequence comprises a first target domain, a second target domain, and an intervening target domain between said first and said second target domains, said method comprising:

a) hybridizing a first nucleic acid containing one or multiple electron donor moieties to said first target domain;
b) hybridizing a second nucleic acid containing one or multiple electron acceptor moieties to said second target domain, wherein said electron donor or acceptor moieties are transition metal complexes covalently attached to the ribose-phosphate backbone of said nucleic acid;
c) hybridizing an intervening nucleic acid to said intervening target domain;
and d) detecting electron transfer between said electron donor and acceptor moieties while said first, second and intervening nucleic acids are hybridized to said first, second and intervening target domains as an indicator of the presence or absence of said target sequence in said nucleic acid sample.

42. A method of detecting a target sequence in a nucleic acid wherein said target sequence comprises a first target domain, a second target domain, and an intervening target domain between said first and said second target domains, said method comprising:
a) hybridizing a first nucleic acid containing one or multiple electron donor moieties to said first target domain;
b) hybridizing a second nucleic acid containing one or multiple electron acceptor moieties to said second target domain, wherein one of said electron donor and acceptor moieties is an electrode and the other is a transition metal complex covalently attached to the ribose-phosphate backbone of said nucleic acid;
c) hybridizing an intervening nucleic acid to said intervening target domain;
and d) detecting electron transfer between said electron donor moiety and said electron acceptor moiety while said first, second and intervening nucleic acids are hybridized to said first, second and intervening target domains as an indicator of the presence or absence of said target sequence in said nucleic acid sample.

43. A method according to claim 41 or 42 wherein said transition metal complex is covalently attached to a ribose of the ribose-phosphate backbone of said nucleic acid.

44. A method according to claim 43 wherein said attachment is at the 2' or 3' position of said ribose.

45. A method according to claim 43 wherein there are no more than 5 unconjugated sigma bonds between said transition metal and the base attached to said ribose.

46. A method according to claim 45 wherein there are no more than 4 unconjugated sigma bonds between said transition metal and the base attached to said ribose.