Nucleic acid mediated electron transfer

IS 2 198 486 T3

DESCRIPTION

Nucleic acid mediated electron transfer.

Field of the invention

The present invention addresses the transfer of electrons through nucleic acids. More specifically, the invention is directed to improvements in site-selective nucleic acid odification with electron transfer groups.

Background of the invention

The detection of specific nucleic acid sequences is an important tool for research in molecular biology and diagnostic medicine. Gene probe assays currently have a role in the identification of infectious organisms such as bacteria and viruses, in verifying the expression of normal genes, and in the identification of mutant genes such as oncogenes, in typing tissues according to their compatibility before transplantation. of tissues, in matching tissue and blood samples in forensic medicine, and to explore homology between genes from different species.

Ideally, a gene probe assay should be sensitive, specific, and easily automatable (for a review, see Nickerson, Current Opinion in Biotechnology 4: 48-51 (1993)). The requirement for sensitivity (i.e., low detection limits) has been greatly mitigated by the development of polymerase chain reaction (PCR) and other amplification technologies that allow researchers to exponentially amplify a specific acid sequence. nucleic acid before analysis (for a review, see Abramson et al., Current Opinion in Biotechnology 4: 41-47 (1993)).

In contrast, specificity remains a problem in many currently available gene probe assays. The degree of molecular complementarity between the probe and the target defines the specificity of the interaction. Variations in the concentrations of probes, targets, and salts in the hybridization medium, in reaction temperature, and in probe length could alter or influence the specificity of the probe / target interaction.

It might be possible, in some limited circumstances, to distinguish perfectly complementary targets from mismatched targets, although this is generally very difficult using traditional technology, since small variations in reaction conditions will alter hybridization. New experimental techniques for the detection of mismatches include DNA ligation assays in which single mismatches prevent ligation, and probe digestion assays, in which mismatches create sites for probe cleavage.

Finally, the automation of gene probe assays remains an area where current technologies fail. Such assays are generally based on hybridization of a labeled probe to a target sequence, followed by removal of the unhybridized free probe. This separation is generally achieved by gel electrophoresis or solid phase capture, and washing of the target DNA, and is generally quite difficult to automate easily.

The time-consuming nature of these separation steps has led to two distinct lines of development. One involves the development of electrophoretic and other high-speed, high-throughput separation techniques. The other involves the development of homogeneous gene probe assays without separation.

For example, Gen-Probe, Inc. (San Diego, CA) has developed a homogeneous protection assay in which hybridized probes are protected from base hydrolysis, and are therefore capable of subsequent chemiluminescence (Okwumabua et al., Res. Microbiol. 143: 183 (1992)). Unfortunately, that this assay is based on a chemiluminescence substrate known for its high photon background emission suggests that this assay will not have high specificity. EPO application number 86116652.8 (EP-A-229,943) describes an attempt to use non-radiant energy transfer, from a donor probe to an acceptor probe, as a homogeneous detection scheme. However, fluorescence energy transfer is highly influenced by both probe topology and topography, and the DNA target itself is capable of significantly attenuating energy, resulting in considerable variability. Thus, there is a need for DNA probes that are specific, capable of detecting mismatches, and that can be incorporated into an automated system for sequence identification.

As outlined above, molecular biology relies quite heavily on modified or labeled oligonucleotides for gene probe assays (Oligonucleotide Synthesis: A Practical Approach. Eds. Gait et al., IRL Press, Oxford, UK, 1984; Oligonucleotide and Analogues: A Practical Approach. Ed. F. Eckstein, Oxford University Press, 1991). As a result, there are currently several techniques for the synthesis of adapted nucleic acid molecules. Since nucleic acids do not naturally contain functional groups to which the molecules of interest could easily and covalently bind, procedures have been developed that allow chemical modification at any one of the terminal phosphates or at the heterocyclic bases (Dreyes et al. ., Proc.

IS 2 198 486 T3

Natl. Acad. Sci. USA 82: 968 (1985)).

For example, deoxyribo- and ribonucleoside analogs containing amino groups at the 2 'or 3' position of the sugar can be prepared using established chemical techniques. (See Imazawa et al., J. Org. Chem. 44: 2039 (1979); 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)). Furthermore, oligonucleotides could be synthesized with 2'-5 'or 3'-5' phosphoamide linkages (Beaucage et al., Tetrahedron 49 (10): 1925 (1992); Letsinger, J. Org. Chem. 35: 3800 (1970 ); Sawai, Chem. Lett. 805 (19845); Oligonucleotide and Analogues: A Practical Approach. Ed. F. Eckstein, Oxford University Press, 1991).

Nucleic acid modification has been done for two reasons: to create non-radioactive DNA markers to serve as probes, and to use chemically modified DNA to obtain site-specific cuts.

To this end, the DNA could be labeled to serve as a probe by altering a nucleotide which then serves as a replacement analog in the resynthesis of the label during translation of the double-stranded DNA. Chemically altered nucleotides could then provide reactive sites for the attachment of immunological labels or other labels such as biotin (Gilliam et al., Anal. Biochem. 157: 199 (1986)). Another example uses ruthenium derivatives that intercalate into DNA to produce photoluminescence under defined conditions (Friedman et al., J. Am. Chem. Soc. 112: 4960 (1990)).

In the second category, there are a number of examples of compounds covalently attached to DNA that subsequently cause DNA strand cutting. For example, 1,10-phenanthroline has been coupled to single chain oligothymidylate through a linker, resulting in 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 performed for EDTA¹-Fe (II), both for double-stranded DNA (Boutorin et al., FEBS Lett. 172: 43-46 (1986) and triple DNA (Strobel et al., Science 249: 73 (1990)), porphyrin-Fe (III) (LeDoan et al., Biochemistry 25: 6736-6739 (1986)), and 1,10-phenanthrone-Cu (I) (Chen et al., Proc. Natl. Acad. Sci. USA 83: 7147 ( 1985)), all resulting in DNA strand cutting in the presence of a reducing agent in aerated solutions. A similar example using porphyrins resulted in DNA strand cutting, and base oxidation or DNA cross-linking under very specific conditions (LeDoan et al., Nucleic Acids Res. 15: 8643 (1987)).

Other work has focused on the chemical modification of heterocyclic bases. For example, the anchoring of an inorganic coordination complex, Fe-EDTA, to a modified internal base resulted in DNA breakage after its hybridization in the presence of dioxygen (Dreyer et al., Proc. Natl. Acad. Sci. USA 82: 968 (1985)), a ruthenium compound has been successfully coupled to an internal base in a DNA octamer, with retention of both the DNA hybridization capabilities and the spectroscopic properties of the ruthenium brand (Telser et al., J. Am. Chem. Soc. 111: 7221 (1989)). Other experiments have successfully added two separate spectroscopic tags to a single double-stranded DNA molecule (Telser et al., J. Am. Chem. Soc. 111: 7226 (1989)).

The study of electron transfer reactions in proteins and DNA has also been explored in search of systems that are capable of transferring electrons over long distances.

To this end, it has been shown that the intramolecular transfer of electrons in protein-protein complexes, such as those found in photosynthetic proteins and proteins in the respiratory tract, takes place over appreciable distances within proteins, at biologically velocities. significant (see Bowler et al., Progress in Inorganic Chemistry: Bioinorganic Chemistry, Vol. 38, Ed. Stephen J. Lippard (1990)). In addition, selective modification of metalloenzymes with transition metals has been achieved, and techniques have been developed to monitor electron transfer in these systems. For example, electron transfer proteins, such as cytochrome c, have been modified with ruthenium through its anchoring on various histidines, and the rate of electron transfer from Fe has been measured.²⁺ from heme to Ru³⁺ United. The results suggest that electron transfer tunnel pathways may exist (Baum, Chemical & Engineering News, February 22, 1993, pages 2023; see also Chang et al., J. Am. Chem. Soc. 113: 7056 ( 1991)). In related work, normal protein isolation, which protects the redox centers of an enzyme or protein from indiscriminate reactions with the external solvent, was "soldered" to transform these systems of electrical insulators into electrical conductors (Heller, Acc. Chem Res. 23: 128 (1990)).

There are a few reports of photoinduced electron transfer in a DNA matrix. In these systems, electron donors and acceptors are not covalently attached to DNA, but randomly associated with DNA, thus making explicit elucidation and control of the donor-acceptor system difficult. For example, the intense fluorescence of certain quaternary diazoaromatic salts is attenuated from their intercalation in DNA, or from their exposure to individual mononucleotides, thus presenting electron donation processes within the same DNA (Brun et al., J Am. Chem. Soc. 113: 8153 (1991)).

Another example of the difficulty of determining electron transfer mechanisms is found in work done with some photoexcitable ruthenium compounds. Early work suggests that certain ruthenium compounds either randomly intersperse between nucleotide bases, or bind to the helix surface (Purugganan et al., Science 241: 1645 (1988)). A recent reference indicates that certain ruthenium compounds do not intercalate in DNA (Satyanarayana et al., Biochemistry 31 (39): 9319 (1992)); instead, they bind non-covalently to the surface of the DNA helix.

IS 2 198 486 T3

In these pioneering work, various electron acceptor compounds, such as cobalt, chromium, or rhodium compounds, were added to certain DNA-associated ruthenium electron donor compounds (Puragganan et al., Science 241: 1645 (1988); Orellana et al. al., Photochem. Photobiol. 499: 54 (1991); Brun et al., J. Am. Chem. Soc. 113: 8153 (1991); Davis, Chem.-Biol. Interactions 62:45 (1987); Tomalia et al., Acc. Chem. Res. 24: 332 (1991)). From the addition of these various electron-accepting compounds, which are randomly non-covalently bound to the helix, the attenuation of the photoexcited state was detected through electron transfer. The rate of attenuation depended on both the individual electron donor and acceptor, as well as their concentrations, thus revealing that the process was bimolecular.

In one set of experiments, the authors postulate that the more mobile surface-bound donor promotes electron transfer more efficiently than intercalated species, and suggests that the sugar phosphate backbone of DNA, and possibly the solvent environment surrounding DNA, they play an important role in electron transport (Purugganan et al., Science 241: 1645 (1988)). In another work, the authors emphasize the dependence of the speed of mobility of the donor and acceptor, and of their local concentrations, and they assign that the role of DNA is mainly to facilitate an increase in the local concentration on the helix of the donor species and acceptor (Orellana et al., see above).

In another experiment, an electron donor was reported randomly intercalated in the DNA base stack, while the acceptor was randomly associated with the DNA surface. The rate of electron transfer attenuation indicated close donor and acceptor contact, and the system also exhibited enhanced electron transfer rate with the addition of salt to the medium (Fromherz et al., J. Am. Chem. Soc. 108: 5361 (1986)).

In all of these experiments, the electron transfer rate for non-covalently bound donors and acceptors is several orders of magnitude lower than that observed in free solution.

An important stimulus for the development of long-distance electron transfer systems is the creation of synthetic light-harvesting systems. Work to date suggests that an artificial light harvesting system contains an energy transfer complex, an energy migration complex, an electron transfer complex, and an electron migrator complex (for a topical review of this area, see Chemical & Engineering News, March 15, 1993, pages 38-48). Two types of molecules have been tested: a) long organic molecules, such as hydrocarbons with covalently linked electron transfer species, or DNA, with interspersed, partially interspersed, or helix-associated electron transfer species, and b) synthetic polymers .

Long organic molecules, although quite rigid, are influenced by a number of factors, which hinder their development. These factors include the polarity and composition of the solvent, the orientation of the donor and acceptor groups, and the chemical character of the covalent bond or association of the electron transferring species to the molecule.

The creation of electron transfer systems has been difficult because the polymers available are too flexible, such that various transfer modes occur. Polymers that are sufficiently rigid often interfere significantly with electron transfer mechanisms or are quite difficult to synthesize.

Therefore, the development of an electron transfer system that is sufficiently rigid, has electron transfer species covalently attached at defined intervals, is easy to synthesize, and does not appreciably interfere with the electron transfer mechanism, would be useful in the process. development of artificial light harvesting systems.

In conclusion, the random distribution and mobility of the donor and electron acceptor pairs, along with potential short distances between donor and acceptor, the loose and presumably reversible association of donors and acceptors, the described solvent dependence, and broad putative electronic pathways, and alteration of the DNA backbone by intercalated compounds, making normal base pairing impossible, all serve as marked limitations on long-distance electron transfer in a DNA matrix. Thus, a process for the production of rigid, covalent, electron donor and acceptor anchors is desirable to provide minimal disturbance to the nucleic acid backbone and retention of its ability to base pair normally. The present invention serves to provide such a system, which allows the development of new bioconductors and diagnostic probes.

Summary of the invention

The present invention provides means for site-specific modification of nucleic acids with redox active groups, such as transition metal complexes. An electron donating group and an electron accepting group are covalently bonded at predetermined positions. The resulting complexes represent a series of new derivatives that are bimolecular templates capable of transferring electrons over very long distances at extremely high speeds. These complexes possess unique structural features that allow the use of an entirely new class of bioconductors and diagnostic probes.

IS 2 198 486 T3

Accordingly, it is an object of the invention to provide nucleic acids with electron transfer species covalently attached to a terminal base of the nucleic acid. It is still another object to provide nucleic acids with covalently linked organic electron transferring species.

Brief description of the figures

Figure 1 illustrates all the possible orientations of electron donor moieties (EDM) and electron acceptor moieties (EAM) on a single-stranded nucleic acid.

Figure 2 illustrates the possible orientations of EDM and EAM electron transfer groups on two adjacent single-stranded nucleic acids. These guidelines will also apply when the two probes are separated by an intermediate sequence.

Figure 3 illustrates a series of amino-modified nucleoside precursors prior to their incorporation into an oligonucleotide.

Figures 4A and 4B illustrate the skeleton of electron transfer groups. Figure 4A illustrates the general formula for a representative class of electron donors and acceptors. Figure 4B illustrates a specific example of a ruthenium electron transfer group using bisbipyridine and imidazole as ligands.

Figure 5 is a schematic showing transition metals attached to the ribose-phosphate backbone at a variety of positions. M is a transition metal. M1 is attached through an amine on the 2 'carbon of ribose; an electron must travel through 4 σ bonds to enter the pi orbitals (“the pi pathway”) of the stacked bases. M2 and M3 are linked through phosphoramid type bonds, electrons must travel respectively through 7 σ bonds to enter the pi pathway. M 4 is attached through an amine on the 3 'carbon of ribose, and an electron travels through 5 σ bonds.

Figures 6A, 6B and 6C illustrate the anchoring of a 2'-amino-modified nucleoside with controlled pore glass (CPG) and the formation of a single-stranded nucleic acid with the elongation and binding of transition metal complexes such as exemplified electron transfer species. Experimental conditions will be outlined in EXAMPLE 9. Figure 6A illustrates the formation of 2'-amino-2'-deoxyuridine derived with controlled pore glass (CPG). Modified uridine 2'-amino is illustrated, although any base could be used. As is known in the art, phosphoramidite nucleosides are added to derived nucleosides, after removing the DMT protecting group, as generally illustrated in Figure 6B, using the sequence UCTCCTACAC as an example. The 5 'terminal addition of a 2'-amino-deoxyuridine phosphoramidite, with a DTM protecting group, results in a single-stranded nucleic acid containing 3' and 5 'a modified 2'-amino nucleoside. Figure 6C illustrates the addition of the electron transfer species, exemplified by two ruthenium transition metal complexes, im (bpy) 2 Ru and Ru (II) (NH3) 4 py.

Figure 7 illustrates the addition of electron transfer groups, exemplified by a transition metal complex, to the C-terminus of PNA. Figure 9 shows a 4-aminomethylpyridine at the C-terminus, to form a ligand that could bind the metal to the nitrogen of the pyridine ring.

Figures 8A and 8B illustrate the binding of amino-modified nucleic acids of the invention to the electrodes. (A) illustrates anchoring to glazed carbon electrodes. R is the oligonucleotide, and GCE is the vitrified carbon electrode. (B) illustrates the anchoring of the amino-modified nucleic acids of the invention to oxidized surfaces using reactions with silane.

Detailed description

Unless otherwise indicated, the term "nucleic acid" or "oligonucleotide", or the grammatical equivalents thereof, means at least two nucleotides covalently linked together. A nucleic acid of the present invention generally contains phosphodiester linkages, although in some instances, as outlined above, nucleic acid analogs are included that could have altered backbones, comprising, for example, phosphoramid linkages (Beaucage et al. , Tetrahedron 49 (10): 1925 (1993) and references therein; Letsinger, J. Org. Chem. 35: 3800 (1970); Sprinzl et al., Eur. J. Biochem. 81: 579 (1977); Letsinger et al., Nucl Acids Res. 14: 3487 (1986); Sawai et al., Chem. Lett. 805 (1984); Letsinger et al., J. Am. Chem. Soc. 110: 4470 (1988); and Pauwels et al., Chemica Scripta 26: 141 (1986)), phosphorothioates, phosphorodithioates, O-methyl-phosphoramidites (see Eckstein, Oligonucleotide and Analogues: A Practica! Approach, Oxford University Press), and peptide-acid linkages and skeletons nucleic (see Egholm, J. Am. Chem. Soc. 114: 1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31: 1008 (1992); Nielsen, Nature 365: 566 (1993); Carisson et al., Nature 380: 207 (1996)). These modifications of the ribose-phosphate backbone could be made to facilitate the addition of electron transfer groups, or to increase the stability and half-life of such molecules in physiological environments.

Peptide nucleic acid (PNA) are particularly preferred. This backbone is substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This results in two advantages. First, this backbone exhibits improved hybridization kinetics. PNAs have large changes in melting temperature (Tm) for mismatched base pairs

ES 2 198 486 T3 regarding the paired perfectly. DNA and RNA typically exhibit a 2-4 ° C drop in Tm for internal mismatch. With the non-ionic backbone of PNA, the drop is close to 7-9 ° C. This allows for better detection of mismatches. Similarly, due to their nonionic nature, hybridization of the bases attached to these backbones is relatively insensitive to salt concentration. These are particularly advantageous in the systems of the present invention, since a reduced salt hybridization solution has a lower Faraday current than a physiological saline solution (in the 150 mM range).

Nucleic acids could be single-stranded or double-stranded, as specified, or contain portions of double-stranded and single-stranded sequences. The nucleic acid could be DNA, both genomic and cDNA, RNA, or a hybrid, wherein the nucleic acid contains any combination of deoxyribo- and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine and hypoxanthine, etc. In the case of an "intermediate nucleic acid", the term refers to one or more nucleosides. As used herein, the term "nucleoside" includes nucleotides.

The terms "electron donating group", "electron accepting group", and "electron transfer groups", or the grammatical equivalents thereof, refer to molecules capable of transferring electrons under certain conditions. It should be understood that the electron donor and acceptor capacities are relative, that is, a molecule that can lose an electron under certain experimental conditions will be able to accept an electron under different experimental conditions. It should be understood that the number of electron donor groups and electron acceptor groups is very high, and that one skilled in the art of electron transfer compounds will be able to use a number of compounds in the present invention. Electron transfer groups include, but are not limited to, transition metal complexes, organic electron transfer groups, and electrodes.

In a preferred embodiment, the electron transfer groups are transition metal complexes. Transition metals are those whose atoms have an incomplete electron shell. Suitable transition metals for use in the invention include, but are not limited to, cadmium (Cd), magnesium (Mg), copper (Cu), cobalt (Co), palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru), rhodium (Ro), osmium (Os), rhenium (Re), platinum (Pt), scandium (Sc), titanium (Ti), vanadium (Va), chromium (Cr), manganese (Mn), nickel (Ni), molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir). That is, the first series of transition metals, the platinum metals (Ru, Rh, Pd, Os, Ir, and Pt) are preferred, along with Re, W, Mo, and Tc. Ruthenium, rhenium, osmium, platinum, and iron are particularly preferred.

Transition metals complex with a variety of ligands to form appropriate transition metal complexes, as is well known in the art. Appropriate ligands include, but are not limited to, -NH2; pyridine; pyrazine; isonicotinamide; imidazole; bipyridine and substituted derivatives of bipyridine; phenanthroline, particularly 1,10-phenanthroline (abbreviated phen) and substituted derivatives of phenanthroline such as 4,7-dimethylphenanthroline; dipyrofenazine; 1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat); 9,10-phenanthrenequinone diimine; 1,4,5,8-tetraazaphenantheneum (abbreviated tap); 1,4,8,11-tetra-azacyclotetradecane; diaminopyridine (abbreviated damp); porphyrins and substituted derivatives of the porphyrin family. A general formula is shown in Figure 4A that is representative of a class of donors and acceptors that could be employed. The R groups¹, R¹, R³, R⁴ and R⁵they could be any coordinating ligand that is capable of covalently binding to the chosen metal, and could include any of the above ligands. The structure of a ruthenium electron transfer species using bisbipyridine and imidazole as ligands is shown in Figure 4B. Examples of useful electron transfer complexes include, but are not limited to, those shown in TABLE 1.

TABLE 1

Donors Acceptors

Ru (bpy) 2im-NH-U Ru (NH3) 5-NH-U

Ru (bpy) 2im-NH-U Ru (NH3) 4py-NH-U

Ru (bpy) 2im-NH-U Ru (NH3) 4im-NH-U tran-Ru (cyclam) py where,

Ru = ruthenium, bpy = bisbipyridine, im = imidazole, py = pyridine, cyclam = 1,4,8,11-tetra-azacyclotetradecane

IS 2 198 486 T3

Other suitable groups include the, bis (phenanthroline) (dipyrophenazine) Ru (II) (abbreviated [Ru (phen) 2 dppz]²⁺ ); bis (9,10-fenthrenequinone diimine) (phenanthroline) Rh (III) (abbreviated [Rh (phi) 2 phen]³⁺ ); tris (phenanthroline) Ru (II) (abbreviated [Ru (o-phen) 3]²⁺ ];

Co (phen)₃³⁺;

Co (bpy)₃³⁺;

Rh (phen)₃³⁺;

Cr (phen)₃³⁺;

Ru (bpy) 2 (dppz)³⁺ ; Y

Ru (bpy) 3²⁺;

In addition to transition metal complexes, other organic electron donors and acceptors could be covalently bound to nucleic acid for use in the invention. These organic molecules include, but are not limited to, riboflavin, xanthene dyes, azine dyes, acridine orange, N, N-dimethyl-2,7-diazapyrene dichloride (DAP²⁺ ), methylviologen, ethidium bromide, quinones such as N, N'-dimethylanthra (2,1,9, -def: 6,5,1,0-d'e'f ') dichloride, diisoquinoline (ADIQ²⁺); porphyrins ([mesotetrakis (N-methyl-x-pyridinium) porphyrin tetrachloride]; varlamine blue B hydrochloride; Bindschedler's green; 2,6-dichloroindophenol; 2,6-dibromophenolindophenol; brilliant crest blue 3-amino-9-dimethyl-amino-10-methylphenoxyazine); methylene blue; Nile blue A (aminoaphthodiethylaminophenoxazine sulfate); indigo-5,5 ', 7,7'-tetrasulfonic acid; indigo-5 acid, 5 ', 7-trisulfonic, fenosaphranine, indigo-5-monosulfonic acid, safranin T; bis (dimethylglyoximate) iron (II) chloride; indulin scarlet; neutral red; and substituted derivatives of these compounds.

In one embodiment, the electron donors and acceptors are redox proteins as is known in the art. However, in many embodiments redox proteins are not preferred.

In a particularly preferred embodiment, an electron transfer group comprises a solid support, such as an electrode to which the nucleic acid has been attached, covalently or otherwise. That is, the electrode serves as both an electron donor and acceptor, and is described in more detail below. The techniques used in this embodiment are analogous to splicing proteins to an electrode, except that the nucleic acids of the present invention are used in place of a redox protein (see, for example, Gregg et al., J. Phys. Chem. 95 : 5970 (1991); Heller et al., Sensors and Actuators R. 13-14: 180 (1993); and Pishko et al., Anal. Chem. 63: 2268 (1991)).

The junction to the electrode is used to initiate an electron transfer pathway through an applied potential, and for electronic electron transfer monitoring procedures.

In a preferred embodiment, the transport of electrons between the electrode and the nucleic acid can be indirect, using mediators of electron transport, which are free in solution or embedded in a gel or polymer, to provide a type of electronic coupling between the electrode and nucleic acids. In a preferred embodiment, nucleic acids modified with an electron transfer group of the invention are attached through such a matrix. Attachment to the array has several advantages for use in a nucleic acid gene sensor. Due to the three-dimensional nature of the polymer, a large number of modified nucleic acid probes can be anchored to a small area of ​​the electrode. Using a highly porous "hydrogel", nucleic acid hybridization rates can be quite high, practically matching those of nucleic acid in solution.

For example, polymers with covalently attached redox molecules behave as highly effective mediators of electron transfer. Siloxane and ethylene oxide polymers, modified with ferrocene molecules, demonstrated electron transfer between enzymes and an electrode; For example, flexible polymers of siloxane and ethylene oxide covalently linked to ferrocene or Os (bpy) 2 have been found to be highly effective redox polymers in mediating electron transfer from various enzymes to an electrode (see Boguslavsky et al., Solid State Ionics V. 60, p. 189 (1993)). Similarly, a redox conductive epoxy cement has been prepared (see Hellar et al., J. Phys. Chem. 95: 5970 (1991)). Cross-linked redox gels have also been prepared, for amperometric biosensor applications, with glucose oxidase electrically connected to electrodes, so that electrons were observed to flow from the enzyme, through the polymer, to the electrode (see Hellar, A. et al., Anal. Chem. 62: 258 (1990)).

In this embodiment, it is preferred that a redox polymer, such as a complex of poly (vinylpyridine) with Os (bby) 2Cl, is crosslinked with an epoxide, such as a diepoxide, to form a redox conductive epoxy cement, which is capable of bonding tightly to electrodes made of conductive material, such as gold, glazed carbon, graphite, and other conductive materials. This strong bond is included in the definition of "covalently bound" for the purposes

ES 2 198 486 T3 of this embodiment. The epoxy crosslinked polymer is then reacted with, for example, an exposed amine, such as the amine of an amino-modified nucleic acid described above, covalently linking the nucleic acid to the complex, forming a "redox hydrogel" on the surface. of the electrode.

In an analogous way, chemically modified DNA can substitute for the redox enzyme or mediator with the result that electron transfer processes are observed from a DNA-modified group with a transition metal, through a coupled redox conducting polymer, to an electrode.

Suitable mediators include water soluble hydroquinones / ferrocene / ferricinium quinones, reducible and oxidizable organic salt components, cobaltocenes, the hexa- and octacyanides of molybdenum, tungsten and iron. In addition, the macrocycles and chelating ligands of transition metals such as cobalt, ruthenium and nickel are used, including Co (ethylenediamine) 3 and Ru (ethylenediamine) 3, and the trisbipyridyl and hexamine complexes of transition metals such as Co, Ru, Fe and Os (see Alyanasundaram, above).

In a preferred embodiment, the electron transport between the electrode and the nucleic acid can be directed through a covalent bond. An advantage of these systems is that the orientation of the DNA probe can be influenced to reduce any back twisting from the probe towards the electrode. Also, more precise control of applied potential and measured current is associated with shorter covalent bonds relative to gels and polymers.

In a preferred embodiment, the covalent bonds should be highly conductive, such as in a redox polymer (Hellar, A., Acc. Chem. Res. Vol. 23, p. 128, 1990). Alternatively, if they are poorly conductive , the length of the bond should be kept short. Consequently, a preferred embodiment has an electron that crosses no more than about five σ bonds, with it being especially preferred when it crosses no more than three. Carbon paste and vitrified charcoal sticks have proven reliable and effective as electrodes in a variety of chemical sensors, including sensitive glucose oxidase enzyme-based biosensors, and could be used in the present invention. Furthermore, flexible ethylene oxide and siloxane polymers covalently bonded to ferrocene or Os (bpy) 2 molecules have been found to be highly effective in mediating electron transfer from various enzymes to an electrode. Modified amino-ribose nucleic acids are attached to carbon electrodes by variations of these techniques from the literature. Finally, the nucleic acids are more directly attached to the oxidized carbon electrodes via guanosine residues, using the known carbodiimide and N-hydroxysuccinimide chemistry.

In a preferred embodiment, vitrified carbon electrodes (GCE) are used. In this embodiment, amine groups are used, as outlined above, on the 2 'or 3' carbon of the ribose ring for anchoring. The reaction progresses through the oxidation of an amine group to a cationic radical, which forms a chemically stable, covalent bond between the amine and the edge of the GCE surface plane (see Deinhammer, R. et al., Langmuir 10: 1306 (1994)). This synthetic strategy has been characterized in detail using X-ray photo-electron spectroscopy and cyclic voltimetry. The yield using this chemistry can be quite high, about 1 x 10¹⁰ molecules / cm². The amine compound forms a stable bond with the carbon surface, and steric effects influence the bonding efficiency. The reactivity of primary amines is substantially greater than that of secondary amines; the binding of tertiary amines has not been observed at all.

Figure 8A illustrates the procedure developed by Deinhammer, R. et al., To prepare GCEs for electrochemical treatment in solution containing amines, using the amino-modified oligonucleotides (primary amino group) described above.

Furthermore, DNA has been immobilized on GCE using a water soluble carodimide (Mikkelsen et al., Electroanalysis 4: 929 (1992)).

In a preferred embodiment, the nucleic acids of the invention are attached to gold electrodes. There are several procedures available for the covalent bonding of redox active species to gold surfaces, and electron transfer reactions have been observed with these materials. Hydroxythiols (OH (CH2) xSH) of different lengths will be prepared by variation of literature procedures (see Miller, C. et al., J. Phys. Chem. 95: 877 (1991) and Chidsey, CED Science V. 251 , p. 919 (1991)). EXAMPLE 8 outlines the preparation of hydroxythiols that are attached to gold electrodes.

Alternative procedures for the preparation of hydroxythiols are known in the art. Au electrodes or surfaces are prepared by literature procedures, and modified hydroxythiols are adsorbed onto Au.

In a further embodiment, the modified nucleic acids of the invention are covalently attached to oxidized thin film surfaces. It has been reported that a variety of compounds can be covalently attached (as monolayers) to SnO thin film electrodes.₂, Uncle₂, RuO₂ and Pt. (see Lenhard, J. and Murray, RJ Electroanal. Chem. 78: 195 (1977)). The reversible electrochemistry of surface-bound complexes, such as 3,5-dinitrobenzamide, has been observed. The complexes described are attached to the electrode through an amide bond anchor. Using these literature procedures, analogous derivatives can be prepared, using amino modified oligonucleotides described in this work, and are schematically represented in Figure 8B.

IS 2 198 486 T3

Consequently, using the above procedures, the oligonucleotides could be attached to a solid support in such a way that the electrode serves as one or the other, or as an electron donor group, or as an electron acceptor group.

Thus, all combinations of electron donors and acceptors can be made: two transition metal complexes; two organic electron transfer species; a transition metal, an organic group; a transition metal and an electrode; and an organic group and an electrode. The choice of electron transfer species will depend in part on the initiation and detection procedure required, as described in more detail below.

The term "target sequence", or grammatical equivalents thereof, refers to a nucleic acid sequence on a single nucleic acid strand. The target sequence could be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, mRNA, or others. It could be of any length, understanding that longer sequences are more specific. As outlined in more detail below, probes are constructed to hybridize to target sequences to determine the presence or absence of the target sequence in the sample. Generally speaking, this term will be understood by those of skill in the art.

The probes of the invention were designed to be complementary to the target sequence, such that hybridization of the target sequence and the probes of the present invention occurs. As outlined above, this complementarity does not have to be perfect; there could be any number of mismatches, which would interfere with hybridization between the target sequence and the single-stranded nucleic acids of the present invention. However, if the number of mutations is such that hybridization cannot occur even under the least stringent hybridization conditions, the sequence is not a complementary target sequence.

A variety of hybridization conditions could be used in the present invention. As is well known in the art, "high" stringency usually refers to conditions such as 0.1XSSC at 65 ° C, low stringency conditions include 2-5XSSC at 25-50 ° C. Hybridization conditions could also vary when using a nonionic backbone, such as PNA, as is well known in the art.

The terms "first target domain" and "second target domain", or grammatical equivalents herein, refer to two portions of a target sequence within a nucleic acid that is under examination. The first target domain could be directly adjacent to the second target domain, or the first and second target domains could be separated by an intermediate target domain. The terms "first" and "second" are not intended to confer an orientation to the sequences with respect to the 5'-3 'orientation of the target sequence. For example, assuming a 5'-3 'orientation of the complementary target sequence, the first target domain could be located either 5' to the second domain, or 3 'to the second domain.

The present invention is directed, in part, to site-selective modification of nucleic acids with active redox groups, such as transition metal complexes, for the preparation of a new series of biomaterials capable of transferring electrons over long distances across nucleic acid matrix. The present invention provides means for the precise placement of electron donor and acceptor groups, at predetermined sites, on a single-stranded or double-stranded nucleic acid. In general, electron transfer between donor and electron acceptor groups in a double-stranded nucleic acid does not occur at an appreciable rate unless there is nucleotide base pairing in sequence between donor and electron acceptor in the sequence. double helix structure.

This difference in electron transfer rate forms the basis of a utility of the present invention for use as a probe. In the system of the present invention, when the electron transfer groups are covalently attached to the nucleic acid backbone, the electrons putatively travel through the π orbitals of the stacked base pairs of the nucleic acid double strand. The rate of electron transfer depends on several factors, including the distance between the electron donor-acceptor pair, the free energy (AG) of the reaction, the rearrangement energy (λ), the contribution of the intervening medium, the orientation and the electronic coupling of the donor and acceptor pair, and the hydrogen bonds between the bases.

The contribution of the intervening medium depends, in part, on the number of sigma (σ) bonds that the electron must traverse, from the electron donor, to reach the base stack, or to exit the stack to reach the electron acceptor. As shown in Figure 5, when the metal is attached to the ribose-phosphate backbone through an amide group on the 2 'carbon of ribose, an electron must travel through four σ bonds to reach the stack: the metal-to-nitrogen bond, the nitrogen-to-carbon 2 'bond, and from the 2' carbon to the base, or vice versa, depending on the direction of the electron flow. Since the base of the nucleotide is conjugated to some degree, the base can be considered to be at the edge of the "π pathway", that is, the conjugated π orbitals of the base pair stack. When the metal is attached to the ribose-phosphate backbone through the 3 'carbon of ribose, an electron must pass through 5 σ bonds. When metal is joined through phosphoramid-type bonds, one electron must pass through 7 σ bonds. In preferred embodiments, the compositions of the invention are designed such that the electron transfer groups are as close to the "π pathway" as possible without significantly altering the secondary and tertiary structure of the nucleic acid double helix, particularly WatsonCrick base pairing.

IS 2 198 486 T3

The effect of hydrogen bonding between bases on the electron transfer rate is a dependence on the particular nucleic acid sequence, since AT pairs contain less hydrogen bonding than CG pairs. However, this sequence dependence is masked by the determination that there is a measurable difference between the rate of electron transfer within a DNA base pair array, and the rate through the ribose-phosphate backbone, the solvent, and other electron tunnels. This speed difference is believed to be at least several orders of magnitude, and could be as high as four orders of magnitude greater across the stacked nucleotide bases compared to other electron transfer pathways. Thus, the presence of double-stranded nucleic acids can be determined, for example, in gene probe assays, by comparing the electron transfer rate of the unhybridized probe with the rates of the hydridated probes.

In one embodiment, the present invention provides new gene probes, which are useful in molecular biology and diagnostic medicine. In this embodiment, single-stranded nucleic acids having a predetermined sequence are synthesized and covalently linked to electron donor and electron acceptor groups. The sequence is selected based on a known target sequence, such that if hybridization occurs with a complementary target sequence in the region between the electron donor and the electron acceptor, the electron transfer progresses at an appreciable and detectable rate. . Thus, the present invention has wide general use, as a new form of labeled gene probe. Furthermore, since non-hybridized probe electron transfer is negligible, the probes of the present invention allow detection of target sequences without suppression of the non-hybridized probe. Thus, the present invention is uniquely suited for automated gene probe assays or field assays.

In a preferred embodiment, the probes can be used in genetic diagnosis. For example, probes can be prepared using the techniques described herein to detect target sequences such as the genes for non-polyposis colon cancer, the BRCA1 breast cancer gene, P53, which is a gene associated with a variety of cancers. , the Apo E4 gene, which indicates an increased risk of Alzheimer's disease, allowing easy presymptomatic examination of patients, mutations in the cystic fibrosis gene, or any of the others well known in the art.

In a further example, bacterial or viral detection is performed using the complexes of the invention. In this embodiment, the probes are designed to detect target sequences from a variety of bacteria and viruses. For example, current blood testing techniques are based on the detection of anti-HIV antibodies. The procedures described herein allow direct examination of clinical samples for HIV nucleic acid sequences, particularly highly conserved HIV sequences. Furthermore, this allows direct monitoring of circulating viruses in a patient as an improved method of verifying the efficacy of anti-viral therapies. Similarly, the viruses associated with leukemia, HTLV-I and HTLV-II, could be detected in this way. Bacterial infections such as tuberculosis may also be detected.

In a preferred embodiment, the nucleic acids of the invention find use as probes for toxic bacteria in the examination of food and water samples. For example, samples could be treated to lyse the bacteria and release their nucleic acid, and then probes designed to recognize bacterial strains, including, but not limited to, pathogenic strains such as Salmonella, Campylobacter, Vibrio cholera, enterotoxic E. . coli, and Legionnaires' disease bacteria. Similarly, bio-healing strategies could be evaluated using the compositions of the invention.

In a further embodiment, the probes are used in "DNA fingerprinting" to match DNA from a crime scene with samples taken from victims and suspects.

The present invention also finds use as a unique methodology for the detection of mutations in target nucleic acid sequences. As a result, if a single-stranded nucleic acid containing electron transfer groups hybridizes to a target sequence with a mutation, the resulting disturbance of nucleoside base pairing will measurably affect the rate of electron transfer. This is the case if the mutation is a substitution, insertion, or deletion. Alternatively, two single-stranded nucleic acids, each with a covalently linked electron transfer species, hybridizing adjacent to a target sequence could be used. Accordingly, the present invention provides means for the detection of mutations in target sequences.

Thus, the present invention provides extremely specific and sensitive probes, which could, in some embodiments, detect target sequences without removing unhybridized probes. This will be useful in generating automated gene probe assays.

In an alternative embodiment, the double-stranded nucleic acids have electron donor and electron acceptor groups covalently attached on opposing strands. Such nucleic acids are useful for detecting successful gene amplification in polymerase chain reactions (PCR), thus allowing successful PCR reactions to be an indication of the presence or absence of a target sequence. PCR could be used in this way in a number of ways. For example, if one of the two PCR primers contains a 5 'end-linked electron donor, and the other contains a 5' end-linked electron electron acceptor, several rounds of PCR will generate double fragments. doubly marked strand (occasionally known as

ES 2 198 486 T3 "amplicons"). After appropriate photoinduction, electron transfer detection provides an indication of successful amplification of the target sequence compared to when no amplification occurs. A particular advantage of the present invention is that the separation of single-stranded primers from amplified double-stranded DNA is not necessary, as outlined above for probe sequences containing electron transfer groups. Alternatively, detection of a target sequence through PCR is accomplished by linking a species with an electron transfer group to one or both of the primers. The other species with an electron transfer group binds individual nucleosides from the whole PCR reaction, as described herein. Incorporation of nucleosides containing the electron transfer group into the nucleic acid during the PCR reaction results in both electron transfer species attached to the same or opposite strands, or both. Allowing the newly synthesized nucleic acid to remain in a hybrid form allows the detection of successful elongation through electron transfer, and thus the detection of a target sequence. Thus, the present invention is used for the PCR detection of target sequences.

In another embodiment the present invention provides double-stranded nucleic acid with electron donor and electron acceptor groups covalently attached to serve as bioconductors or "molecular splices". Electron transport could occur over distances up to and in excess of 28 x 10^-10 m (28 Angstroms) per pair of donor and electron acceptor. Furthermore, the electron transfer rate is very fast, although it depends on the distance between the electron donor and acceptor groups. By modifying nucleic acid at regular intervals with electron donor and / or electron acceptor groups, it might be possible to transport electrons over great distances, thus creating bioconductors. These bioconductors are useful in a large number of applications, including traditional applications for conductors, such as reaction mediators and electrochemical processes.

In addition, these bioconductors could be useful as probes for photosynthesis reactions, as well as in the construction of artificial light harvesting systems. Current models for the electron transfer component of an artificial light harvesting system have several problems, as outlined above, including a dependence on solvent polarity and composition, and an absence of sufficient rigidity without arduous synthesis. Thus, the present invention is useful as both a novel form of bioconductor, as well as a new gene probe.

The present invention provides nucleic acids with covalently attached electron transfer groups. Electron transfer groups could be attached to nucleic acid at a variety of positions.

In one embodiment, the electron donor and acceptor groups are added to the 3 'and / or 5' ends of the nucleic acid, on one of the two, the sugar-phosphate backbone or on a terminal base. In alternative embodiments, the electron donor and acceptor groups are added to the backbone of one or more internal nucleosides, that is, any nucleoside that is not the 3 'or 5' nucleoside. In a further embodiment, the electron donor and acceptor groups are added to the backbone of both the internal and terminal nucleosides.

In a preferred embodiment, the electron transfer groups are added to the ribose phosphate backbone at a number of positions. As shown in Figure 5, several positions are possible, with ribose binding of a ribose-phosphate backbone being particularly preferred. Accordingly, in Figure 5, the most preferred site for attachment of an electron transferring group is M1, followed by M4, M2, and M3, in that order. In a preferred embodiment, the electron transfer groups are attached to the 2 'or 3' positions of the ribose, with the 2 'being particularly preferred.

In a preferred embodiment, the electron transfer groups do not intersperse, and are linked so that they do not intersperse. Thus, while it is possible to use a "linker", such as alternating double bonds, to attach the electron transfer group to the nucleic acid, the linker is preferably no longer than the equivalent of one or two nucleosides in length, or not it is significantly flexible to allow for collation. Preferably, if linkers are used, they are attached through the ribose of the nucleic acid backbone.

In one embodiment, the electron transfer groups are attached to the bases of the terminal nucleosides. Thus, when the target sequence to be detected is n nucleosides in length, a probe can be prepared that has an extra terminal nucleoside at one or both nucleic acid ends (n + 1 or n + 2), which is used to covalently link the nucleosides. electron transfer groups, but does not participate in base pair hybridization. This extra terminal nucleoside is important since the attachment of electron transfer groups to a base of an internal nucleoside is expected to disrupt the Watson-Crick base pairing. That is, the base used for covalent attachment should be outside the region used to identify the target sequence. Additionally, it is preferred that, upon probe hybridization, the terminal nucleoside containing the electron transfer group covalently attached to the base is directly adjacent to the nucleosides of the Watson-Crick base pairs; that is, the electron transfer group should be as close as possible to the stacked π orbitals of the bases, such that an electron travels through a minimum of σ bonds to reach the “π path”, or alternatively, you can contact the π path electronically.

In one embodiment, a single-stranded nucleic acid is labeled with an electron transfer group through the terminal bases at both ends. Alternative embodiments use a terminal base and a 5 'or 3' ribosephosphate anchor as described above. In other embodiments, compositions are provided that comprise

ES 2 198 486 T3 ignite a first single-stranded nucleic acid containing an electron donor covalently attached to a terminal base, and a second single-stranded nucleic acid containing an electron acceptor covalently attached at a position as described above. , that is, in a 5 ', 3' or internal position; alternatively, the electron donor and the electron acceptor could be exchanged. A particularly preferred embodiment uses an electrode as one of the electron transfer groups, the other electron transfer group being attached to a terminal base, preferably on the same strand.

The present invention further provides methods for site-specific addition of electron transfer groups to nucleic acids. As outlined above, electron transfer groups could be added at the 2 'or 3' position of a ribose on the ribose phosphate backbone, to a 3 'or 5' terminal base, or to an internal nucleoside using linkages. peptide-nucleic acid, phosphoramidate linkages, phosphorothioate linkages, phosphorodithioate linkages, or O-methyl phosphoramidate linkages.

Molecular mechanism calculations indicate that disturbances due to ribose modification of nucleic acid terminal nucleosides are minimal, and that Watson-Crick base pairings are not altered (unpublished data using Biograf from Molecular Simulations Inc. , San Diego, CA).

For alaribose linkages, a preferred embodiment uses nucleosides modified to link the electron transfer group. Preferably amino-modified nucleosides and nucleosides are used. In an alternative embodiment, thio-modified nucleosides are used to anchor the electron transfer groups of this invention.

The modified nucleosides are then used to specifically add a transition metal electron transfer group at a site, at either one of the 3 'or 5' ends of the nucleic acid, or to any internal nucleoside. Or the 2 'or 3' position of ribose could be altered for binding at the 3 'end; for binding to an internal ribose or to the 5 'end, the 2' position is preferred. Thus, for example, the ribose 2 'position of the deoxyribo- or ribonucleoside is modified prior to the addition of the electron transfer species, leaving the ribose 3' position unchanged for subsequent linkages to the chain if necessary. . In a preferred embodiment, an amino group is added to the 2 'or 3' carbon of the sugar using established chemical techniques (Imazawa et al., J. Org. Chem. 44: 2039 (1979); Hobbs et al., J. Org. Chem. 42 (4): 714 (1977); Verheyden et al., J. Org. Chem. 36 (2): 250 (1971)).

The amino-modified nucleosides prepared as described above are converted to the 2 'or 3' modified nucleotide triphosphate form using standard biochemical procedures (Fraser et al., Proc. Natl. Acad. Sci. USA 4: 2671 (1973)).

Modified nucleosides for the attachment of electron transfer groups to bases are prepared as outlined in Telser, see above. These modified nucleosides are then incorporated at one of the 3 'or 5' ends as outlined above.

Once the modified nucleosides have been prepared, protected, and activated, they could be incorporated in various ways into a growing oligonucleotide using standard synthetic techniques (Gait, Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, UK, 1984; Eckstein) . In one embodiment, one or more modified nucleosides are incorporated into a growing oligonucleotide strand using standard molecular biology techniques such as with the use of DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, DNA Taq polymerase, reverse transcriptase, and RNA polymerases. For the incorporation of a 3 'modified nucleoside into a nucleic acid, terminal deoxynucleotidyl transferase (Ratliff, Terminal deoxynucleotidyltransferase. In "The Enzymes", Vol 14A, Ed. PD Boyer, pp. 105-118, Academic Press , San Diego, CA, 1981). Alternatively, and preferably, the amino-nucleoside is converted to the phosphoramidite or H-phosphonate form, which are then used in solid phase or solution oligonucleotide synthesis. In this way the modified nucleoside, either for binding to ribose (ie amino- or thiol-modified nucleosides) or to the base, is incorporated into the oligonucleotide at either an internal position or at the 5 'end. This is generally done by protecting the 5 'position of the ribose with 4', 4-dimethylxytrityl (DMT) followed by reaction with 2-cyanoethoxy-bis-diisopropylaminophosphine, in the presence of diisopropylammonium tetrazolide, to yield the phosphoramidite, as is known in the art. ; although other techniques could be used, as will be appreciated by those skilled in the art. See Gait, above; Caruthers, Science 230: 281 (1985).

For attachment of an electron transfer group to the 3 'end, a preferred procedure uses anchoring the modified nucleoside to controlled pore glass (CPG) or other polymeric supports. In this embodiment, the modified nucleoside is protected at the 5 'end with DMT, and then reacted with succinic anhydride with activation. The resulting succinyl compound is attached to CPG or other polymeric supports as is known in the art. Other nucleoside phosphoramidites, whether modified or not, are added to the 5 'end after deprotection.

In another embodiment, the electron transfer group (s) are added to the middle of the nucleic acid, that is, to an internal nucleoside. This could be achieved in three ways.

In a preferred embodiment, a modified nucleoside is incorporated at the 5 'end as described above. In this embodiment, the synthesis of the oligonucleotide simply extends the 5 'end from the nucleoside

ES 2 198 486 T3 modified using standard techniques. This results in an internally amino-modified oligonucleotide.

In one embodiment, the nucleosides are modified to contain an aromatic amine capable of binding an electron transfer group at one of the 2 'or 3' positions of ribose. For example, one of the imidazole nitrogens can be attached at the 2 'or 3' position of ribose, and thus used to attach the electron transferring group, such as a transition metal complex. This could effectively reduce the number of σ bonds an electron must travel to reach the "pi pathway" since imidazole offers substantially less resistance to electron transfer compared to a σ bond. In a preferred embodiment, the imidazole is attached to the 2 'position of ribose. In an alternative embodiment, the imidazole is attached to the 3 'position. The imidazole modified nucleoside could be incorporated into an oligonucleotide as outlined herein for amino modified nucleosides.

In an alternative embodiment, the electron transfer groups are added to the backbone at a site other than ribose, resulting in an internal anchor. For example, phosphoramide linkages can be used instead of phosphodiester as the site for transition metal modification. These transition metals serve as donors and acceptors for electron transfer reactions. Although structural deviations from native phosphodiester bonds do occur and have been studied using CD and NMR (Heller, Acc. Chem. Res. 23: 128 (1990); Schuhmann et al., J. Am. Chem. Soc. 113: 1394 (1991)), the inter-nucleotide phosphoramidite bond has been reported to bind complementary polynucleotides and to be stable (Beaucage et al., See above, and references therein; Letsinger, see above; Sawai, see above; Jager, Biochemistry 27: 7237 (1988)). In this embodiment, nucleotide dimers with phosphoramid linkages at one of the 2'-5 'or 3'-5' positions are created. A preferred embodiment uses the 3'-5 'position for the phosphoramide bond, such that subsequent Watson-Crick base pair structural alteration is minimized. These dimer units are incorporated into a growing oligonucleotide chain, as above, at defined intervals, as outlined below.

Thus, the present invention provides methods for preparing nucleic acid with covalently attached electron transfer groups. In a preferred embodiment, the method is to prepare a nucleic acid with an electron transfer group attached at the 3 'end of said nucleic acid. The method comprises attaching a 2 'modified nucleoside to controlled pore glass, and adding phosphoramidite nucleosides to the 5' end of the modified nucleoside to form a nucleic acid. The nucleic acid is then optionally cut from the CPG using known procedures. The nucleic acid could hybridize with its complement, if required, to protect the bases from modification, and the electron transfer group is added to the 2'-amino modified nucleoside.

In a preferred embodiment, methods are provided for preparing a nucleic acid with an electron transfer group attached at the 5 'end. The method comprises attaching a nucleoside to controlled pore glass, and adding phosphoramidite nucleosides to the 5 'end of the nucleoside to form a nucleic acid. A 2 'or 3' amino modified nucleoside is added to the 5 'end, and the nucleic acid is optionally cut from the CPG. The nucleic acid could hybridize to its complement, if required, and the electron transfer group added to the 2'-amino or 3'-amino modified nucleoside.

In a preferred embodiment, a process is provided for preparing a single-stranded nucleic acid with electron transfer groups attached to both the 3 'and 5' ends. The method comprises attaching a modified nucleoside to controlled pore glass. The modified nucleoside could be modified with modified for binding through ribose as described therein, or modified at the base. Additional phosphoramidite nucleosides are added to the 5 'end of the modified nucleoside to form a nucleic acid. An extra modified phosphoramidite nucleoside is added to the 5 'end of the nucleic acid, which is then optionally separated from the controlled pore glass, and could hybridize to its complement. An electron donor group is added to a modified nucleoside and an electron acceptor group is added to another modified nucleoside.

Cutting of the CPG could occur either before the transition metal modification or after.

It should be understood that it is important that the base pairing is not significantly disturbed in order to allow hybridization, good electron transfer rates, and detection of mismatches. Thus, for example, transition metal groups, when attached to nucleic acids of the invention, do not intersperse, that is, they do not insert and stack between the base pairs of the nucleic acid double strand. Intercalation of transition metals with their accompanying ligands disrupts base pairing, thus preventing electron transfer and identification of mismatches. Similarly, with the exception of terminal bases, as outlined above, docking of transition metal complexes to nucleoside bases (Telser et al., See above) also alters base pairing and prevents the identification of mismatches.

It should be noted that, when using the above techniques for the modification of internal residues, it is possible to create a nucleic acid that has an electron transfer species in the nucleoside next to the last one at the 3 'end, thus eliminating the need for the extra steps. required to produce the 3 'terminally labeled nucleoside.

In a further embodiment for modification of internal residues, 2 'or 3' modified nucleoside triphosphates are generated using the techniques described above for 3 'nucleoside modification. Nucleosides

Modified ES 2 198 486 T3 are inserted internally into nucleic acid using standard molecular biology techniques for DNA and RNA labeling. Enzymes used for such labeling include DNA polymerases, such as polymerase I, T4 DNA polymerase, T7 DNA polymerase, DNA Taq polymerase, reverse transcriptase, and RNA polymerases such as polymerase. RNA from E. coli or RNA polymerases from Sp6, T7 or T3 phages (Short Protocols in Molecular Biology. 1992, Ed. Ausubel et al., pp. 3.11-3.30).

As described above, the electron transfer group, preferably a transition metal complex, could bind to any of the five bases (adenine, thymine, uracil, cytosine, guanine, and other non-naturally occurring bases, such as inosine, xanthine, and hypoxanthine, among others). This is done using well known techniques; see Telser et al., J. Am. Chem. Soc. 111: 7226-7232 (1989); Telser et al., J. Am. Chem. Soc. 111: 72217226 (1989). As outlined herein, these terminally modified nucleosides could be linked to nucleic acid enzymatically as is known in the art, using DNA polymerases; alternatively, the modified nucleosides could be incorporated into a growing oligonucleotide chain using traditional phosphoramidite chemistry during oligonucleotide synthesis, as outlined herein.

The exposed amine or other ligand at the 2 'or 3' position of ribose, the phosphoramid linkages, or the other linkages useful in the present invention, are easily modified with a variety of electron transfer groups, and particularly with metal complexes. transition, with techniques readily known in the art (see for example, Millet et al., in Metals in Biological Systems, Eds. Sigel et al., Vol. 27, pp. 223-264, Marcell Dekker Inc., New York, 1991, Durham et al., In ACS Advances in Chemistry Series, Eds. Johnson et al., Vol. 226, pp. 180-193, American Chemical Society, Washington DC; and Meade et al., J. Am. Chem. Soc. 111: 4353 (1989)). Generally, these techniques involve contacting a partially chelated transition metal complex with the amino group of the modified nucleoside.

The electron transfer species are also added to the functional group of the modified nucleosides, such as an amino group, using techniques known in the art.

When peptide nucleic acids (PNAs) are used, the docking of the electron transfer groups proceeds as follows. A partially chelated transition metal or organic electron transfer group will be attached to the amino group at the N-terminus of the PNA in a manner similar to amino modified ribose. The addition to the carboxy-terminal end can proceed in a number of ways, one of which is illustrated in Figure 7. Additionally, for single-stranded PNAs, one electron transferring group could be attached to the N-terminal end, and the other electron transferring group could be attached to the base terminal at the carboxy-terminal end. Alternatively, both transfer groups are attached to the terminal bases. Similar combinations could be made for two single-stranded nucleic acids, each containing an electron transferring group.

Furthermore, the present invention provides a novel method for site-specific addition of a nucleic acid of electron donor and electron acceptor groups to a previously modified nucleoside at a ribose-phosphate backbone.

In one embodiment, the electron donor and electron acceptor groups are attached to the modified nucleoside by procedures using a single protective hybridization step. In this embodiment, the modified single-stranded nucleic acid hybridizes to an unmodified complementary sequence. This blocks sites on heterocyclic bases that are susceptible to being attacked by transition metal electron transfer species.

When the terminal bases have been labeled with electron transfer species, the complementary sequence does not extend to the base to be labeled. That is, a complementary sequence of n nucleosides in length is chosen for hybridization with a probe sequence of n + 1 or n + 2, such that the terminal base is not protected. Thus, the unprotected base is exposed to the electron transfer group, such that the group binds to the base.

After successful addition of the desired metal complex, the modified nucleic acid duplex is separated into single strands using techniques well known in the art.

In a preferred embodiment, single-stranded nucleic acids are prepared that contain an electron donating group and an electron accepting group. The electron donor and electron acceptor groups could be attached at either the 5 'or 3' ends of the single-stranded nucleic acid. Alternatively, the electron transfer groups could be attached to internal nucleosides, or one to an internal nucleoside and the other to a terminal nucleoside. It should be understood that the orientation of the electron transfer species relative to the 5'-3 'orientation of the nucleic acid is not determinative. Thus, as outlined in Figure 1, any combination of internal and external nucleosides could be used in this embodiment.

In an alternative preferred embodiment, single-stranded nucleic acids with at least one electron donating group and at least one electron accepting group are used to detect mutations in a complementary target sequence. A mutation, whether it is a substitution, insertion, or deletion of a nucleoside or nucleosides, results in base mismatch in a hybridized nucleic acid double helix. Consequently, if the path of an electron from an electron donor group to an electron acceptor group spans the region where the mismatch is found, electron transfer will be eliminated or reduced so that a change will be observed.

ES 2 198 486 T3 in relative speed. Thus, in this embodiment, the electron donor group is attached to the nucleic acid at a 5 'position to the mutation, and the electron acceptor group is attached at a 3' position, or vice versa.

In this embodiment it is also possible to use an additional label on the modified single-stranded nucleic acid to detect hybridization when there are one or more mismatches. If the complementary nucleic acid contains a mutation, electron transfer is reduced or eliminated. For current as a control, the modified single-stranded nucleic acid could be radioactively or fluorescently labeled so that hybridization with the target sequence could be detected, according to traditional techniques of molecular biology. This allows the determination that the target sequence exists, but contains a substitution, insertion, or deletion of one or more nucleosides. Alternatively, single-stranded nucleic acids, with at least one electron donor group and one electron acceptor group, that hybridize to exactly matched regions can be used as a control for the presence of the target sequence.

It should be understood that the rate of electron transfer through a double-stranded nucleic acid helix depends on the nucleoside distance between the electron donor and electron acceptor groups. Longer lengths will have slower speeds, and the consideration of speeds will be a parameter in the design of probes and bioconductors. Thus, while it is possible to measure velocities for distances in excess of 100 nucleosides, a preferred embodiment has the electron donor group and the electron acceptor group separated by at least 3 and not more than 100 nucleosides. More preferably, the groups are separated by 8 to 64 nucleosides, with 15 being the most preferred distance.

Furthermore, it should be noted that certain distances could allow the use of different detection systems. For example, the sensitivity of some detection systems could allow the detection of extremely high speeds; that is, the electron transfer groups could be closer together.

Other detection systems might require slightly slower speeds, and thus allow the electron transfer groups to be further apart.

In an alternative embodiment, a single-stranded nucleic acid is modified with more than one electron donor or electron acceptor group. For example, to increase the signal obtained from these probes, or decrease the required detector sensitivity, multiple sets of electron donor-electron acceptor pairs could be used.

As outlined above, in some embodiments different electron transfer groups are added to a single-stranded nucleic acid. For example, when an electron donor group and an electron acceptor group, or several electron donors or electron acceptors, must be added, the synthesis of the single-stranded nucleic acid proceeds in several steps. Nucleic acid sequences are constructed first, each containing a single electron transfer species, ie, either a single electron transfer group or several of the same transfer groups, using the techniques outlined above. These partial nucleic acid sequences are then ligated together using techniques common in the field, such as hybridization of the individual modified nucleic acid partials to a single complementary strand, followed by ligation with a commercially available ligase.

Alternatively, the single-stranded nucleic acid could be constructed by incorporating an amino-modified nucleoside at two positions using the above techniques. As a result of the synthesis, one of the amino modified nucleosides temporarily has a protecting group on the amine, such as DMT. Upon hybridization to the unmodified complementary strand, the deprotected amine is exposed to the first electron transferring group, ie, either a donor or an acceptor, resulting in covalent anchoring. The protecting group of the protected amino modified nucleoside is then removed, and the hybrid is contacted with the second electron transfer species, and the strands are separated, resulting in a single strand that is labeled with both the donor and the acceptor. The single strand containing the appropriate electron transfer group is then purified using traditional techniques.

In a preferred embodiment, single-stranded nucleic acids are prepared that contain an electron donating group or an electron accepting group. The electron donor or electron acceptor groups are attached at either the 5 'or 3' ends of the single-stranded nucleic acid. Alternatively, the electron donor group is attached to an internal nucleoside.

It should be understood that different species of electron donor and electron acceptor groups could be attached to a single stranded nucleic acid. Thus, more than one type of electron donor group or electron acceptor group could be added to any single stranded nucleic acid.

In a preferred embodiment, a first single-stranded nucleic acid is prepared with one or more electron donor groups attached. A second single-stranded nucleic acid has one or more electron-withdrawing groups attached. In this embodiment, single-stranded nucleic acids are prepared for use as probes for a complementary target sequence. In one embodiment, the complementary target sequence is constructed up to a first target domain and a second target domain, where the first and second sequences are directly adjacent to each other. In this embodiment, the first single-stranded nucleic acid, containing only electron donor groups or

ES 2 198 486 T3 electron withdrawing groups but not both, hybridizes to the first target domain, and the second single-stranded nucleic acid, containing only the corresponding electron transferring species, binds to the second target domain. The relative orientation of the electron transferring species is not important, as outlined in Figure 2, and the present invention is intended to include all possible orientations.

In designing a probe consisting of two single-stranded nucleic acids that hybridize to adjacent first and second target sequences, several factors should be considered. These factors include the distance between the electron donating group and the electron accepting group in the hybridized form, and the length of the individual single-stranded probes. For example, it might be desirable to synthesize only 5 'terminally labeled probes. In this case, the single-stranded nucleic acid that hybridizes to the first sequence could be relatively short, such that the desirable distance between the probes could be achieved. For example, if the optimal distance between the electron transfer groups is 15 nucleosides, the first probe could be 15 nucleosides in length.

In one aspect of this embodiment, the two single-stranded nucleic acids that have hybridized to the adjacent first and second domains are ligated together prior to the electron transfer reaction. This could be done using standard molecular biology techniques using a DNA ligase, such as T4 DNA ligase.

In an alternative embodiment, the complementary target sequence will have a first target domain, an intervening target domain, and a second target domain. In this embodiment, the first modified single-stranded nucleic acid, containing only electron donating groups or electron accepting groups, but not both, hybridizes to the first target domain, and the second single-stranded modified nucleic acid, containing only the corresponding electron transfer species binds to the second target domain. When an intervening single-stranded nucleic acid hybridizes to the intervening target sequence, transfer between the electron donor and the electron acceptor is possible. The intervening sequence could be any length, and could comprise a single nucleoside. However, its length should take into account the desirable distances between the electron donor and electron acceptor groups on the first and second modified nucleic acids. Intervening sequences with lengths greater than 14 are desirable since, if longer sequences are used, the intervening sequence is more likely to remain hybridized to form a double-stranded nucleic acid. The presence or absence of an intervening sequence can be used to detect insertions or deletions.

In one aspect of this embodiment, the first single-stranded nucleic acid hybridized to the first target domain, the intervening nucleic acid hybridized to the intervening domain, and the second single-stranded nucleic acid hybridized to the second target domain, could be ligated together prior to removal. electron transfer reaction. This could be done using standard techniques in molecular biology. For example, when the nucleic acids are DNA, a DNA ligase can be used, such as T4 DNA ligase.

The complementary single strand nucleic acid of the present invention could take many forms. For example, the complementary target single-stranded nucleic acid sequence could be contained in a larger nucleic acid sequence, ie, all or part of a gene or mRNA, a restriction fragment of a plasmid, or genomic DNA, among others. One trained in the field of molecular biology will understand how to construct useful probes for a variety of target sequences using the present invention.

In one embodiment, two single-stranded nucleic acids with covalently attached electron transfer groups have complementary sequences so that they can hybridize together to form a bioconductor. In this embodiment, the hybridized duplex is capable of transferring at least one electron from the electron donating group to the electron accepting group. In a preferred embodiment, the individual single-stranded nucleic acids are aligned so that they have blunt ends; In alternative embodiments, the nucleic acids are aligned such that the double helix has sticky ends. In either embodiment it is preferred that you find an unbroken double helix base pairing between the electron donating group and the electron accepting group such that the electrons travel through the base pair stack.

In a bioconductor embodiment, the double-stranded nucleic acid has a single-stranded nucleic acid that carries all of the electron transfer groups.

In another embodiment, the electron transfer groups could be on one of the two strands, and in either orientation. For example, one strand could carry only electron donors, the other only electron acceptors, or both strands could carry both.

In one embodiment, the double-stranded nucleic acid could have different electron transfer groups covalently attached in a fixed orientation, to facilitate electron transfer over long distances. This type of system takes advantage of the fact that electron transfer species can act as both electron donors and electron acceptors, depending on their oxidative state. In this way, an electron donor group, after the loss of an electron, could act as an electron acceptor, and vice versa. Thus, the electron transfer groups could be oriented sequentially on any of the strands of the double-stranded nucleic acid, such that directional transfer of an electron over very long distances could be achieved. For example, a double-stranded nucleic acid could contain a single electron donor group at one end and electron acceptor groups, of the same or different composition, throughout the molecule. In this way a cascade effect of electron transfer could be achieved, the

ES 2 198 486 T3 which could result in extremely long electron transfer ranges. This could be achieved, for example, by incorporating transition metal complexes that possess a range in oxidation potentials due to ligand substitutions made at the metal center.

The choice of specific electron donor and electron acceptor pairs will be influenced by the type of electron transfer measurement used; for a review, see Winkler et al., Chem. Rev. 92: 369379 (1992). When a long-lived excited state can be prepared at one of the redox sites, direct measurement of the electron transfer rate after photoinduction can be measured, using, for example, the flash quenching procedure of Chang et al., J. Am. Chem. Soc. 113: 7057 (1991). In this preferred embodiment, the excited redox site, being both a better acceptor and donor than the species in the ground state, can transfer electrons to or from the redox pair. An advantage of this procedure is that two electron transfer rates could be measured: photoinduced electron transfer rates and thermal electron-hole recombination reactions. In this way they could differential rates for hybridized nucleic acids with perfect complementarity and nucleic acids with mismatches.

In alternate embodiments, no redox site has a long-lived excitation state, and the measurement of electron transfer depends on the biomolecular generation of a kinetic intermediate. For a review, see Winkler et al., Above. This intermediate is then relaxed to the thermodynamic product through intramolecular transfer using a quencher, as seen below:

DA + hv -> DA^-

GIVES^- + Q -> DA⁺ + Q^-

GIVES⁺ -> D⁺-TO

D⁺-A + Q^- -> DA + Q

The upper limit of measurable intramolecular electron transfer rates using this procedure is approximately 10⁴ per second.

Alternative embodiments use radiolytic pulse generation of oxidizing and reducing radicals, which inject electrons into a donor or withdraw electrons from a donor, as reviewed in Winkler et al., See above.

As will be appreciated in the art, there are a variety of ways to initiate and detect electron transfer.

Electron transfer can be initiated and detected using a wide variety of procedures, including, but not limited to, electrical, electrochemical, electromagnetic (optical) radiation, and chemical procedures. It is possible to prepare a variety of compositions using different electron transfer groups depending on the desired electron transfer initiation and transfer detection procedure. TABLE 2 illustrates a variety of preferred combinations for initiation and detection of electron transfer in complexes of the invention.

TABLE 2

<td>Initiation</td><td>Detection</td><td>Description</td>

<td>light</td><td>light</td><td>absorbance, fluorescence, phosphorescence, refractive index, surface plasmon resonance, electron spin resonance.</td>

<td>light</td><td>current</td><td>amperimetry, voltimetry, capacitance, impedance, opto-electronic sensing, photo-amperimetry</td>

<td>light plus electronic initiation</td><td>light</td><td>absorbance, fluorescence, phosphorescence, refractive index, surface plasmon resonance, electron spin resonance.</td>

<td>light plus electronic initiation</td><td>current</td><td>amperimetry, voltmeter, capacitance, impedance, opto-electronic detection, photo-amperimetry, amperometric detection, cyclic voltmeter.</td>

<td>initiation electronics</td><td>current</td><td>amperimetry, voltmeter, capacitance, impedance, amperometric detection, cyclic voltmeter.</td>

<td>initiation electronics</td><td>light</td><td>chemiluminescence, electrochemiluminescence, electroluminescence.</td>

IS 2 198 486 T3

By "light" herein is meant electromagnetic radiation, with light in the UV, visible, and infrared range being preferred, and UV and infrared being most preferred.

In a preferred embodiment, the initiation of electron transfer is through direct or indirect photoactivation ("light ingress"). Simply, electromagnetic radiation of the appropriate wavelength strikes the redox molecule at one end of the DNA, causing the excitation of an electron from the donor group, which immediately decays or is involved in intramolecular electron transfer. The efficiency with which electron transfer is induced depends on the electronic coupling between the donor and the electron acceptor, and therefore depends on whether the nucleic acid is single-stranded or double-stranded. Furthermore, the efficiency of electron transfer depends on the extinction coefficient of the electron donor at the wavelength used (longer is better) and the lifetime of the excited state of the electron donor (longer duration is better). Thus, preferred donor complexes include acridine orange, N, N'-dimethyl-2,7-diazapyrene dichloride (DAP²⁺), methylviologen, ethidium bromide, quinones such as N, N'-dimethylanthra (2, 1,9-def: 6.5, 10-d'e'f ') dichloride, diisoquinoline (ADIQ²⁺); porphyrins ([meso-tetrakis (N-methyl-x-pyridinium) porphyrin] tetrachloride). Transition metal donors and acceptors include the ruthenium, rhenium, and osmium complexes (most preferred), where at least one of the ligands is a chromophore.

Photoactivation can also be used to excite "mediators" that transfer energy to the electron donor group on the DNA through an intermolecular process. Such mediators include stable, water-soluble complexes of the transition metals, including the halides of molybdenum and tungsten, the trisbipyridyl complexes of rhenium, osmium, and ruthenium. Furthermore, other examples include bipyridyl and pyridyl complexes such as Re (bpy) (CO) 3X, where X is a halide and Re (py) 4O2. Other examples include transition metal dimers such as [Re2Cl8]²and [Pt2 (P2O5H2) 4]^4-. Ruthenium trisbipyridine (Ru²⁺(bpy) 3) is the most preferred.

In the preferred embodiment, electron transfer occurs after photoinduction with a laser. In this embodiment, the electron donor groups could, after donating an electron, serve as electron acceptors under certain circumstances. Similarly, electron acceptor groups could serve as an electron donor under certain conditions.

A preferred embodiment uses electronic actuation, with voltage being preferred. A potential is applied to a sample containing modified nucleic acid probes, either through direct binding of the modified nucleic acid to an electrode, or using mediators of electron transport. Direct bonding may involve a redox active polymer to transfer the electrons from (and to, if the electrode is also used for detection) the electrode. Such polymers are outlined below. Alternatively, the direct connection may involve a relatively poor conductive link, provided the link is kept reasonably short (less than six sigma links). Preferred bonds will be three or fewer sigma bonds in length to allow efficient transfer of electrons from the electrode, as outlined above.

Indirect initiation of electron transfer involves electron transfer mediators , or diffusionally efficient electron donor and electron acceptor, such as water-soluble ferrocene / ferricinium, hydroquinones / quinones, reducible and oxidizable organic salt components, cobaltocenes, the hexa- and octacyanides of molybdenum, tungsten and iron. In addition, other examples include macrocycles and chelating ligands of transition metals such as cobalt, ruthenium and nickel, including Co (ethylenediamine) 3 and Ru (ethylenediamine) 3, and the trisbipyridyl and hexamine complexes of transition metals such as Co, Ru, Fe, and Os. See K. Alyanasundaram, Coord. Chem. Rev. V. 46, p. 159, 1982. Finally, organic molecules such as 4,4-bipyridine and 4-mercaptopyridine are examples where ferrocene is most preferred.

Precise control and variations in applied potential can be done through a potentiostat and a system of three electrodes (one for reference, one for the sample, and a counter electrode). This allows the applied potential to be equalized to the peak electron transfer potential of the system, which depends in part on the choice of electron acceptors attached to the nucleic acid. The driving forces are achieved using transition metal bisbipyridyl complexes, eg, ruthenium rhenium bisbipyridyl complexes, such as (Ru (bpy) 2im-), as electron acceptors.

Alternatively, electrochemical initiation of electron transfer could be used. The redox states of the electron donor and electron acceptor groups attached to nucleic acid can be changed electrochemically using water soluble chemical reducing and oxidizing agents, with or without electrical activation or photoactivation. Such compounds include numerous derivatives known in the art (T. Kuwana, Electrochemical Studies of Biological Systems, Ed. DT Sawyer, ACS Symp. Series, # 38 (1977)), and include the hexacyan complexes of iron, zinc-mercury amalgam, and the trisphenanthroline complexes of ruthenium and iron.

Electron transfer through nucleic acid can be detected in a variety of ways. A variety of detection procedures could be used, including, but not limited to, optical detection, including fluorescence, phosphorescence, and refractive index; and electronic sensing, including, but not limited to, amperimetry, voltmeter, capacitance, and impedance. These procedures include time or frequency dependent procedures based on AC or DC currents, pulsed procedures, fixation techniques, filtering techniques (high pass, low pass, band pass) and time resolved, including fluorescence resolved in time. In some embodiments, all that is required is electron transfer detection;

ES 2 198 486 T3 in others, the electron transfer rate could be determined.

In one embodiment, efficient transfer of electrons from one end of a double-stranded nucleic acid to the other results in stereotyped changes in the redox state of both the electron donor and the electron acceptor. With many electron transfer groups including the ruthenium complexes containing bipyridine, pyridine, and imidazole rings, these changes in the redox state are associated with changes in spectral properties ("lights out"). Significant differences in absorbance are observed between the reduced and oxidized states of these molecules. These differences can be monitored using a spectrophotometer or a simple photomultiplier tube apparatus.

In this embodiment, potential electron donors and acceptors include all derivatives listed above for photoactivation or initiation. Preferred electron donors and acceptors have characteristically large spectral changes from oxidation and reduction (large "deltas" in extinction coefficients), resulting in highly sensitive monitoring of electron transfer. Such examples include Ru (NH3) 4 py and Ru (bpy) 2im as preferred examples. It should be understood that only the donor or acceptor being monitored by absorbance has to have ideal spectral characteristics. That is, the electron acceptor can be optically invisible if only the electron donor is monitored for changes in absorbance.

In a preferred embodiment, electron transfer is detected fluorometrically. Many transition metal complexes, including those of ruthenium, have characteristic fluorescent properties. Thus, the change in the redox state of electron donors and electron acceptors bound to nucleic acid can be monitored very sensitively using fluorescence. The highly efficient transfer of electrons through double-stranded nucleic acid can, for example, result in the production of Ru (4,7-biphenyl2-phenanthroline) 3²⁺ at one end of a nucleic acid probe when the electron transfer group at the other end is excited. The production of this compound can be easily measured using standard fluorescence assay techniques. For example, laser-induced fluorescence can be recorded on a standard single-cell fluorimeter, a flow-through “in-line” fluorimeter (such as those attached to a chromatography system), or on a multi-cell “plate reader”. Samples similar to those marketed for 96-well immunoassays.

Alternatively, fluorescence can be measured using fiber optic sensors with nucleic acid probes in solution or attached to fiber optic. Fluorescence is monitored using a photomultiplier tube, or other light detecting instrument attached to the optical fiber. The advantage of this system is that extremely small sample volumes can be tested.

Furthermore, scanning fluorescence detectors, such as the FluorImager sold by Molecular Dynamics, are ideally suited for monitoring the fluorescence of ordered modified nucleic acid molecules on solid surfaces. The advantage of this system is the large number of electron transfer probes that can be scanned at once using chips coated with thousands of different nucleic acid probes.

Many transition metal complexes fluoresce with large Stokes shifts. Appropriate examples include the bis- and trisphenanthroline complexes and the bis- and trisbipyridyl complexes of transition metals such as ruthenium (see Juris, A., Balzani, V., et al., Coord. Chem. Rev. V 84, p. 85-277, 1988). Preferred examples show efficient fluorescence (reasonably high quantum yield) as well as low rearrangement energy. These include Ru (4,7-biphenyl2-phenanthroline) 3²⁺ and Ru (4,4'-biphenyl2,2'-bipyridine) 3²⁺.

Alternatively, a reduction in fluorescence associated with hybridization can be measured using these systems. An electron transfer “donor” molecule that readily fluoresces when on a single-stranded nucleic acid (with an “acceptor” at the other end), will experience a reduction in fluorescence intensity when a complementary nucleic acid binds to the probe. , allowing efficient transfer of the electron from the excited state. This decrease in fluorescence can easily be monitored as an indicator of the presence of a target sequence using the same procedures as above.

In a further embodiment, electrochemiluminescence is used as the basis for electron transfer detection. With some electron transfer groups such as Ru²⁺(bpy) 3, direct luminescence accompanies the decay of the excited state. Changes in this property are associated with nucleic acid hybridization and can be monitored with a single photomultiplier tube arrangement (see Blackburn, GF, Clin. Chem. 37: 1534-1539 (1991); and Juris et al., See higher.

In a preferred embodiment electronic sensing is used, including ammeter, voltmeter, capacitance, and impedance. Appropriate techniques include, but are not limited to, electrogravimetry; coulometry (including controlled potential coulometry and constant current coulometry); voltimetry (cyclic voltimetry, pulse voltimetry (normal pulse voltimetry, rectangular pulse voltmeter, differential pulse voltmeter, Osteryoung square wave voltmeter, and coulostatic pulse techniques); stripping analysis (anodic stripping analysis, cathodic stripping analysis, square wave stripping voltmeter); Conductance measurements (electrolytic conductance, direct analysis); time-dependent electrochemical analyzes (chronoamperimetry, chronopotentiometry, cyclic chronopotentiometry and amperimetry, AC polargraphy, chronogalvametry, and chronocoulometry); AC impedance measurement, AC impedance measurement

ES 2 198 486 T3 capacitance; and photoelectrochemistry.

In a preferred embodiment, the monitoring of electron transfer through nucleic acid is by amperometric detection, either directly using a covalently attached electrode, or indirectly using electron transport "mediators" to send electrons from the nucleic acid to an electrode. . The ways of anchoring the nucleic acids to the electrodes and the possible mediators are described below. In amperometric detector it would resemble the numerous enzyme-based biosensors currently used, for example, to monitor blood glucose. This detection procedure involves applying a potential (compared to a separate reference electrode) between the nucleic acid-conjugated electrode and an auxiliary (counter) electrode, in the sample containing target genes of interest. Electron transfer with different efficiencies is induced in the sample in the presence or absence of the target nucleic acid; that is, the single-stranded probe exhibits a different rate than the probe hybridized to the target sequence. Different efficiencies of electron transfer result in the generation of different currents at the electrode.

The apparatus for measuring electron transfer amperimetrically involves sensitive sensing of current (nanoamps to picoamps), and includes a means of controlling the voltage potential, using a potentiostat. This voltage is optimized with reference to the potential of the electron donor complex on the nucleic acid. Possible electron donor complexes include those previously mentioned, with ruthenium complexes being preferred, and rhenium complexes being most preferred.

In a preferred embodiment, alternative modes of electron transfer detection are used. For example, potentiometric (or voltometric) measurements that involve non-faradaic processes (no net current flow) and are traditionally used in pH and other ion detectors. Similar sensors are used to monitor electron transfer through nucleic acid. In addition, other properties of insulators (such as resistance) and conductors (such as conductivity, impedance, and capacitance) could be used to monitor electron transfer through nucleic acid. Finally, any system that generates a current (such as electron transfer) also generates a small magnetic field, which could be monitored in some embodiments.

It should be understood that a benefit of the high electron transfer rates observed in the compositions of the invention is that temporal resolution can greatly improve the signal-noise results of monitors based on absorbance, fluorescence, and electrical current. The fast electron transfer rates of the present invention result in both high signals and stereotyped delays between initiation and completion of electron transfer. By amplifying signals with particular delays, such as through the use of pulsed initiation of electron transfer and fixed detection amplifiers, improvements of two to four orders of magnitude in signal-to-noise ratio could be achieved.

In a preferred embodiment, DNA is modified by adding electron donor and electron acceptor groups. In an alternative embodiment RNA is modified. In a further embodiment, a double-stranded nucleic acid for use as a bioconductor will contain some deoxyribose nucleosides, some ribose nucleosides, and a mixture of adenosine, thymidine, cytosine, guanine, and uracil bases.

According to a further aspect of the present invention, preferred formulations for donors and acceptors will possess a transition metal covalently attached to a series of ligands, and further covalently attached to an amino group as part of the ribose ring (position 2 'or 3 '), or a nitrogen or sulfur atom as part of a nucleoside dimer linked by a peptide bond, phosphoramidate bond, phosphoraothioate bond, phosphorodithioate bond, or O-methyl-phosphoramidate bond.

In a preferred embodiment, an oligonucleotide containing at least one electron transfer group is anchored to an electrode, which also serves as an electron transfer group, thereby forming a single-stranded nucleic acid with both, an electron donating group. and an electron acceptor group attached in the manner outlined above. Preferably, the single-stranded nucleic acid containing an electron transfer group is bound covalently, or in such a way as to allow transfer of electrons from the electrode to the single-stranded nucleic acid in order to allow transfer of electrons between the donor. electron and acceptor. Preferably, the non-electrode electron transfer group is attached at or near the end of the oligonucleotide, such that the probe sequence to hybridize to the target sequence is between the donor and the acceptor. The electrode could be immersed in a sample containing the target sequence, such that the target sequence hybridizes to the probe, and an electron transfer can be detected using the techniques outlined above.

In a further embodiment, two nucleic acids are used as a probe as previously described. For example, one nucleic acid is covalently attached to a solid electrode, which serves as an electron transfer group, and the other with a covalently attached electron transfer group is freely in solution. Upon hybridization to the target sequence, the two nucleic acids are aligned in such a way that electron transfer occurs between the electron transfer group of the hybridized nucleic acid and the electrode. Electron transfer is detected as outlined above, using techniques well known in the art.

The following examples serve to describe in more detail how to use the invention described further

ES 2 198 486 T3 above, as well as to detail the best contemplated modes of carrying out various aspects of the invention. It will be understood that these examples in no way serve to limit the true scope of this invention, but are presented for illustrative purposes.

Examples

The amino-modified monomer units are prepared by a variation of published procedures, and incorporated into the growing oligonucleotide by standard synthetic techniques. The procedure is applicable to both DNA and RNA derivatives.

Example 1

Synthesis of an oligonucleotide duplex with electron transfer groups at the 5 'ends

In this example an eight nucleotide double stranded nucleic acid was produced, each single strand having a single electron transfer group covalently attached to the 5 'terminal uridine nucleotide at the 2' carbon of the ribose sugar.

Step 1

Synthesis of 5'-di (p-methoxyphenyl) methyl ether-2 '- (trifluoroacetamido) -2'-deoxyuridine

2 '- (trifluoroacetamido) -2'-deoxyuridine (2.0 g, 5.9 mmol), prepared by minor modification of published procedures (Imazawa, supra), was repeatedly dissolved in minimal very dry CH3CN and was rotary evaporated to dryness, then transferred to a vacuum line with inert atmosphere and further dried over a period of 1 hour. The following procedure for the synthesis of the material was adapted from Gait (above): under positive argon pressure, the material was dissolved in freshly dried and distilled pyridine, and with stirring, 0.05 equivalents ( by weight) of 4-dimethylaminopyridine (DMAP), 1.5 equivalents of triethylamine (TEA), and 1.2 equivalents of 4,4'-dimethoxytrityl chloride (DMTr-Cl). The progress of the reaction was monitored by silica gel TLC (98: 2 methyl chloride: methanol, mobile phase). After 30 minutes, an additional 0.5 equivalents of each of DMTr-Cl and TEA were added, and the reaction was allowed to progress for an additional three hours. An equal volume of water was added to this reaction mixture and the solution was extracted several times with diethyl ether. The ether layers were rotary evaporated to dryness, redissolved in a minimal amount of methyl chloride, and purified by flash chromatography (99: 1 methyl chloride: methanol, mobile phase), to obtain the product 5'- di (p-methoxyphenyl) methyl ether-2 '- (trifluoroacetamido) -2'-deoxyuridine.

Step 2

5'-2'-aminouridine-GCTACGA and 5'-2'-aminouridine-CGTAGCA

The 5'-di (p-methoxyphenyl) methyl ether-2 '- (trifluoroacetamido) -2'-deoxyuridine was dried under reduced pressure (glass) and dissolved in freshly dried and distilled CH3CN, and placed in a specially conical vial constructed, and placed on an ABI DNA synthesizer. The schedule for the preparation of standard (ie unmodified) oligonucleotides was altered during the addition of the final base (amino-modified) up to a coupling time of 15-30 minutes. The oligonucleotide was separated from the column by standard procedures and purified by reverse phase HPLC on C-18. In this way the 5'-2'-aminouridine-GCTACGA and 5'-2'-aminouridineCGTAGCA were prepared. In addition, complementary strands of both products were prepared for use in the synthesis of electron transfer groups below.

Step 3

5'-2'-ruthenium bisbipridineimidazole-aminouridine-GCTACGA

The 5'-2'-aminouridine-GCTACGA produced in the previous step was associated with the unmodified complementary strand using standard techniques. All manipulations of the associated duplex, prior to the addition of the transition metal complex, were carried out at 4 ° C. In order to ensure that the DNA remained associated during the modification, the reactions were carried out in 1 M salt. The 5 'amino modified DNA duplex was dissolved in 0.2M HEPES, 0.8M NaCl, pH 6.8, and repeatedly evacuated on a Schlenk line. The previously prepared bisbipyridine ruthenium carbonate was dissolved in the above buffer and oxygen was removed by repeated evacuation and argon purging through a Schlenk line. The ruthenium complex was transferred to DNA solution via cannulation (argon / vacuum) and the reaction was allowed to proceed under positive argon pressure, with stirring, for 24 hours. To this reaction, 50 equivalents of imidazole were added in the reaction flask, and the reaction was allowed to proceed for an additional 24 hours. The reaction mixture was removed from the vacuum line and applied to a PD-10 gel filtration column, and eluted with water to remove excess ruthenium complex. The volume of the collected fractions was reduced to dryness through a speed-vac, and the solid was dissolved in 0.1M trimethylammonium acetate (TEAC), pH 6.0. The DNA duplex was heated to 60 ° C for 15 minutes with 50% formamide to denature the duplex. The single-stranded DNA was purified using reverse phase HPLC, with a C-18 column, equipped with a diode array detector, and using a gradient from 3% to 35% acetonitrile in

IS 2 198 486 T3

0.1 M TEAC, pH 6.0.

Step 4

5'-2'-ruthenium tetraminepyridine aminouridine-CGTAGCA

The 5'-2'-aminouridine-CGTAGCA (0.3 µM) was dissolved in 0.2 M HEPES buffer, 0.8 M NaCl, pH 6.8, and degassed in the vacuum line. Ru (III) tetraaminopyridine chloride slurry (10 µM) in the same buffer was added to a 10 ml conical shaped flask equipped with a stir bar and a septum. In a separate flask, Zn / Hg amalgam was prepared and dried under reduced pressure, and the ruthenium (III) solution was transferred (via cannulation) to the Zn / I Ig amalgam. The immediate formation of a light yellow solution (λ ^ = 406 nm) indicated that the reduced form of ruthenium had been achieved, and the reaction was allowed to progress for 30 minutes. This solution was transferred to the flask containing the amino-modified DNA, and the reaction was allowed to proceed at room temperature for 24 hours under argon. The reaction mixture was removed from the vacuum line and a 50-fold excess of cobalt-EDTA was added to the solution (Kirschner, Inorganic Synthesis (1957), pp. 186). The solution was applied to a Sephadex® G-25 gel filtration column to remove excess ruthenium complex and further purified by reverse phase HPLC as described above. The two ruthenium-modified nucleotides were associated by standard techniques and characterized (see EXAMPLE 5).

Example 2

Synthesis of long DNA duplexes with electron transfer groups at the 5 'ends

In this example, an in vitro DNA amplification technique, PCR (reviewed in Abramson et al., Curr. Op. In Biotech. 4: 41-47 (1993)), is used to generate modified duplex DNA by polymerization of nucleotides from modified primer strands (Saiki et al., Science 239: 487 (1988)). Two oligonucleotides 18 bases long, and not complementary to each other, are synthesized with amino modification at the 2'-ribose position of the 5 'nucleotides, as in EXAMPLE 1.

A series of oligonucleotides of increasing lengths, starting with 40 bases, are chemically synthesized using standard chemistry. Each of the PCR templates shares a 5 'sequence identical to a modified 18-mer. The 3 'end of the template oligonucleotide shares a sequence complementary to another 18-mer.

PCR rapidly generates modified duplex DNA by catalyzing the 5'-3 'synthesis of DNA from each of the modified 18-mers, using the unmodified strand as a template. One hundred nanomoles of each of the modified 18-mers are mixed in 1 ml of an aqueous solution containing 2000 units of Taq polymerase, deoxyribonucleoside triphosphates at 0.2 M each, KCl 50 mM, Tris-HCl 10 mM, pH 8.8, 1.5 mM MgCl2, 3 mM dithiothreitol, and 0.1 mg / ml bovine serum albumin. A femtomol of the template strand 40 bases in length is added to the mixture. The sample is heated at 94 ° C for one minute for denaturation, two minutes at 55 ° C for association, and three minutes at 72 ° C for extension. This cycle was repeated 30 times using an automated thermal cycler.

The template sequences amplified with transition metal complexes at both 5 'ends were purified by agarose gel electrophoresis, and used directly in electron transfer applications. Example 3

Synthesis of electron transfer groups covalently linked to duplex DNA internucleotide bonds

In this example, alternative backbones to the phosphodiester linkages of the oligonucleotides are used. The functional groups incorporated into these internucleotide linkages serve as the covalent attachment site for the electron transfer groups. These alternative internucleotide linkages include, but are not limited to, peptide linkages, phosphoramidate linkages, phosphorothioate linkages, phosphorodithioate linkages, and O-methylphosphoramidate linkages.

The nucleic acid peptide (PNA) preparation follows literature procedures (see Engholm, above), with the synthesis of Boc-protected pentafluorophenyl ester of the chosen base (thymidine). The resulting PNA could be prepared using Merrifield's solid phase strategy (Merrifield, Science 232: 341 (1986)), using a single coupling protocol with 0.1 M of the thyminyl monomer in 30% (v / v) DMF in CH₂Cl₂. The progress of the reaction is followed by quantitative analysis with ninhydrin (Sarin, Anal. Biochem. 117: 147 (1981)). The resulting PNA could be modified with an appropriate transition metal complex as outlined in EXAMPLE

1.

The synthesis of phosphoramidate internucleotide linkages (Beaucage, above; Letsinger, above; Sawai, above) and N-alkylphosphoramidates (Jager, above) follows standard literature procedures, with only slight modification (procedures are they stop after the addition of a single base to the solid support, and are then separated to obtain a dinucleotide phosphoramidate). A typical example is the preparation of the phenylester of 5O-isobutyloxy-carbonylthimidyl- (3'-5 ') - 5'-amino-5'-deoxythymidine (Letsinger, J. Org. Chem., Supra). The dimer units are replaced by standard oligonucleotides at chosen intervals during the preparation of the

IS 2 198 486 T3

DNA using established automated techniques. Transition metal modification of the modified bonds occurs as described in EXAMPLE 1.

The synthesis of phosphorothioate and phosphorodithioate linkages (Eckstein, supra, and references cited therein) is well documented. A published protocol uses an Applie Biosystems DNA synthesizer using a modified β-cyanoethylphosphoramidite cycle that is protected after sulfurization with tetraethylthiuram disulfide (TETD) (Lyerm J. Org. Chem. 55: 4693 (1990)). Phosphorothioate and phosphorodithioate analogs are prepared as dimers and separated from the solid support and purified by HPLC (mobile phase: acetonitrile / triethylammonium acetate). Example 4

Synthesis of two oligonucleotides each with an electron transfer group at the 5 'end

In this example, two oligonucleotides are prepared that hybridize to a single target sequence, with no intervening sequences. One oligonucleotide has a covalently attached electron donor group at the 5 'end, and the other has a covalently attached electron acceptor group at the 5' end. In this example, the electron transfer species bind through a uridine nucleotide, but one of ordinary skill in the art will understand that current procedures can be used to modify any of the nucleotides. Furthermore, one of skill in the art will recognize that the method is not limited to the generation of 8-mer, but is useful in the generation of oligonucleotide probes of different lengths.

The procedure is exactly as in EXAMPLE 1, except that the generated 8-mers are not complementary to each other, and instead are complementary to a 16 nucleotide target sequence. Therefore, the final association step of step 4 of EXAMPLE 1 is not performed. Instead, the two modified oligonucleotides associate with the target sequence, and the resulting complex is characterized as in EXAMPLE 8.

Example 5

Characterization of modified nucleic acids

Enzymatic digestion

The modified oligonucleotides of EXAMPLE 1 were subjected to enzymatic digestion using established protocols, and converted to their constituent nucleosides by sequential reaction with phosphodiesterase and alkaline phosphatase. By comparison with standards of the experimentally obtained HPLC profiles and of the UV-visible spectra of the digested oligonucleotides (including 2'-aminouridine and 2'-aminoadenine), the presence of the amino-modified base with time was confirmed. retention and UV-visible spectrum. An identical procedure was carried out with the transition metal modified duplex DNA, and the assignments of the constituent nucleosides demonstrated the modification of a single site at the predicted site.

Fluorescent labeled amino-modified oligonucleotides

Fluorochrome isothiocyanate fluorescein (FITC) has been shown to be specific for labeling primary amines on modified oligonucleotides, whereas it does not bind to amines or amides present on nucleotide bases (Haugland, Handbook of Fluorescent Probes and Research Chemicals, 5^to edition, (1992)). This reaction was carried out using the synthesized amino-oligonucleotide as described in EXAMPLE 1, and on an identical base sequence without the 2'-amino-ribose group being present. Fluorescence spectroscopic measurements were obtained from both oligonucleotides and the results confirm the presence of the amine on the 5'-terminal ribose ring.

Thermodynamic melting curves of modified duplex DNA

A well-established technique for measuring the thermodynamic parameters of duplex DNA is the acquisition of melting curves of DNA. A series of melting curves were measured as a function of the concentration of the temperature-controlled UV-visible modified duplex DNA (Hewlett-Packard), using techniques well known in the art. These results confirm that hybridization of amino-modified and transition-metal-modified DNA had taken place. Furthermore, the results indicate that the modified DNA forms a stable duplex comparable to the stability of the standard unmodified oligonucleotides.

Two-dimensional nuclear magnetic resonance (NMR) spectroscopy

The amino-modified oligonucleotides synthesized as part of this work were prepared in sufficient quantities (6 micromoles) to allow assignment of the proton NMR spectra. ¹H using a Varian 600 MHz NMR spectrometer.

Measurement of electron transfer rate

An excellent review of measurement techniques is found in Winkler et al., Chem. Rev. 92: 369-379 (1992).

IS 2 198 486 T3

The donor is Ru (bpy) 2 (NHuridine) im, E⁰ 1 V, and the acceptor is Ru (NH3) 4 py (NHuridine) im, E⁰ 330 mV. The purified transition metal modified oligonucleotides (UNHRu (bpy) 2im GCATCGA and UNHRu (NH3) 4 (py) im CGATGCA) were associated by heating an equimolar mixture of the oligonucleotides (30 μΜ: 60 nmoles of DNA in 2 ml of buffer ) at pH 6.8 (100 mM NaPi, 900 mM NaCl) up to 60 ° C for 10 minutes and slowly cooling to room temperature over a 4 hour period. The solution was transferred to an inert atmosphere cuvette equipped with adapters for the attachment of a vacuum line and a magnetic stir bar. The solution was degassed several times and the sealed apparatuses were repeatedly filled with Ar gas.

The entire apparatus was inserted into a cuvette holder as part of the setup, using a pumped dye excimer XeCl laser, and data was acquired over various wavelengths, including 360, 410, 460 and 480 nm. The transfer rate of photoinduced electrons is 1.6 x 10⁶ s^-1 over a distance of 28 Angstroms.

Example 6

Synthesis of a single-stranded nucleic acid labeled with two electron transfer groups

This example uses the basic procedure described above to generate two modified oligonucleotides each with an electron transfer group attached. Ligation of the two modified strands together produces a double-labeled nucleic acid with any of four configurations: labeled 5 'and 3' ends, labeled 5 'end and labeled internal nucleotide, labeled 3' end and labeled internal nucleotide, and double labels. in internal nucleotides. Specifically, the synthesis of a 24 base long oligonucleotide with an electron transfer donor group at the 5 'end and an internal electron transfer group is described.

Five hundred nanomoles of each of two oligonucleotides of 12 bases in length were synthesized, as detailed above, labeled 5 'with ruthenium (II) bisbipyridine imizadol in an oligonucleotide "D", and with ruthenium (III) tetraamine pyridine on a second oligonucleotide "A".

An unmodified oligonucleotide, 24 bases in length, and complementary was produced by standard synthetic techniques with the juxtaposition of oligonucleotide "D" followed in the 5 'to 3' direction by oligonucleotide "A". Five hundred nanomoles of this hybridization template were added to a mixture of oligonucleotides "A" and "D" in 5 ml of an aqueous solution containing 500 mM Tris-Cl, pH 7.5, 50 mM MgCl2, 50 mM dithiothreitol, and 5 mg / ml of gelatin. To promote maximum hybridization of labeled oligonucleotides with the complementary strand, the mixture was incubated at 60 ° C for 10 minutes, then slowly cooled at a rate of approximately 10 ° C per hour, to a final temperature of 12 ° C. Enzymatic ligation of the two labeled strands is achieved with T4 DNA ligase at 12 ° C to prevent ligation and oligomerization of duplexed DNA with other duplexes (blunt end ligation). Alternatively, E. coli DNA ligase can be used since it does not catalyze blunt end ligation.

One hundred Weiss units of T4 DNA ligase are added to the associated DNA and adenosine triphosphate is added to a final concentration of 0.5 mM. The reaction that catalyzes the formation of a phosphodiester bond between the 5 'terminal phosphate of oligonucleotide "A" and the 3' terminal hydroxyl group of oligonucleotide "D" is allowed to progress for 18 hours at 12 ° C. The reaction is terminated by heat inactivation of the enzyme, at 75 ° C for 10 minutes. The doubly labeled oligonucleotide is separated from the uniquely labeled oligonucleotides, and from the complementary unlabeled oligonucleotide by HPLC in the presence of urea, as in the previous examples. The double-labeled oligonucleotide of this example is ideally suited for use as a photoactive gene probe, as detailed below.

Example 7

Use of an Electron Transferring Group Double Modified Oligonucleotide as a Photoactive Probe for the Detection of Homologous Nucleic Acid Sequences

This example uses the 24-mer oligonucleotide of EXAMPLE 6 in a single-type gene probe assay, where removal of the unlabeled probe is not required prior to signal detection. In the test procedure, a region of the gag gene of the human immunodeficiency virus type I (HIV-I) is amplified by the polymerase chain reaction (Saiki et al., Science 239: 487-491 (1988) ). This region of HIV-I is highly conserved among clinical isolates.

The amplified target DNA, relative to controls lacking HIV-I DNA, is added to a 6XSSC hybridization solution (0.9 M NaCl, 0.09 M sodium citrate, pH 7.2) containing 50 nanomoles of the probe. of 24-mer doubly labeled from EXAMPLE 6. Hybridization is allowed to progress at 60 ° C for 10 minutes with moderate shaking. Electron transfer detection following laser excitation is performed as in EXAMPLE 5. Control samples lacking the hybridized probe show negligible electron transfer rates. Probes hybridized to the gag sequence show efficient and rapid electron transfer through the DNA double helix, providing a highly specific, homogeneous, and automatable HIV-I detection assay.

IS 2 198 486 T3

A similar homogeneous gene probe assay involves the use of two probes, one an electron donor and the other an electron acceptor, which hybridize to the gag region of HIV-I in a tandem configuration, one probe adjoining the other. In this assay, the electronic coupling between the two electron transfer groups is entirely dependent on hybridization with the target DNA. If appropriate, the transfer of electrons from one probe to the other is enhanced by ligation of the juxtaposed ends using the T4 DNA ligand as in EXAMPLE 6.

Example 8

Preparation of a hydroxythiol for anchoring to a gold electrode

OH (CH2) OH was purchased from Aldrich, and the monoacetate form was prepared by mixing the slurry with dry CH2Cl2, 0.5 equivalents of dimethylaminopyridine were added along with 1.4 equivalents of triethylamine and 1 equivalent of acetic anhydride. The reaction was allowed to progress for 2 hours and was purified by flash chromatography (80:20 hexane: diethyl ether).

The monoacetate compound was converted to the monosylate monoacetate using p-TOSCl by literature procedures, and then treated with triphenyl methyl mercaptan. To remove the monoacetate, the product was dissolved in MeOH (1 mmol, 9 ml), cooled to 0 ° C, and an aqueous solution of NaOH (1 mmol, in 2 ml of water) was added. The temperature was allowed to rise to room temperature slowly, and the reaction was followed by TLC (5% MeOH / CH₂Cl₂). When the ester had been removed, the mixture was re-cooled to 0 ° C, and acidified with KHSO₄up to pH 5-6 using p} H paper. MeOH was evaporated, and the residue was extracted with CH2Cl2 (200 mL), dried (Na2SO4), evaporated, and checked via TLC. The material was "phosphoramidite" by standard procedures. This material was inserted into the DNA synthesizer and a modified oligonucleotide was produced. The phosphoramidite oligonucleotide was modified with a ruthenium complex by adding Ru (bpy) 2CO3 to it, followed by imidazole, to yield Ru (bpy)₂im-oligonucleotide. The triethyl protecting group was removed by dissolving the nucleotide in 200 µl of 0.1 M triethylammonium acetate (TEEA) buffer, pH 7.5, 30 µl of 1 M silver nitrate solution was added, and the mixture was stirred with vortexing, and incubated at room temperature for 30 minutes. 50 µl of 1 M dithiothreitol (DTT) was added, the mixture was vortexed, and incubated for 15 minutes, at which time it was microcentrifuged for 15 minutes to remove the Ag + DTT precipitate. The supernatant was collected and the precipitate was washed with 100 µl of TEAA buffer, and the solutions pooled. The resulting oligonucleotide was then attached to the gold surface by standard techniques.

Example 9

Synthesis of a single-stranded nucleic acid containing both an electron acceptor group and an electron donor group

In order to assess the path-dependent nature of the electron transfer process through duplex DNA, an oligonucleotide with an electron donor at the 3 'end and an electron acceptor at the 50 end was prepared. Multiple was prepared by synthesizing a derivative with an amine at the 2 'position of the terminal ribose at both ends.

Synthesis of bis-3 ', 5', 2'-deoxyuridine oligonucleotides

A DMT-2'-N-trifluoroacetyl protected 2'-amino-2'-deoxyuridine phosphoramidite (UNH2) was prepared as described above and reacted with succinic anhydride. This material was reacted with p-nitrophenol to produce the precursor for anchoring to controlled pore glass resin (GPC) as in Figure 6A. The modified oligonucleotides were assembled by standard automated solid phase DNA synthesis techniques, and the bis-3 ', 5', 2'-deoxyuridine oligonucleotide was isolated and characterized by mass spectrometry and HPLC digest analysis. Furthermore, the aminoribose oligomers and their complements were reacted with FITC under conditions that favor the labeling of primary amines. As expected, only the 2'-amino-2'-deoxyribose site was labeled, verifying the presence of a primary amine on the DNA. As in the example, an 11 base pair sequence was prepared (calculated for UNH2CTCCTACACUNH2 3229, found 3229.1), and the subsequent digest map was consistent with the proposed structure. Metal modification of the bis-amino modified oligonucleotide was performed similarly. The new metal-modified oligonucleotides were characterized by fluorescent labeling, enzymatic digestion, and duplex melting temperature studies. Denaturation and thermal association experiments show similar melting temperatures for both the ruthenium and amino-ribose oligomers. Furthermore, the amino-modified duplex DNA has been characterized by 2D NMR. These data confirm that donors and acceptors are covalently attached to the 2'-aminodeoxyribose position, and indicate that the DNA structure is not disturbed by the presence of the ruthenium complexes.