U.S. patent application number 10/636371 was filed with the patent office on 2004-05-27 for nucleic acid mediated electron transfer.
Invention is credited to Fraser, Scott E., Kayyem, Jon Faiz, Meade, Thomas J..
Application Number | 20040101890 10/636371 |
Document ID | / |
Family ID | 46253490 |
Filed Date | 2004-05-27 |
United States Patent
Application |
20040101890 |
Kind Code |
A1 |
Meade, Thomas J. ; et
al. |
May 27, 2004 |
Nucleic acid mediated electron transfer
Abstract
The present invention provides for the selective covalent
modification of nucleic acids with redox active moieties such as
transition metal complexes. Electron donor and electron acceptor
moieties are covalently bound to the ribose-phosphate backbone of a
nucleic acid at predetermined positions. The resulting complexes
represent a series of new derivatives that are bimolecular
templates capable of transferring electrons over very large
distances at extremely fast rates. These complexes possess unique
structural features which enable the use of an entirely new class
of bioconductors and photoactive probes.
Inventors: |
Meade, Thomas J.;
(Willamette, IL) ; Kayyem, Jon Faiz; (Pasadena,
CA) ; Fraser, Scott E.; (La Canada, CA) |
Correspondence
Address: |
Robin M. Silva
DORSEY & WHITNEY LLP
Suite 3400
Four Embarcadero Center
San Francisco
CA
94111-4187
US
|
Family ID: |
46253490 |
Appl. No.: |
10/636371 |
Filed: |
August 7, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10636371 |
Aug 7, 2003 |
|
|
|
09306749 |
May 7, 1999 |
|
|
|
09306749 |
May 7, 1999 |
|
|
|
08873598 |
Jun 12, 1997 |
|
|
|
5952172 |
|
|
|
|
08873598 |
Jun 12, 1997 |
|
|
|
08660534 |
Jun 7, 1996 |
|
|
|
5770369 |
|
|
|
|
08660534 |
Jun 7, 1996 |
|
|
|
08475051 |
Jun 7, 1995 |
|
|
|
5824473 |
|
|
|
|
08475051 |
Jun 7, 1995 |
|
|
|
08166036 |
Dec 10, 1993 |
|
|
|
5591578 |
|
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/287.2; 435/91.2; 536/23.1; 536/25.3 |
Current CPC
Class: |
C12Q 1/6818 20130101;
C07H 21/00 20130101; Y02A 50/30 20180101; C12Q 1/6825 20130101;
C07H 23/00 20130101; C12Q 1/6818 20130101; C12Q 2565/607 20130101;
C12Q 2565/401 20130101; C12Q 2563/137 20130101; C12Q 1/6818
20130101; C12Q 2563/137 20130101; C12Q 2563/113 20130101; C12Q
1/6818 20130101; C12Q 2565/607 20130101; C12Q 2563/113 20130101;
C12Q 1/6818 20130101; C12Q 2563/113 20130101; C12Q 1/6825 20130101;
C12Q 2565/101 20130101; C12Q 1/6818 20130101; C12Q 2565/1015
20130101; C12Q 2563/137 20130101; C12Q 2563/113 20130101; C12Q
1/6818 20130101; C12Q 2565/101 20130101; C12Q 2563/137 20130101;
C12Q 2563/113 20130101 |
Class at
Publication: |
435/006 ;
435/091.2; 536/023.1; 536/025.3 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 019/34 |
Claims
1. A phosphoramidite nucleoside comprising a covalently attached
electron transfer moiety.
2. A nucleoside comprising: a) a covalently attached electron
transfer moiety; and b) a phosphoramidite moiety.
3. A nucleoside according to claims 1 or 2 wherein said electron
transfer moiety is attached to the ribose of said nucleoside.
4. A nucleoside according to claim 3 wherein said electron transfer
moiety is attached to said ribose via a linker at the 2'
position.
5. A nucleoside according to claims 1 or 2 wherein said electron
transfer moiety is a transition metal complex.
6. A nucleoside according to claim 5 wherein said transition metal
complex comprises ruthenium.
7. A nucleoside according to claim 5 wherein said transition metal
complex comprises iron.
8. A nucleoside according to claim 5 wherein said transition metal
complex comprises cobalt.
9. A method of making a nucleic acid comprising a covalently
attached electron transfer moiety, said method comprising: a)
providing a phosphoramidite nucleoside comprising a covalently
attached electron transfer moiety; and b) incorporating said
phosphoramidite nucleoside in a synthetic reaction to form a
nucleic acid with a covalently attached electron transfer
moiety.
10. A method according to claim 9 wherein said synthetic reaction
is a solid-phase synthetic reaction.
11. A method according to claim 9 wherein said electron transfer
moiety is attached to the ribose of said nucleoside.
12. A method according to claim 9 wherein said electron transfer
moiety is attached to said ribose via a linker at the 2'
position.
13. A method according to claim 9 wherein said electron transfer
moiety is a transition metal complex.
14. A method according to claim 13 wherein said transition metal
complex comprises ruthenium.
15. A method according to claim 13 wherein said transition metal
complex comprises iron.
16. A method according to claim 13 wherein said transition metal
complex comprises cobalt.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuing application of U.S. Ser. No.
09/306,749, filed May 7, 1999, which is a continuation of
application Ser. No. 08/873,598, filed Jun. 12, 1997, now U.S. Pat.
No. 5,952,172, which is an continuation-in-part of application Ser.
No. 08/660,534, filed Jun. 7, 1996, now U.S. Pat. No. 5,770,369,
which is a continuation-in-part of application Ser. No. 08/475,051,
filed Jun. 7, 1995, now U.S. Pat. No. 5,824,473, which is a
continuation-in-part of application Ser. No. 08/166,036, filed Dec.
10, 1993, now U.S. Pat. No. 5,591,578.
FIELD OF THE INVENTION
[0002] The present invention is directed to electron transfer via
nucleic acids. More particularly, the invention is directed to
improvements in the site-selective modification of nucleic acids
with electron transfer moieties.
BACKGROUND OF THE INVENTION
[0003] The detection of specific nucleic acid sequences is an
important tool for diagnostic medicine and molecular biology
research. Gene probe assays currently play roles in identifying
infectious organisms such as bacteria and viruses, in probing the
expression of normal genes and identifying mutant genes such as
oncogenes, in typing tissue for compatibility preceding tissue
transplantation, in matching tissue or blood samples for forensic
medicine, and for exploring homology among genes from different
species.
[0004] Ideally, a gene probe assay should be sensitive, specific
and easily automatable (for a review, see Nickerson, Current
Opinion in Biotechnology 4:48-51 (1993)). The requirement for
sensitivity (i.e. low detection limits) has been greatly alleviated
by the development of the polymerase chain reaction (PCR) and other
amplification technologies which allow researchers to amplify
exponentially a specific nucleic acid sequence before analysis (for
a review, see Abramson et al., Current Opinion in Biotechnology,
4:41-47 (1993)).
[0005] Specificity, in contrast, remains a problem in many
currently available gene probe assays. The extent of molecular
complementarity between probe and target defines the specificity of
the interaction. Variations in the concentrations of probes, of
targets and of salts in the hybridization medium, in the reaction
temperature, and in the length of the probe may alter or influence
the specificity of the probe/target interaction.
[0006] It may be possible under some limited circumstances to
distinguish targets with perfect complementarity from targets with
mismatches, although this is generally very difficult using
traditional technology, since small variations in the reaction
conditions will alter the hybridization. New experimental
techniques for mismatch detection with standard probes include DNA
ligation assays where single point mismatches prevent ligation and
probe digestion assays in which mismatches create sites for probe
cleavage.
[0007] Finally, the automation of gene probe assays remains an area
in which current technologies are lacking. Such assays generally
rely on the hybridization of a labeled probe to a target sequence
followed by the separation 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.
[0008] The time consuming nature of these separation steps has led
to two distinct avenues of development. One involves the
development of high-speed, high-throughput automatable
electrophoretic and other separation techniques. The other involves
the development of nonseparation homogeneous gene probe assays.
[0009] For example, Gen-Probe Inc., (San Diego, Calif.) has
developed a homogeneous protection assay in which hybridized probes
are protected from base hydrolysis, and thus are capable of
subsequent chemiluminescence. (Okwumabua et al. Res. Microbiol.
143:183 (1992)). Unfortunately, the reliance of this approach on a
chemiluminescent substrate known for high background photon
emission suggests this assay will not have high specificity. EPO
application number 86116652.8 describes an attempt to use
non-radiative energy transfer from a donor probe to an acceptor
probe as a homogeneous detection scheme. However, the fluorescence
energy transfer is greatly influenced by both probe topology and
topography, and the DNA target itself is capable of significant
energy quenching, resulting in considerable variability. Therefore
there is a need for DNA probes which are specific, capable of
detecting target mismatches, and capable of being incorporated into
an automated system for sequence identification.
[0010] As outlined above, molecular biology relies quite heavily on
modified or labeled oligonucleotides for traditional gene probe
assays (Oligonucleotide Synthesis: A Practical Approach. Gait et
al., Ed., IRL Press: Oxford, UK, 1984; Oligonucleotides and
Analogues: A Practical Approach. Ed. F. Eckstein, Oxford University
Press, 1991). As a result, several techniques currently exist for
the synthesis of tailored nucleic acid molecules. Since nucleic
acids do not naturally contain functional groups to which molecules
of interest may easily be attached covalently, methods have been
developed which allow chemical modification at either of the
terminal phosphates or at the heterocyclic bases (Dreyer et al.
Proc. Natl. Acad. Sci. USA, 1985, 82:968).
[0011] For example, analogues of the common deoxyribo- and
ribonucleosides which contain amino groups at the 2' or 3' position
of the sugar can be made using established chemical techniques.
(See Imazawa et al., J. Org. Chem., 1979, 44:2039; Imazawa et al.,
J. Org. Chem. 43(15):3044 (1978); Verheyden et al., J. Org. Chem.
36(2):250 (1971); Hobbs et al., J. Org. Chem. 42(4):714 (1977)). In
addition, oligonucleotides may be synthesized with 2'-5' or 3'-5'
phosphoamide linkages (Beaucage et al., Tetrahedron 49(10):1925
(1992); Letsinger, J. Org. Chem., 35:3800 (1970); Sawai, Chem.
Lett. 805 (1984); Oligonucleotides and Analogues: A Practical
Approach, F. Eckstein, Ed. Oxford University Press (1991)).
[0012] The modification of nucleic acids has been done for two
general reasons: to create nonradioactive DNA markers to serve as
probes, and to use chemically modified DNA to obtain site-specific
cleavage.
[0013] To this end, DNA may be labeled to serve as a probe by
altering a nucleotide which then serves as a replacement analogue
in the nick translational resynthesis of double stranded DNA. The
chemically altered nucleotides may then provide reactive sites for
the attachment of immunological or other labels such as biotin.
(Gilliam et al., Anal. Biochem. 157:199 (1986)). Another example
uses ruthenium derivatives which intercalate into DNA to produce
photoluminescence under defined conditions. (Friedman et al., J.
Am. Chem. Soc. 112:4960 (1990)).
[0014] In the second category, there are a number of examples of
compounds covalently linked to DNA which subsequently cause DNA
chain cleavage. For example 1,10-phenanthroline has been coupled to
single-stranded oligothymidylate via a linker which results in the
cleavage of poly-dA oligonucleotides in the presence of Cu.sup.2+
and 3-mercaptopropionic acid (Francois et al., Biochemistry 27:2272
(1988)). Similar experiments have been done for EDTA.sup.1-Fe(II)
(both for double stranded DNA (Boutorin et al., FEBS Lett.
172:43-46 (1986)) and triplex DNA (Strobel et al., Science 249:73
(1990)), porphyrin-Fe(III) (Le Doan et al., Biochemistry
25:6736-6739 (1986)), and 1,10-phenanthronine-Cu(I) (Chen et al.,
Proc. Natl. Acad. Sci USA, 83:7147 (1985)), which all result in DNA
chain cleavage in the presence of a reducing agent in aerated
solutions. A similar example using porphyrins resulted in DNA
strand cleavage, and base oxidation or cross-linking of the DNA
under very specific conditions (Le Doan et al., Nucleic Acids Res.
15:8643 (1987)).
[0015] Other work has focused on chemical modification of
heterocyclic bases. For example, the attachment of an inorganic
coordination complex, Fe-EDTA, to a modified internal base resulted
in cleavage of the DNA after hybridization in the presence of
dioxygen (Dreyer et al., Proc. Natl. Acad. Sci. USA 82:968 (1985)).
A ruthenium compound has been coupled successfully to an internal
base in a DNA octomer, with retention of both the DNA hybridization
capabilities as well as the spectroscopic properties of the
ruthenium label (Telser et al., J. Am. Chem. Soc. 111:7221 (1989)).
Other experiments have successfully added two separate
spectroscopic labels to a single double-stranded DNA molecule
(Telser et al., J. Am. Chem. Soc. 111:7226 (1989)).
[0016] The study of electron transfer reactions in proteins and DNA
has also been explored in pursuit of systems which are capable of
long distance electron transfer.
[0017] To this end, intramolecular electron transfer in
protein-protein complexes, such as those found in photosynthetic
proteins and proteins in the respiration pathway, has been shown to
take place over appreciable distances in protein interiors at
biologically significant rates (see Bowler et al., Progress in
Inorganic Chemistry: Bioinorganic Chemistry, Vol. 38, Ed. Stephen
J. Lippard (1990). In addition, the selective modification of
metalloenzymes with transition metals has been accomplished and
techniques to monitor electron transfer in these systems developed.
For example, electron transfer proteins such as cytochrome c have
been modified with ruthenium through attachment at several
histidines and the rate of electron transfer from the heme
Fe.sup.2+ to the bound Ru.sup.3+ measured. The results suggest that
electron transfer "tunnel" pathways may exist. (Baum, Chemical
& Engineering News, Feb. 22, 1993, pages 2023; see also Chang
et al., J. Am. Chem. Soc. 113:7056 (1991)). In related work, the
normal protein insulation, which protects the redox centers of an
enzyme or protein from nondiscriminatory reactions with the
exterior solvent, was "wired" to transform these systems from
electrical insulators into electrical conductors (Heller, Acc.
Chem. Res. 23:128 (1990)).
[0018] There are a few reports of photoinduced electron transfer in
a DNA matrix. In these systems, the electron donors and acceptors
are not covalently attached to the DNA, but randomly associated
with the DNA, thus rendering the explicit elucidation and control
of the donor-acceptor system difficult. For example, the intense
fluorescence of certain quaternary diazoaromatic salts is quenched
upon intercalation into DNA or upon exposure to individual
mononucleotides, thus exhibiting electron donor processes within
the DNA itself. (Brun et al., J. Am. Chem. Soc. 113:8153
(1991)).
[0019] Another example of the difficulty of determining the
electron transfer mechanism is found in work done with some
photoexcitable ruthenium compounds. Early work suggested that
certain ruthenium compounds either randomly intercalate into the
nucleotide bases, or bind to the helix surface. (Purugganan et al.,
Science 241:1645 (1988)). A recent reference indicates that certain
ruthenium compounds do not intercalate into the DNA (Satyanarayana
et al., Biochemistry 31 (39):9319 (1992)); rather, they bind
non-covalently to the surface of the DNA helix.
[0020] In these early experiments, various electron acceptor
compounds, such as cobalt, chromium or rhodium compounds were added
to certain DNA-associated ruthenium electron donor compounds.
(Puragganan et al., Science 241:1645 (1988); Orellana et al.,
Photochem. Photobiol. 499:54 (1991); Brun et al., J. Am. Chem. Soc.
113:8153 (1991); Davis, Chem.-Biol. Interactions 62:45 (1987);
Tomalia et al., Acc. Chem. Res., 24:332 (1991)). Upon addition of
these various electron acceptor compounds, which randomly bind
non-covalently to the helix, quenching of the photoexcited state
through electron transfer was detected. The rate of quenching was
dependent on both the individual electron donor and acceptor as
well as their concentrations, thus revealing the process as
bimolecular.
[0021] In one set of experiments, the authors postulate that the
more mobile surface bound donor promotes electron transfer with
greater efficiency than the intercalated species, and suggest that
the sugar-phosphate backbone of DNA, and possibly the solvent
medium surrounding the DNA, play a significant role in the electron
transport. (Purugganan et al., Science 241:1645 (1988)). In other
work, the authors stress the dependence of the rate on the mobility
of the donor and acceptor and their local concentrations, and
assign the role of the DNA to be primarily to facilitate an
increase in local concentration of the donor and acceptor species
on the helix. (Orellana et al., supra).
[0022] In another experiment, an electron donor was reportedly
randomly intercalated into the stack of bases of DNA, while the
acceptor was randomly associated with the surface of the DNA. The
rate of electron transfer quenching indicated a close contact of
the donor and the acceptor, and the system also exhibits
enhancement of the rate of electron transfer with the addition of
salt to the medium. (Fromherz et al., J. Am. Chem. Soc. 108:5361
(1986)).
[0023] In all of these experiments, the rate of electron transfer
for non-covalently bound donors and acceptors is several orders of
magnitude less than is seen in free solution.
[0024] An important stimulus for the development of long distance
electron transfer systems is the creation of synthetic light
harvesting systems. Work to date suggests that an artificial light
harvesting system contains an energy transfer complex, an energy
migration complex, an electron transfer complex and an electron
migration complex (for a topical review of this area, see Chemical
& Engineering News, Mar. 15, 1993, pages 38-48). Two types of
molecules have been tried: a) long organic molecules, such as
hydrocarbons with covalently attached electron transfer species, or
DNA, with intercalated, partially intercalated or helix associated
electron transfer species, and b) synthetic polymers.
[0025] The long organic molecules, while quite rigid, are
influenced by a number of factors, which makes development
difficult. These factors include the polarity and composition of
the solvent, the orientation of the donor and acceptor groups, and
the chemical character of either the covalent linkage or the
association of the electron transfer species to the molecule.
[0026] The creation of acceptable polymer electron transfer systems
has been difficult because the available polymers are too flexible,
such that several modes of transfer occur. Polymers that are
sufficiently rigid often significantly interfere with the electron
transfer mechanism or are quite difficult to synthesize.
[0027] Thus the development of an electron transfer system which is
sufficiently rigid, has covalently attached electron transfer
species at defined intervals, is easy to synthesize and does not
appreciably interfere with the electron transfer mechanism would be
useful in the development of artificial light harvesting
systems.
[0028] In conclusion, the random distribution and mobility of the
electron donor and acceptor pairs, coupled with potential short
distances between the donor and acceptor, the loose and presumably
reversible association of the donors and acceptors, the reported
dependence on solvent and broad putative electron pathways, and the
disruption of the DNA structure of intercalated compounds rendering
normal base pairing impossible all serve as pronounced limitations
of long range electron transfer in a DNA matrix. Therefore, a
method for the production of rigid, covalent attachment of electron
donors and acceptors to provide minimal perturbations of the
nucleic acid structure and retention of its ability to base pair
normally, is desirable. The present invention serves to provide
such a system, which allows the development of novel bioconductors
and diagnostic probes.
SUMMARY OF THE INVENTION
[0029] The present invention provides for the modification of
nucleic acids at specific sites with redox active moieties such as
transition metal complexes. An electron donor and/or electron
acceptor moiety are covalently bound at predetermined positions.
The resulting complexes represent a series of new derivatives that
are biomolecular templates capable of transferring electrons over
very large distances at extremely fast rates. These complexes
possess unique structural features which enable the use of an
entirely new class of bioconductors and diagnostic probes.
[0030] 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 a further object to
provide nucleic acids with covalently attached organic electron
transfer species, and modified nucleic acids attached to control
pore glass.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIGS. 1A to 1H illustrates all the possible orientations of
electron donor (EDM) and electron acceptor (EAM) moieties on a
single stranded nucleic acid.
[0032] FIGS. 2A-1 to 2A-9 and 2B-1 to 2B-9 illustrate the possible
orientations of electron transfer moieties EDM and EAM on two
adjacent single stranded nucleic acids. These orientations also
apply when the two probes are separated by an intervening
sequence.
[0033] FIG. 3 illustrates a series of amino-modified nucleoside
precursors prior to incorporation into an oligonucleotide.
[0034] FIGS. 4A and 4B depict the structure of electron transfer
moieties. FIG. 4A depicts the general formula of a representative
class of electron donors and acceptors. FIG. 4B depicts a specific
example of a ruthenium electron transfer moiety using bisbipyridine
and imidazole as the ligands.
[0035] FIG. 5 is a schematic showing transition metals bound to the
ribose-phosphate backbone in a variety of positions. M is a
transition metal. M.sub.1 is bound via an amine on the 2' carbon of
the ribose; an electron must travel through 4 .sigma. bonds to
enter the pi-orbitals (the "pi-way") of the stacked bases. M.sub.2
and M.sub.3 are bound via a phosphoramide-type linkages, and
electrons must travel through 7 .sigma. bonds to enter the pi-way,
respectively. M.sub.4 is bound via an amine on the 3' carbon of the
ribose, and an electron traverses through 5 .sigma. bonds.
[0036] FIGS. 6A, 6B and 6C depict the attachment of a
2'-amino-modified nucleoside to control pore glass (CPG) and the
formation of a single stranded nucleic acid with elongation and
attachment of transition metal complexes as the exemplified
electron transfer species. The experimental conditions are outlined
in Example 9. FIG. 6A depicts the formation of
2'-amino-2'-deoxyuridine derivatized to control pore glass (CPG).
2'-amino modified uridine is depicted, although any base may be
used. As is known in the art, phosphoramidite nucleosides are added
to the derivatized nucleoside, after removal of the DMT protecting
group, as generally depicted in FIG. 6B, using the UCTCCTACAC
sequence as an example. The addition of a 5' terminal
phosphoramidite 2-amino-deoxyuridine, with a DMT protecting group,
results in a single stranded nucleic acid containing a 3' and 5'
2'-amino modified nucleoside. FIG. 6C depicts the addition of the
electron transfer species, exemplified by two ruthenium transition
metal complexes, im(bpy).sub.2Ru and Ru(II)(NH.sub.3).sub.4py.
[0037] FIG. 7 depicts the addition of electron transfer moieties,
exemplified by a transition metal complex, to the C-terminus of
PNA. FIG. 9 attaches 4-aminomethylpyridine to the carboxy terminus,
to form a ligand which may bind the metal at the nitrogen of the
pyridine ring.
[0038] FIGS. 8A and 8B depicts attachment of the amino-modified
nucleic acids of the invention to electrodes. (A) depicts the
attachment to glassy carbon electrodes. R is the oligonucleotide,
and GCE is a glassy carbon electrode. (B) depicts the attachment of
the amino-modified nucleic acids of the invention to oxidized
surfaces using silane reactions.
DETAILED DESCRIPTION
[0039] Unless otherwise stated, the term "nucleic acid" or
"oligonucleotide" or grammatical equivalents herein means at least
two nucleotides covalently linked together. A nucleic acid of the
present invention will generally contain phosphodiester bonds,
although in some cases, as outlined below, a nucleic acid analogs
are included that may have alternate backbones, comprising, for
example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925
(1993) and references therein; Letsinger, J. Org. Chem. 35:3800
(1970); 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 91986)),
phosphorothioate, phosphorodithioate, O-methylphophoroamidite
linkages (see Eckstein, Oligonucleotides and Analogues: A Practical
Approach, Oxford University Press), and peptide nucleic acid
backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895
(1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen,
Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all
of which are incorporated by reference). These modifications of the
ribose-phosphate backbone may be done to facilitate the addition of
electron transfer moieties, or to increase the stability and
half-life of such molecules in physiological environments.
[0040] Particularly preferred are peptide nucleic acids (PNA). 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
larger changes in the melting temperature (Tm) for mismatched
versus perfectly matched basepairs. DNA and RNA typically exhibit a
2-4.degree. C. drop in Tm for an internal mismatch. With the
non-ionic backbone of PNA, the drop is closer to 7-9.degree. C.
This allows for better detection of mismatches. Similarly, due to
their non-ionic nature, hybridization of the bases attached to
these backbones is relatively insensitive to salt concentration.
This is particularly advantageous in the systems of the present
invention, as a reduced salt hybridization solution has a lower
Faradaic current than a physiological salt solution (in the range
of 150 mM).
[0041] The nucleic acids may be single stranded or double stranded,
as specified, or contain portions of both double stranded or single
stranded sequence. The nucleic acid may be DNA, both genomic and
cDNA, RNA or a hybrid, where the nucleic acid contains any
combination of deoxyribo- and ribo-nucleotides, and any combination
of bases, including uracil, adenine, thymine, cytosine, guanine,
inosine, xathanine and hypoxathanine, etc. In some instances, e.g.
in the case of an "intervening nucleic acid", the term nucleic acid
refers to one or more nucleosides. As used herein, the term
"nucleoside" includes nucleotides.
[0042] The terms "electron donor moiety", "electron acceptor
moiety", and "electron transfer moieties" or grammatical
equivalents herein refers to molecules capable of electron transfer
under certain conditions. It is to be understood that electron
donor and acceptor capabilities are relative; that is, a molecule
which can lose an electron under certain experimental conditions
will be able to accept an electron under different experimental
conditions. It is to be understood that the number of possible
electron donor moieties and electron acceptor moieties is very
large, and that one skilled in the art of electron transfer
compounds will be able to utilize a number of compounds in the
present invention. Preferred electron transfer moieties include,
but are not limited to, transition metal complexes, organic
electron transfer moieties, and electrodes.
[0043] In a preferred embodiment, the electron transfer moieties
are transition metal complexes. Transition metals are those whose
atoms have an incomplete d shell of electrons. Suitable transition
metals for use in the invention include, but are not limited to,
cadmium (Cd), magnesium (Mg), copper (Cu), cobalt (Co), palladium
(Pd), zinc (Zn), iron (Fe), ruthenium (Ru), rhodium (Rh), osmium
(Os), rhenium (Re), platinium (Pt), scandium (Sc), titanium (Ti),
Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni),
Molybdenum (Mo), technetium (Tc), tungsten (W), and iridium (Ir).
That is, the first series of transition metal, the platinum metals
(Ru, Rh, Pd, Os, Ir and Pt), along with Re, W, Mo and Tc, are
preferred. Particularly preferred are ruthenium, rhenium, osmium,
platinium and iron.
[0044] The transition metals are complexed with a variety of
ligands to form suitable transition metal complexes, as is well
known in the art. Suitable ligands include, but are not limited to,
--NH.sub.2; pyridine; pyrazine; isonicotinamide; imidazole;
bipyridine and substituted derivative of bipyridine;
phenanthrolines, particularly 1,10-phenanthroline (abbreviated
phen) and substituted derivatives of phenanthrolines such as
4,7-dimethylphenanthroline; dipyridophenazine;
1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);
9,10-phenanthrenequinone diimine; 1,4,5,8-tetraazaphenanthrene
(abbreviated tap); 1,4,8,11-tetra-azacyclotetradecane;
diaminopyridine (abbreviated damp); porphyrins and substituted
derivatives of the porphyrin family. A general formula that is
representative of a class of donors and acceptors that may be
employed is shown in FIG. 4A. The groups R.sup.1, R.sup.2, R.sup.3,
R.sup.4, and R.sup.5 may be any coordinating ligand that is capable
of covalently binding to the chosen metal and may include any of
the above ligands. The structure of a ruthenium electron transfer
species using bisbipyridine and imidazole as the ligands is shown
in FIG. 4B. Specific examples of useful electron transfer complexes
include, but are not limited to, those shown in Table 1.
1 TABLE 1 Donors Acceptors Ru(bpy).sub.2im-NH.sub.2--U
Ru(NH.sub.3).sub.5--NH.sub.2--U Ru(bpy).sub.2im-NH.sub.2--U
Ru(NH.sub.3).sub.4py-NH.sub.2--U Ru(bpy).sub.2im-NH.sub.2--U
Ru(NH.sub.3).sub.4im-NH.sub.2--U trans-Ru(cyclam)py Where: Ru =
ruthenium bpy = bisbipyridine im = imidazole py = pyridine cyclam =
1,4,8,11-tetra-azacyclotetradecane Other suitable moieties include
bis(phenanthroline) (dipyridophenazine)Ru(II) (abbreviated
[Ru(phen).sub.2dppz].sup.+2); bis(9,10-phenthrenequinone
diimine)(phenanthroline)Rh(III), abbreviated
[Rh(phi).sub.2phen].sup.+3; tris(phenanthroline)Ru(II) (abbreviated
[Ru(o-phen).sub.3].sup.+2), Co(phen).sub.3.sup.+3,
Co(bpy).sub.3.sup.+3; Rh(phen).sub.3.sup.+3; Cr(phen).sub.3.sup.+3;
Ru(bpy).sub.2(dppz).sup.+2; # and Ru(bpy).sub.3.sup.+2.
[0045] In addition to transition metal complexes, other organic
electron donors and acceptors may be covalently attached to the
nucleic acid for use in the invention. These organic molecules
include, but are not limited to, riboflavin, xanthene dyes, azine
dyes, acridine orange, N,N'-dimethyl-2,7-diazapyrenium dichloride
(DAP.sup.2+), methylviologen, ethidium bromide, quinones such as
N,N'-dimethylanthra(2,1,9-def:6,5,10-d- 'e'f')diisoquinoline
dichloride (ADIQ.sup.2+); porphyrins
([meso-tetrakis(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-ami- no-10-methylphenoxyazine
chloride), methylene blue; Nile blue A
(aminoaphthodiethylaminophenoxazine sulfate),
indigo-5,5',7,7'-tetrasulfo- nic acid, indigo-5,5',7-trisulfonic
acid; phenosafranine, indigo-5-monosulfonic acid; safranine T;
bis(dimethylglyoximato)-iron(II) chloride; induline scarlet,
neutral red, and subsitituted derivatives of these compounds.
[0046] In one embodiment, the electron donors and acceptors are
redox proteins as are known in the art. However, redox proteins in
many embodiments are not preferred.
[0047] In a particularly preferred embodiment, an electron transfer
moiety comprises an solid support such as an electrode to which the
nucleic acid is attached, covalently or otherwise. That is, the
electrode serves as either the electron donor or acceptor, as is
more fully described below. The techniques used in this embodiment
are analogous to the wiring of proteins to an electrode except that
the nucleic acids of the present invention are used rather than a
redox protein (see for example Gregg et al., J. Phys. Chem. 95:5970
(1991); Heller et al., Sensors and Actuators R., 13-14:180 (1993);
and Pishko et al., Anal. Chem., 63:2268 (1991)).
[0048] Electrode attachment is utilized in initiating electron
transfer via an applied potential and for electronic methods of
electron transfer monitoring.
[0049] In a preferred embodiment, electron transport between the
electrode and the nucleic acid can be indirect, utilizing electron
transport mediators which are free in solution or imbedded in a gel
or polymer to provide a type of electronic coupling between the
electrode and the nucleic acids. In a preferred embodiment, the
electron transfer moiety-modified nucleic acids of the invention
are attached via such a matrix. Matrix attachment has several
advantages for use in a nucleic acid gene sensor. Because of the
3-dimensional nature of the polymer, large numbers of modified
nucleic acid probes can be attached to a small surface area of
electrode. Using a highly porous "hydrogel," rates of nucleic acid
hybridization can be quite high, nearly matching that of nucleic
acid in solution.
[0050] For example, polymers with covalently attached redox
molecules behave as highly effective electron transfer mediators.
Siloxane and ethylene oxide polymers, modified with ferrocene
molecules, demonstrated electron transfer between enzymes and an
electrode; for example, flexible siloxane and ethylene oxide
polymers covalently attached to ferrocene or Os(bpy).sub.2 have
been shown to be highly effective redox polymers for mediating
electron transfer from several enzymes to an electrode. (see
Boguslavsky et al., Solid State Ionics, V. 60, p. 189, (1993)).
Similarly, a redox-conducting epoxy cement has been prepared (see
Hellar et al., J. Phys. Chem., 95:5970 (1991)). Cross linked redox
gels for amperometric biosensors applications have also been
prepared with glucose oxidase electrically connected to electrodes
so that electrons were shown to flow from the enzyme, through the
polymer and to the electrode (see Hellar, A., et. al., Anal. Chem.,
62, 258, (1990)).
[0051] In this embodiment, it is preferred that a redox polymer
such as a poly-(vinylpyridine) complex of Os(bby).sub.2Cl be
cross-linked with an epoxide such as diepoxide to form a
redox-conducting epoxide cement which is capable of strongly
binding to electrodes made of conductive material such as gold,
vitreous carbon, graphite, and other conductive materials. This
strong attachment is included in the definition of "covalently
attached" for the purposes of this embodiment. The epoxide
cross-linking polymer is then reacted with, for example, an exposed
amine, such as the amine of an amino-modified nucleic acid
described above, covalently attaching the nucleic acid to the
complex, forming a "redox hydrogel" on the surface of the
electrode.
[0052] In an analogous fashion, chemically modified DNA can be
substituted for the redox enzyme or mediator with the result of
electron transfer processes being observed from a transition
metal-modified DNA moiety through a coupled redox conducting
polymer to an electrode.
[0053] Suitable mediators include water soluble
ferrocene/ferricinium hydroquinones/quinones, reducible and
oxidizable components of organic salts, cobaltocenes, the hexa- and
octacyanides of molybdenum, tungsten and iron. In addition,
macrocycles and chelating ligands of transition metals such as
cobalt, ruthenium and nickel are used, including
Co(ethylenediamine).sub.3 and Ru(ethylenediamine).sub.3 and the
trisbypyridyl and hexamine complexes of transition metals such as
Co, Ru, Fe, and Os (see Alyanasundaram, supra). In a preferred
embodiment, electron transport between the electrode and the
nucleic acid can be direct via a covalent bond. One advantage of
these systems is that the orientation of the DNA probe can be
influenced to reduce any bending back of the probe onto the
electrode. Also, more precise control of applied potential and
measured current is associated with short covalent linkages versus
gels and polymers.
[0054] In a preferred embodiment, the covalent bonds must be highly
conducting such as in a redox polymer (Hellar, A. Acc. Chem. Res.
Vol. 23, p. 128, 1990). Alternatively, if they are poorly
conducting, the length of the linkage must be kept short.
Accordingly, a preferred embodiment has an electron traversing no
more than about five a bonds, with no more than three being
especially preferred. Carbon paste and glassy carbon rods have
proven reliable and effective as electrodes in a variety of
chemical sensors, including sensitive glucose oxidase enzyme-based
biosensors, and may be used in the present invention. In addition,
flexible siloxane and ethylene oxide polymers covalently attached
to ferrocene or Os(bpy).sub.2 molecules have been shown to be
highly effective redox polymers for mediating electron transfer
from several enzymes to an electrode. Amino-ribose modified nucleic
acids are attached to carbon electrodes by variations of these
literature techniques. Finally, nucleic acids are more directly
attached to oxidized carbon electrodes via guanosine residues,
using known carbodiimide and N-hydroxysuccinimide chemistry.
[0055] In a preferred embodiment, glassy carbon electrodes (GCEs)
are used. In this embodiment, amine groups such as outlined above
on the 2' or 3' carbon of the ribose ring are used for attachment.
The reaction proceeds via the oxidation of an amine group to a
cation radical which forms a chemically stable and covalent bond
between the amine and the edge plane of the GCE surface (see
Deinhammer, R, et al. Langmuir 10: 1306 (1994)) This synthetic
approach has been well characterized using X-ray photo-electron
spectroscopy and cyclic voltammetry. The yield using this chemistry
can be quite high, approximately 1.times.10.sup.10
molecules/cm.sup.2. The amine compound forms a stable bond to the
carbon surface, and steric effects influence binding efficiency.
The reactivity of primary amines is substantially higher than
secondary amines; the binding of tertiary amines is not observed at
all.
[0056] Employing the amino-modified (primary amine group)
oligonucleotides described earlier, the procedure developed by
Deinhammer, R, et al. to prepare the GCEs for electrochemical
treatment in amine containing solution is depicted in FIG. 8A.
[0057] In addition, DNA has been immobilized onto GCEs using a
water soluble carodimide (Mikkelsen et al., Electroanalysis 4:929
(1992)).
[0058] In a preferred embodiment, the nucleic acids of the
invention are attached to gold electrodes. Several methods are
available for the covalent attachment of redox active species to
gold surfaces and electron transfer reactions with these materials
have been observed. Hydroxy thiols (OH(CH.sub.2).sub.xSH) of
varying lengths are prepared by variation of literature procedures
(see Miller, C. et al. J. Phys. Chem. 95: 877 (1991) and Chidsey,
C. E. D., Science, V. 251, p. 919, (1991)). Example 8 outlines the
preparation of hydroxyl thiols which are attached to gold
electrodes.
[0059] Alternative procedures for the preparation of hydroxythiols
are known in the art. Au electrodes or surfaces are prepared by
literature procedures and the modified hydroxythiols adsorbed onto
the Au.
[0060] In an additional embodiment, the modified nucleic acids of
the invention are covalently attached to thin film oxidized
surfaces. It has been reported that a variety of compounds can be
covalently bonded (in the form of monolayers) to thin-film
SnO.sub.2, TiO.sub.2, and RuO.sub.2 and Pt electrodes (see Lenhard,
J. and Murray, R. J. Electroanal. Chem. 78:195 (1977)). Reversible
electrochemistry of surface bound complexes such as
3,5-dinitrobenzamide to electrodes has been observed. The reported
complexes are attached to the electrode via an amide bond linkage.
Employing these literature procedures, analogous derivatives using
amino-modified oligonucleotides described in this work can be
prepared and are schematically represented in FIG. 8B.
[0061] Accordingly, using the above methods, oligonucleotides may
be attached to a solid support such that the electrode serves as
either the electron donor moiety or the electron acceptor
moiety.
[0062] Thus, all combinations of electron donors and acceptors may
be made: two transition metal complexes; two organic electron
transfer species; one transition metal, one organic moiety; one
transition metal and an electrode; and one organic moiety and an
electrode. The choice of the electron transfer species will depend
in part on the method of initiation and detection required, as is
more fully described below.
[0063] The term "target sequence" or grammatical equivalents herein
means a nucleic acid sequence on a single strand of nucleic acid.
The target sequence may be a portion of a gene, a regulatory
sequence, genomic DNA, cDNA, mRNA, or others. It may be any length,
with the understanding that longer sequences are more specific. As
is outlined more fully below, probes are made to hybridize to
target sequences to determine the presence or absence of the target
sequence in a sample. Generally speaking, this term will be
understood by those skilled in the art.
[0064] The probes of the present invention are designed to be
complementary to the target sequence, such that hybridization of
the target sequence and the probes of the present invention occurs.
As outlined below, this complementarity need not be perfect; there
may be any number of base pair mismatches which will interfere with
hybridization between the target sequence and the single stranded
nucleic acids of the present invention. However, if the number of
mutations is so great that no hybridization can occur under even
the least stringent of hybridization conditions, the sequence is
not a complementary target sequence.
[0065] A variety of hybridization conditions may be used in the
present invention. As is known in the art, "high" stringency
usually refers to conditions such as 0.1.times.SSC at 65.degree.
C., reduced stringency conditions include 2-5.times.SSC at
25-50.degree. C. The hybridization conditions may also vary when a
non-ionic backbone such as PNA is used, as is known in the art.
[0066] The terms "first target domain" and "second target domain"
or grammatical equivalents herein means two portions of a target
sequence within a nucleic acid which is under examination. The
first target domain may be directly adjacent to the second target
domain, or the first and second target domains may be separated by
an intervening target domain. The terms "first" and "second" are
not meant to confer an orientation of the sequences with respect to
the 5'-3' orientation of the target sequence. For example, assuming
a 5'-3' orientation of the complementary target sequence, the first
target domain may be located either 5' to the second domain, or 3'
to the second domain.
[0067] The present invention is directed, in part, to the
site-selective modification of nucleic acids with redox active
moieties such as transition metal complexes for the preparation of
a new series of biomaterials capable of long distance electron
transfer through a nucleic acid matrix. The present invention
provides for the precise placement of electron transfer donor and
acceptor moieties at predetermined sites on a single stranded or
double stranded nucleic acid. In general, electron transfer between
electron donor and acceptor moieties in a double helical nucleic
acid does not occur at an appreciable rate unless nucleotide base
pairing exists in the sequence between the electron donor and
acceptor in the double helical structure.
[0068] This differential in the rate of electron transfer forms the
basis of a utility of the present invention for use as probes. In
the system of the present invention, where electron transfer
moieties are covalently bound to the backbone of a nucleic acid,
the electrons putatively travel via the .pi.-orbitals of the
stacked base pairs of the double stranded nucleic acid. The
electron transfer rate is dependent on several factors, including
the distance between the electron donor-acceptor pair, the free
energy (.DELTA.G) of the reaction, the reorganization energy
(.lambda.), the contribution of the intervening medium, the
orientation and electronic coupling of the donor and acceptor pair,
and the hydrogen bonding between the bases.
[0069] The contribution of the intervening medium depends, in part,
on the number of sigma (.sigma.) bonds the electron must traverse
from the electron donor to reach the bases stack, or to exit the
stack to reach the electron acceptor. As is shown in FIG. 5, when
the metal is bound to the ribose-phosphate backbone via an amine
moiety at the 2' carbon of the ribose, an electron must travel
through four .sigma. bonds to reach the stack: the metal to
nitrogen bond, the nitrogen to 2' carbon bond, and from the 2'
carbon to the base, or vice versa depending on the direction of the
electron flow. Since the base of the nucleotide is conjugated in
some degree, the base can be considered to be the edge of the
".pi.-way"; that is, the conjugated .pi. orbitals of the stacked
base pairs. When the metal is bound to the ribose-phosphate
backbone via the 3' carbon of the ribose, an electron must traverse
through 5 .sigma. bonds. When the metal is bound via
phosphoramide-type linkages, an electron must traverse through 7
.sigma. bonds. In the preferred embodiments, the compositions of
the invention are designed such that the electron transfer moieties
are as close to the "pi-way" as possible without significantly
disturbing the secondary and tertiary structure of the double
helical nucleic acid, particularly the Watson-Crick
basepairing.
[0070] The effect on the electron transfer rate by the hydrogen
bonding between the bases is a dependence on the actual nucleic
acid sequence, since A-T pairs contain one less hydrogen bond than
C-G pairs. However, this sequence dependence is overshadowed by the
determination that there is a measurable difference between the
rate of electron transfer within a DNA base-pair matrix, and the
rate through the ribose-phosphate backbone, the solvent or other
electron tunnels. This rate differential is thought to be at least
several orders of magnitude, and may be as high as four orders of
magnitude greater through the stacked nucleotide bases as compared
to other electron transfer pathways. Thus the presence of double
stranded nucleic acids, for example in gene probe assays, can be
determined by comparing the rate of electron transfer for the
unhybridized probe with the rate for hybridized probes.
[0071] In one embodiment, the present invention provides for novel
gene probes, which are useful in molecular biology and diagnostic
medicine. In this embodiment, single stranded nucleic acids having
a predetermined sequence and covalently attached electron donor and
electron acceptor moieties are synthesized. The sequence is
selected based upon a known target sequence, such that if
hybridization to a complementary target sequence occurs in the
region between the electron donor and the electron acceptor,
electron transfer proceeds at an appreciable and detectable rate.
Thus, the present invention has broad general use, as a new form of
labeled gene probe. In addition, since detectable electron transfer
in unhybridized probes is not appreciable, the probes of the
present invention allow detection of target sequences without the
removal of unhybridized probe. Thus, the present invention is
uniquely suited to automated gene probe assays or field
testing.
[0072] In a preferred embodiment, the probes are used in genetic
diagnosis. For example, probes can be made using the techniques
disclosed herein to detect target sequences such as the gene for
nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which
is a gene associated with a variety of cancers, the Apo E4 gene
that indicates a greater risk of Alzheimer's disease, allowing for
easy presymptomatic screening of patients, mutations in the cystic
fibrosis gene, or any of the others well known in the art.
[0073] In an additional embodiment, viral and bacterial detection
is done using the complexes of the invention. In this embodiment,
probes are designed to detect target sequences from a variety of
bacteria and viruses. For example, current blood-screening
techniques rely on the detection of anti-HIV antibodies. The
methods disclosed herein allow for direct screening of clinical
samples to detect HIV nucleic acid sequences, particularly highly
conserved HIV sequences. In addition, this allows direct monitoring
of circulating virus within a patient as an improved method of
assessing the efficacy of anti-viral therapies. Similarly, viruses
associated with leukemia, HTLV-I and HTLV-II, may be detected in
this way. Bacterial infections such as tuberculosis may also be
detected.
[0074] In a preferred embodiment, the nucleic acids of the
invention find use as probes for toxic bacteria in the screening of
water and food samples. For example, samples may be treated to lyse
the bacteria to release its nucleic acid, and then probes designed
to recognize bacterial strains, including, but not limited to, such
pathogenic strains as, Salmonella, Campylobacter, Vibrio cholerae,
enterotoxic strains of E. coli, and Legionnaire's disease bacteria.
Similarly, bioremediation strategies may be evaluated using the
compositions of the invention.
[0075] In a further embodiment, the probes are used for forensic
"DNA fingerprinting" to match crime-scene DNA against samples taken
from victims and suspects.
[0076] The present invention also finds use as a unique methodology
for the detection of mutations in target nucleic acid sequences. As
a result, if a single stranded nucleic acid containing electron
transfer moieties is hybridized to a target sequence with a
mutation, the resulting perturbation of the base pairing of the
nucleosides will measurably affect the electron transfer rate. This
is the case if the mutation is a substitution, insertion or
deletion. Alternatively, two single stranded nucleic acids each
with a covalently attached electron transfer species that hybridize
adjacently to a target sequence may be used. Accordingly, the
present invention provides for the detection of mutations in target
sequences.
[0077] Thus, the present invention provides for extremely specific
and sensitive probes, which may, in some embodiments, detect target
sequences without removal of unhybridized probe. This will be
useful in the generation of automated gene probe assays.
[0078] In an alternate embodiment double stranded nucleic acids
have covalently attached electron donor and electron acceptor
moieties on opposite strands. Such nucleic acids are useful to
detect successful gene amplification in polymerase chain reactions
(PCR), thus allowing successful PCR reactions to be an indication
of the presence or absence of a target sequence. PCR may be used in
this manner in several ways. For example, if one of the two PCR
primers contains a 5' terminally attached electron donor, and the
other contains a 5' terminally attached electron acceptor, several
rounds of PCR will generate doubly labeled double stranded
fragments (occasionally referred to as "amplicons"). After
appropriate photoinduction, the detection of electron transfer
provides an indication of the successful amplification of the
target sequence as compared to when no amplification occurs. A
particular advantage of the present invention is that the
separation of the single stranded primers from the amplified double
stranded DNA is not necessary, as outlined above for probe
sequences which contain electron transfer moieties. Alternatively,
the detection of a target sequence via PCR is done by attaching one
electron transfer moiety species to one or both of the primers. The
other electron transfer moiety species is attached to individual
nucleosides of the PCR reaction pool, as is described herein.
Incorporation of the nucleosides containing the electron transfer
moiety into the nucleic acid during the PCR reaction results in
both electron transfer species being attached either to the same
single strand or to opposite strands, or both. Allowing the newly
synthesized nucleic acid to remain in a hybridized form allows the
detection of successful elongation via electron transfer, and thus
the detection of a target sequence. In this way, the present
invention is used for PCR detection of target sequences
[0079] In another embodiment the present invention provides for
double stranded nucleic acids with covalently attached electron
donor and electron acceptor moieties to serve as bioconductors or
"molecular wire". The electron transport may occur over distances
up to and in excess of 28 .ANG. per electron donor and acceptor
pair. In addition, the rate of electron transfer is very fast, even
though dependent on the distance between the electron donor and
acceptor moieties. By modifying the nucleic acid in regular
intervals with electron donor and/or electron acceptor moieties, it
may be possible to transport electrons over long distances, thus
creating bioconductors. These bioconductors are useful in a large
number of applications, including traditional applications for
conductors such as mediators for electrochemical reactions and
processes.
[0080] In addition, these bioconductors may be useful as probes for
photosynthesis reactions as well as in the construction of
synthetic light harvesting systems. The current models for the
electron transfer component of an artificial light harvesting
system have several problems, as outlined above, including a
dependence on solvent polarity and composition, and a lack of
sufficient rigidity without arduous synthesis. Thus the present
invention is useful as both a novel form of bioconductor as well as
a novel gene probe.
[0081] The present invention provides nucleic acids with covalently
attached electron transfer moieties.
[0082] The electron transfer moieties may be attached to the
nucleic acid at a variety of positions. In one embodiment, the
electron donor and acceptor moieties are added to the 3' and/or 5'
termini of the nucleic acid on either the sugar-phosphate backbone
or a terminal base. In alternative embodiments, the electron donor
and acceptor moieties are added to the backbone of one or more
internal nucleosides, that is, any nucleoside which is not the 3'
or 5' terminal nucleoside. In a further embodiment, the electron
donor and acceptor moieties are added to the backbone of both
internal and terminal nucleosides.
[0083] In a preferred embodiment, the electron transfer moieties
are attached to the ribose-phosphate backbone in a number of
positions. As shown in FIG. 5, several positions are possible, with
attachment to a ribose of the ribose-phosphate backbone being
particularly preferred. Accordingly, in FIG. 5, the most preferred
site of attachment of a electron transfer moiety is M.sub.1,
followed by M.sub.4, M.sub.2 and M.sub.3, in that order. In a
preferred embodiment, the electron transfer moieties are attached
at the 2' or 3' position on the ribose, with 2' being particularly
preferred.
[0084] In a preferred embodiment, the electron transfer moieties do
not intercalate, and are attached such that do not intercalate.
Thus, while it is possible to utilize a "linker", such as
alternating double bonds to attach the electron transfer moiety to
the nucleic acid, the linker is either preferably not longer than
the equivalent of one or two nucleosides in length, or is not
significantly flexible to allow intercalation. Preferably, if
linkers are used, they are attached via the ribose of the nucleic
acid backbone.
[0085] In one embodiment, the electron transfer moieties are added
to the bases of the terminal nucleosides. Thus, when the target
sequence to be detected is n nucleosides long, a probe can be made
which has an extra terminal nucleoside at one or both of the ends
of the nucleic acid (n+1 or n+2), which are used to covalently
attach the electron transfer moieties but which do not participate
in basepair hybridization. This extra terminal nucleoside is
important since attachment of electron transfer moieties to an
internal nucleoside base is expected to perturb Watson-Crick
basepairing. That is, the base used for covalent attachment should
be outside of the region used to identify the target sequence.
Additionally, it is preferred that upon probe hybridization, the
terminal nucleoside containing the electron transfer moiety
covalently attached at the base be directly adjacent to
Watson-Crick basepaired nucleosides; that is, the electron transfer
moiety should be as close as possible to the stacked n-orbitals of
the bases such that an electron travels through a minimum of
.sigma. bonds to reach the ".pi.-way", or alternatively can
otherwise electronically contact the .pi.-way.
[0086] In one embodiment, a single stranded nucleic acid is labeled
with an electron transfer moiety via the terminal bases at both
ends. Alternate embodiments utilize a terminal base and a 5' or a
3' terminal ribose-phosphate attachment as described above. In
further embodiments, compositions are provided comprising a first
single stranded nucleic acid containing an electron donor
covalently attached at a terminal base and a second single stranded
nucleic acid containing an electron acceptor covalently attached at
a position as described above, that is, at a 5', 3' or internal
position; alternatively, the electron donor and acceptor may be
switched. A particularly preferred embodiment utilizes an electrode
as one of the electron transfer moieties with the other electron
transfer moiety being attached to a terminal base, preferably on
the same single strand.
[0087] The present invention further provides methods for the
site-specific addition of electron transfer moieties to nucleic
acids. As outlined above, the electron transfer moieties may be
added at the 2' or 3' position of a ribose of the ribose-phosphate
backbone, to a 3' or 5' terminal base, or to an internal nucleoside
using peptide nucleic acid linkages, phosphoramidate bonds,
phosphorothioate bonds, phosphorodithioate bonds, or O-methyl
phosphoramidate bonds.
[0088] Molecular mechanics calculations indicate that perturbations
due to the modification of at the ribose of the terminal
nucleosides of nucleic acids are minimal, and Watson-Crick base
pairing is not disrupted (unpublished data using Biograf from
Molecular Simulations Inc., San Diego, Calif.).
[0089] For attachment to a ribose, a preferred embodiment utilizes
modified nucleosides to attach the electron transfer moieties.
Preferably amino-modified nucleosides and nucleosides are used. In
an alternate embodiment, thio-modified nucleosides are used to
attach the electron transfer moieties of the invention.
[0090] The modified nucleosides are then used to site-specifically
add a transition metal electron transfer moiety, either to the 3'
or 5' termini of the nucleic acid, or to any internal nucleoside.
Either the 2' or 3' position of the ribose may be altered for
attachment at the 3' terminus; for attachment to an internal ribose
or the 5' terminus, the 2' position is preferred. Thus, for
example, the 2' position of the ribose of the deoxyribo- or
ribonucleoside is modified prior to the addition of the electron
transfer species, leaving the 3' position of the ribose unmodified
for subsequent chain attachment 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)).
[0091] The amino-modified nucleosides made as described above are
converted to the 2' or 3' modified nucleotide triphosphate form
using standard biochemical methods (Fraser et al., Proc. Natl.
Acad. Sci. USA, 4:2671 (1973)).
[0092] Modified nucleosides for the attachment of the electron
transfer moieties to the bases, is done as outlined in Telser,
supra, both of which are expressly incorporated by reference. These
modified nucleosides are then incorporated at either the 3' or 5'
terminus as outlined below.
[0093] Once the modified nucleosides are prepared, protected and
activated, they may be incorporated into a growing oligonucleotide
by standard synthetic techniques (Gait, Oligonucleotide Synthesis:
A Practical Approach, IRL Press, Oxford, UK 1984; Eckstein) in
several ways. In one embodiment, one or more modified nucleosides
are incorporated into a growing oligonucleotide chain by using
standard molecular biology techniques such as with the use of the
enzyme DNA polymerase 1, T4 DNA polymerase, T7 DNA polymerase, Taq
DNA polymerase, reverse transcriptase, and RNA polymerases. For the
incorporation of a 3' modified nucleoside to a nucleic acid,
terminal deoxynucleotidyltransfera- se may be used. (Ratliff,
Terminal deoxynucleotidyltransferase. In The Enzymes, Vol 14A. P.
D. Boyer ed. pp 105-118. Academic Press, San Diego, Calif. 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 syntheses of oligonucleotides. In this way
the modified nucleoside, either for attachment at the ribose (i.e.
amino- or thiol-modified nucleosides) or the base, is incorporated
into the oligonucleotide at either an internal position or the 5'
terminus. This is generally done by protecting the 5' position of
the ribose with 4',4-dimethoxytrityl (DMT) followed by reaction
with 2-cyanoethoxy-bis-diisopropylaminophosphine in the presence of
diisopropylammonium tetrazolide to give the phosphoramidite as is
known in the art; although other techniques may be used as will be
appreciated by those in the art. See Gait, supra; Caruthers,
Science 230:281 (1985), both of which are expressly incorporated
herein by reference.
[0094] For attachment of an electron transfer moiety to the 3'
terminus, a preferred method utilizes the attachment of 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.
Further phosphoramidite nucleosides are added, either modified or
not, to the 5' end after deprotection.
[0095] In other embodiments, the electron transfer moiety or
moieties are added to the middle of the nucleic acid, i.e. to an
internal nucleoside. This may be accomplished in three ways.
[0096] In a preferred embodiment, a modified nucleoside is
incorporated at the 5' terminus as described above. In this
embodiment, oligonucleotide synthesis simply extends the 5' end
from the modified nucleoside using standard techniques. This
results in an internally amino modified oligonucleotide.
[0097] In one embodiment, the nucleosides are modified to contain
an aromatic amine capable of binding an electron transfer moiety at
either the 2' or 3' position of the ribose. For example, one of the
nitrogens of imidazole can be attached at the 2' or 3' position of
the ribose and thus used to attach the electron transfer moiety
such as a transition metal complex. This may effectively reduce the
number of a bonds an electron must travel through to reach the
"pi-way" since the imidazole offers substantially less resistance
to electron transfer as compared to a .sigma. bond. In a preferred
embodiment, the imidazole is attached at the 2' position of the
ribose. In an alternate embodiment, the imidazole is attached at
the 3' position. The imidazole-modified nucleoside may be
incorporated into an oligonucleotide as outlined herein for
amino-modified nucleosides.
[0098] In an alternate embodiment, electron transfer moieties are
added to the backbone at a site other than ribose, resulting in an
internal attachment. For example, phosphoramide rather than
phosphodiester linkages can be used as the site for transition
metal modification. These transition metals serve as the donors and
acceptors for electron transfer reactions. While structural
deviations from native phosphodiester linkages do occur and have
been studied using CD and NMR (Heller, Acc. Chem. Res. 23:128
(1990); Schuhmann et al. J. Am. Chem. Soc. 113:1394 (1991)), the
phosphoramidite internucleotide link has been reported to bind to
complementary polynucleotides and is stable (Beaucage et al.,
supra, and references therein; Letsinger, supra; Sawai, supra;
Jager, Biochemistry 27:7237 (1988)). In this embodiment, dimers of
nucleotides are created with phosphoramide linkages at either the
2'-5' or 3'-5' positions. A preferred embodiment utilizes the 3'-5'
position for the phosphoramide linkage, such that structural
disruption of the subsequent Watson-Crick basepairing is minimized.
These dimer units are incorporated into a growing oligonucleotide
chain, as above, at defined intervals, as outlined below.
[0099] Thus, the present invention provides methods for making a
nucleic acid with covalently attached electron transfer moieties.
In a preferred embodiment, the method is for making a nucleic acid
with an electron transfer moiety attached at the 3' terminus of
said nucleic acid. The method comprises attaching a 2'-amino
modified nucleoside to control pore glass, and adding
phosphoramidite nucleosides to the 5' terminus of the modified
nucleoside to form a nucleic acid. The nucleic acid is then
optionally cleaved from the CPG using known methods. The nucleic
acid may be hybridized to its complement, to protect the bases from
modification, if required, and the electron transfer moiety is
added to the 2'-amino modified nucleoside.
[0100] In a preferred embodiment, methods for making a nucleic acid
with an electron transfer moiety attached at the 5' terminus are
provided. The method comprises attaching a nucleoside to control
pore glass, and adding phosphoramidite nucleosides to the 5'
terminus of the nucleoside to form a nucleic acid. A 2' or 3' amino
modified nucleoside is added to the 5' terminus, and the nucleic
acid is optionally cleaved from the CPG. The nucleic acid may be
hybridized to its complement if required, and the electron transfer
moiety is added to the 2' or 3'-amino modified nucleoside.
[0101] In a preferred embodiment, a method for making a single
stranded nucleic acid with electron transfer moieties attached at
both the 3' and 5' terminus. The method comprises attaching a
modified nucleoside to control pore glass. The modified nucleoside
may be either amino-modified, for attachment via the ribose as
described herein, or modified at the base. Additional
phosphoramidite nucleosides are added to the 5' terminus of the
modified nucleoside to form a nucleic acid. A modified
phosphoramidite nucleoside is further added to the 5' terminus of
the nucleic acid, which is then optionally cleaved off the control
pore glass and may be hybridized to its complement. An electron
donor moiety is added to one modified nucleoside and an electron
acceptor moiety is added to the other modified nucleoside.
[0102] The cleavage from the CPG may occur either prior to
transition metal modification or afterwards.
[0103] It should be understood that it is important that the
basepairing of the nucleoside bases is not significantly perturbed
in order to allow hybridization, good electron transfer rates, and
the detection of mismatches. Thus, for example, the transition
metal moieities, when attached to the nucleic acids of the
invention, do not intercalate, i.e. insert and stack between the
basepairs of the double stranded nucleic acid. Intercalation of the
transition metals with the accompanying ligands disturbs the
basepairing, and thus hinders the transfer of electrons and the
identification of mismatches. Similarly, with the exception of
terminal bases, as is outlined below, attaching the transition
metal complexes at the nucleoside bases (Telser et al., supra) also
disturbs the basepairing and impedes the identification of
mismatches.
[0104] It should be noted that when using the above techniques for
the modification of internal residues it is possible to create a
nucleic acid that has an electron transfer species on the
next-to-last 3' terminal nucleoside, thus eliminating the need for
the extra steps required to produce the 3' terminally labeled
nucleoside.
[0105] In a further embodiment for the modification of internal
residues, 2' or 3' modified nucleoside triphosphates are generated
using the techniques described above for the 3' nucleoside
modification. The modified nucleosides are inserted internally into
nucleic acid using standard molecular biological techniques for
labeling DNA and RNA. Enzymes used for said labelling include DNA
polymerases such as polymerase I, T4 DNA polymerase, T7 DNA
polymerase, Taq DNA polymerase, reverse transcriptase and RNA
polymerases such as E. coli RNA polymerase or the RNA polymerases
from phages SP6, T7 or T3 (Short Protocols in Molecular Biology,
1992. Ausubel et al. Ed. pp 3.11-3.30).
[0106] As described above, the electron transfer moiety, preferably
a transition metal complex, may be attached 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. 111:7221-7226 (1989). As
outlined herein, these terminally modified nucleosides may be
attached to the nucleic acid enzymatically as is known in the art,
using DNA polymerases; alternatively, the modified nucleosides may
be incorporated into a growing oligonucleotide chain using
traditional phosphoramidite chemistry during oligonucleotide
synthesis as is outlined herein.
[0107] The exposed amine or other ligand at the 2' or 3' position
of the ribose, the phosphoramide linkages, or the other linkages
useful in the present invention, are readily modified with a
variety of electron transfer moieties, and particularly transition
metal complexes with techniques readily known in the art (see for
example Millet et al, in Metals in Biological Systems, Sigel et al.
Ed. Vol. 27, pp 223-264, Marcell Dekker Inc. New York, 1991 and
Durham, et al. in ACS Advances in Chemistry Series, Johnson et al.
Eds., Vol. 226, pp 180-193, American Chemical Society, Washington
D.C.; and Meade et al., J. Am. Chem. Soc. 111:4353 (1989)).
Generally, these techniques involve contacting a partially chelated
transition metal complex with the amine group of the modified
nucleoside.
[0108] The organic electron transfer species are also added to the
functional group of the modified nucleoside such as an amine group,
using techniques known in the art.
[0109] When peptide nucleic acids (PNA) are used, attachment of the
electron transfer moieties proceeds as follows. The amino group at
the N-terminus of the PNA will bind a partially chelated transition
metal or organic electron transfer moiety similar to the
amino-modified ribose. Addition to the carboxy terminus can proceed
in a variety of ways, one of which is depicted in FIG. 7.
Additionally, for single stranded PNAs, one electron transfer
moiety may be attached to the N-terminus, and the other electron
transfer moiety is attached to the terminal base at the carboxy
terminus. Alternatively, both transfer moieties are attached to
terminal bases. Similar combinations may be made for two single
stranded nucleic acids, each containing an electron transfer
moiety.
[0110] In addition, the present invention provides a novel method
for the site specific addition to the ribose-phosphate backbone of
a nucleic acid of electron donor and electron acceptor moieties to
a previously modified nucleoside.
[0111] In one embodiment, the electron donor and acceptor moieties
are attached to the modified nucleoside by methods which utilize a
unique protective hybridization step. In this embodiment, the
modified single strand nucleic acid is hybridized to an unmodified
complementary sequence. This blocks the sites on the heterocyclic
bases that are susceptible to attack by the transition metal
electron transfer species.
[0112] When the terminal bases are to be 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 to a probe
sequence of n+1 or n+2, such that the terminal base is not
protected. Thus the unprotected base is exposed to the electron
transfer moiety such that the moiety is attached to the base.
[0113] After successful addition of the desired metal complex, the
modified duplex nucleic acid is separated into single strands using
techniques well known in the art.
[0114] In a preferred embodiment, single stranded nucleic acids are
made which contain one electron donor moiety and one electron
acceptor moiety. The electron donor and electron acceptor moieties
may be attached at either the 5' or 3' end of the single stranded
nucleic acid. Alternatively, the electron transfer moieties may be
attached to internal nucleosides, or one to an internal nucleoside
and one to a terminal nucleoside. It should be understood that the
orientation of the electron transfer species with respect to the
5'-3' orientation of the nucleic acid is not determinative. Thus,
as outlined in FIG. 1, any combination of internal and terminal
nucleosides may be utilized in this embodiment.
[0115] In an alternate preferred embodiment, single stranded
nucleic acids with at least one electron donor moiety and at least
one electron acceptor moiety are used to detect mutations in a
complementary target sequence. A mutation, whether it be a
substitution, insertion or deletion of a nucleoside or nucleosides,
results in incorrect base pairing in a hybridized double helix of
nucleic acid. Accordingly, if the path of an electron from an
electron donor moiety to an electron acceptor moiety spans the
region where the mismatch lies, the electron transfer will be
eliminated or reduced such that a change in the relative rate will
be seen. Therefore, in this embodiment, the electron donor moiety
is attached to the nucleic acid at a 5' position from the mutation,
and the electron acceptor moiety is attached at a 3' position, or
vice versa.
[0116] In this embodiment it is also possible to use an additional
label on the modified single stranded nucleic acid to detect
hybridization where there is one or more mismatches. If the
complementary target nucleic acid contains a mutation, electron
transfer is reduced or eliminated. To act as a control, the
modified single stranded nucleic acid may be radio- or
fluorescently labeled, such that hybridization to the target
sequence may be detected, according to traditional molecular
biology techniques. This allows for the determination that the
target sequence exists but contains a substitution, insertion or
deletion of one or more nucleosides. Alternatively, single stranded
nucleic acids with at least one electron donor moiety and one
electron acceptor moiety which hybridize to regions with exact
matches can be used as a controls for the presence of the target
sequence.
[0117] It is to be understood that the rate of electron transfer
through a double stranded nucleic acid helix depends on the
nucleoside distance between the electron donor and acceptor
moieties. Longer distances will have slower rates, and
consideration of the rates will be a parameter in the design of
probes and bioconductors. Thus, while it is possible to measure
rates for distances in excess of 100 nucleosides, a preferred
embodiment has the electron donor moiety and the electron acceptor
moiety separated by at least 3 and no more than 100 nucleosides.
More preferably the moieties are separated by 8 to 64 nucleosides,
with 15 being the most preferred distance.
[0118] In addition, it should be noted that certain distances may
allow the utilization of different detection systems. For example,
the sensitivity of some detection systems may allow the detection
of extremely fast rates; i.e. the electron transfer moieties may be
very close together. Other detection systems may require slightly
slower rates, and thus allow the electron transfer moieties to be
farther apart.
[0119] In an alternate embodiment, a single stranded nucleic acid
is modified with more than one electron donor or acceptor moiety.
For example, to increase the signal obtained from these probes, or
decrease the required detector sensitivity, multiple sets of
electron donor-acceptor pairs may be used.
[0120] As outlined above, in some embodiments different electron
transfer moieties are added to a single stranded nucleic acid. For
example, when an electron donor moiety and an electron acceptor
moiety are to be added, or several different electron donors and
electron acceptors, the synthesis of the single stranded nucleic
acid proceeds in several steps. First partial nucleic acid
sequences are made, each containing a single electron transfer
species, i.e. either a single transfer moiety or several of the
same transfer moieties, using the techniques outlined above. Then
these partial nucleic acid sequences are ligated together using
techniques common in the art, such as hybridization of the
individual modified partial nucleic acids to a complementary single
strand, followed by ligation with a commercially available
ligase.
[0121] Alternatively, single stranded nucleic acid may be made 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 has a temporary protecting group on the
amine such as DMT. Upon hybridization to the complementary
unmodified strand, the unprotected amine is exposed to the first
electron transfer moiety, i.e. either a donor or an acceptor,
resulting in covalent attachment. The protecting group of the
protected amino-modified nucleoside is then removed, and the hybrid
is contacted with the second electron transfer species, and the
strands separated, resulting in a single strand being labeled with
both a donor and acceptor. The single strand containing the proper
electron transfer moieties is then purified using traditional
techniques.
[0122] In a preferred embodiment, single stranded nucleic acids are
made which contain one electron donor moiety or one electron
acceptor moiety. The electron donor and electron acceptor moieties
are attached at either the 5' or 3' end of the single stranded
nucleic acid. Alternatively, the electron transfer moiety is
attached to an internal nucleoside.
[0123] It is to be understood that different species of electron
donor and acceptor moieties may be attached to a single stranded
nucleic acid. Thus, more than one type of electron donor moiety or
electron acceptor moiety may be added to any single stranded
nucleic acid.
[0124] In a preferred embodiment, a first single stranded nucleic
acid is made with on or more electron donor moieties attached. A
second single stranded nucleic acid has one or more electron
acceptor moieties attached. In this embodiment, the single stranded
nucleic acids are made for use as probes for a complementary target
sequence. In one embodiment, the complementary target sequence is
made up of a first target domain and a second target domain, where
the first and second sequences are directly adjacent to one
another. In this embodiment, the first modified single stranded
nucleic acid, which contains only electron donor moieties or
electron acceptor moieties but not both, hybridizes to the first
target domain, and the second modified single stranded nucleic
acid, which contains only the corresponding electron transfer
species, binds to the second target domain. The relative
orientation of the electron transfer species is not important, as
outlined in FIG. 2, and the present invention is intended to
include all possible orientations.
[0125] In the design of probes comprised of two single stranded
nucleic acids which hybridize to adjacent first and second target
sequences, several factors should be considered. These factors
include the distance between the electron donor moiety and the
electron acceptor moiety in the hybridized form, and the length of
the individual single stranded probes. For example, it may be
desirable to synthesize only 5' terminally labeled probes. In this
case, the single stranded nucleic acid which hybridizes to the
first sequence may be relatively short, such that the desirable
distance between the probes may be accomplished. For example, if
the optimal distance between the electron transfer moieties is 15
nucleosides, then the first probe may be 15 nucleosides long.
[0126] In one aspect of this embodiment, the two single stranded
nucleic acids which have hybridized to the adjacent first and
second target domains are ligated together prior to the electron
transfer reaction. This may be done using standard molecular
biology techniques utilizing a DNA ligase, such as T4 DNA
ligase.
[0127] In an alternative embodiment, the complementary target
sequence will have a first target domain, an intervening target
domain, and a second target domain. In this embodiment, the first
modified single stranded nucleic acid, which contains only electron
donor moieties or electron acceptor moieties but not both,
hybridizes to the first target domain, and the second modified
single stranded nucleic acid, which contains only the corresponding
electron transfer species, binds to the second target domain. When
an intervening single stranded nucleic acid hybridizes to the
intervening target sequence, electron transfer between the donor
and acceptor is possible. The intervening sequence may be any
length, and may comprise a single nucleoside. Its length, however,
should take into consideration the desirable distances between the
electron donor and acceptor moieties on the first and second
modified nucleic acids. Intervening sequences of lengths greater
than 14 are desirable, since the intervening sequence is more
likely to remain hybridized to form a double stranded nucleic acid
if longer intervening sequences are used. The presence or absence
of an intervening sequence can be used to detect insertions and
deletions.
[0128] In one aspect of this embodiment, the first single stranded
nucleic acid hybridized to the first target domain, the intervening
nucleic acid hybridized to the intervening domain, and the second
single stranded nucleic acid hybridized to the second target
domain, may be ligated together prior to the electron transfer
reaction. This may be done using standard molecular biology
techniques. For example, when the nucleic acids are DNA, a DNA
ligase, such as T4 DNA ligase can be used.
[0129] The complementary target single stranded nucleic acid of the
present invention may take many forms. For example, the
complementary target single stranded nucleic acid sequence may be
contained within a larger nucleic acid sequence, i.e. all or part
of a gene or mRNA, a restriction fragment of a plasmid or genomic
DNA, among others. One skilled in the art of molecular biology
would understand how to construct useful probes for a variety of
target sequences using the present invention.
[0130] In one embodiment, two single stranded nucleic acids with
covalently attached electron transfer moieties have complementary
sequences, such that they can hybridize together to form a
bioconductor. In this embodiment, the hybridized duplex is capable
of transferring at least one electron from the electron donor
moiety to the electron acceptor moiety. In a preferred embodiment,
the individual single stranded nucleic acids are aligned such that
they have blunt ends; in alternative embodiments, the nucleic acids
are aligned such that the double helix has cohesive ends. In either
embodiment, it is preferred that there be uninterrupted double
helix base-pairing between the electron donor moiety and the
electron acceptor moiety, such that electrons may travel through
the stacked base pairs.
[0131] In one bioconductor embodiment, the double stranded nucleic
acid has one single strand nucleic acid which carries all of the
electron transfer moieties.
[0132] In another embodiment, the electron transfer moieties may be
carried on either strand, and in any orientation. For example, one
strand may carry only electron donors, and the other only electron
acceptors or both strands may carry both.
[0133] In one embodiment, the double stranded nucleic acid may have
different electron transfer moieties covalently attached in a fixed
orientation, to facilitate the long range transfer of electrons.
This type of system takes advantage of the fact that electron
transfer species may act as both electron donors and acceptors
depending on their oxidative state. Thus, an electron donor moiety,
after the loss of an electron, may act as an electron acceptor, and
vice versa. Thus, electron transfer moieties may be sequentially
oriented on either strand of the double stranded nucleic acid such
that directional transfer of an electron over very long distances
may be accomplished. For example, a double stranded nucleic acid
could contain a single electron donor moiety at one end and
electron acceptor moieties, of the same or different composition,
throughout the molecule. A cascade effect of electron transfer
could be accomplished in this manner, which may result in extremely
long range transfer of electrons. This may be accomplished, for
example, by incorporating transition metal complexes that possess a
range in oxidation potentials due to ligand substitutions made at
the metal center.
[0134] The choice of the specific electron donor and acceptor pairs
will be influenced by the type of electron transfer measurement
used; for a review, see Winkler et al., Chem. Rev. 92:369-379
(1992). When a long-lived excited state can be prepared on one of
the redox sites, direct measurement of the electron transfer rate
after photoinduction can be measured, using for example the
flash-quench method of Chang et al., J. Amer. Chem. Soc. 113:7057
(1991). In this preferred embodiment, the excited redox site, being
both a better acceptor and donor than the ground-state species, can
transfer electrons to or from the redox partner. An advantage of
this method is that two electron transfer rates may be measured:
the photoinduced electron transfer rates and thermal electron-hole
recombination reactions. Thus differential rates may be measured
for hybridized nucleic acids with perfect complementarity and
nucleic acids with mismatches.
[0135] In alternative embodiments, neither redox site has a long
lived excited state, and electron transfer measurements depend upon
bimolecular generation of a kinetic intermediate. For a review, see
Winkler et al., supra. This intermediate then relaxes to the
thermodynamic product via intramolecular electron transfer using a
quencher, as seen below:
D-A+hv.fwdarw.D-A*
D-A*+Q.fwdarw.D-A.sup.++Q.sup.-
D-A*+.fwdarw.D.sup.+-A
D.sup.+-A+Q.sup.-.fwdarw.D-A+Q
[0136] The upper limit of measurable intramolecular electron
transfer rates using this method is about 10.sup.4 per second.
[0137] Alternative embodiments use the pulse-radiolytic generation
of reducing or oxidizing radicals, which inject electrons into a
donor or remove electrons from a donor, as reviewed in Winkler et
al., supra.
[0138] As is appreciated in the art, there are a variety of ways to
initiate and detect the electron transfer.
[0139] Electon transfer can be initiated and detected using a wide
variety of methods, including, but not limited to, electrical,
electrochemical, electromagnetic radiation (optical) and chemical
methods. It is possible to make a variety of compositions utilizing
different electron transfer moieties depending on the desired
methods of initiating electron transfer and detection of electron
transfer. Table 2 depicts a variety of preferred combinations for
initiation and detection of electron transfer in the complexes of
the invention.
2TABLE 2 Initiation Detection Description light light absorbance,
fluorescence, phosphorescence, refractive index, surface plasmon
resonance, electron spin resonance light current amperommetry,
voltammetry, capacitance, impedence, opto-electronic detection,
photo- amperometry light plus light absorbance, fluorescence,
phosphorescence, electronic refractive index, surface plasmon
resonance, initiation electron spin resonance light plus current
amperommetry, voltammetry, capacitance, electronic impedence,
opto-electronic detection, photo- initiation amperommetry,
amperommetric detection, cyclic voltammetry electronic current
amperommetry, voltammetry, capacitance, initiation impedence,
amperommetric detection, cyclic voltammetry electronic light
chemiluminescence, initiation electrochemiluminescence,
electroluminescence
[0140] By "light" herein is meant electromagnetic radiation, with
light in the UV, visible and infrared range being preferred, and UV
and visible being the most preferred.
[0141] In a preferred embodiment, initiation of electron transfer
is via direct or indirect photoactivation ("light in"). Simply,
electromagnetic radiation of appropriate wavelength strikes the
redox molecule on one end of the DNA causing excitation of a donor
moiety electron which either decays immediately or is involved in
intramolecular electron transfer. The efficiency with which
electron transfer is induced depends upon the electronic coupling
between the electron donor and acceptor and therefore depends on
whether the nucleic acid is single or double stranded. In addition,
the efficiency of electron transfer depends upon the extinction
coefficient of the electron donor at the wavelength of light used
(higher is better) and upon the lifetime of the donor electron
excited state (longer is better). Preferred donor complexes
therefore include acridine orange, N,N'-dimethyl-2,7-diazapyrenium
dichloride (DAP.sup.2+), methylviologen, ethidium bromide, quinones
such as N,N'-dimethylanthra(2,1,9-def:6,5,10-d'e'f')diisoquinoline
dichloride (ADIQ.sup.2+); porphyrins
([meso-tetrakis(N-methyl-x-pyridinium)porphyrin tetrachloride].
Transition metal donors and acceptors include complexes of
ruthenium, rhenium and osmium (most preferred) where at least one
of the ligands is a chromophore.
[0142] Photoactivation can also be used to excite "mediators" that
transfer energy to the electron donor moiety on the DNA via an
inter-molecular process. Such mediators include water soluble and
stable complexes of the transition metals, including molybdenum and
tungsten halides, trisbipyridyl complexes of rhenium, osmium and
ruthenium. In addition, other examples include bipyridyl and
pyridyl complexes such as Re(bpy)(CO).sub.3X where X is a halide
and Re(py).sub.4O.sub.2. Other examples include transition metal
dimers such as [Re.sub.2Cl.sub.8].sup.2- - and
[Pt.sub.2(P.sub.2O.sub.5H.sub.2).sub.4].sup.4-. Ruthenium
trisbypyridine (Ru.sup.2+(bpy).sub.3) is most preferred.
[0143] In the preferred embodiment, electron transfer occurs after
photoinduction with a laser. In this embodiment, electron donor
moieties may, after donating an electron, serve as electron
acceptors under certain circumstances. Similarly, electron acceptor
moieties may serve as electron donors under certain
circumstances.
[0144] A preferred embodiment utilizes electronic activation, with
voltage being preferred. A potential is applied to a sample
containing modified nucleic acid probes either via a direct linkage
of the modified nucleic acid to an electrode, or using electron
transport mediators. Direct linkage can involve a redox active
polymer to shuttle electrons from (and to, if the electrode is also
used for detection) the electrode. Such polymers are outlined
below. Alternatively, the direct connection can involve a
relatively poorly conducting linkage provided the linkage is kept
reasonably short (less than six sigma bonds). Preferred linkages
will be three or fewer sigma bonds in length to allow efficient
transfer of electrons from the electrode, as is outlined below.
[0145] Indirect electron transfer initiation involves electron
transfer mediators or effective diffusional electron donors and
acceptors such as water soluble ferrocene/ferricinium,
hydroquinones/quinones, reducible and oxidizable components of
organic salts, cobaltocenes, the hexa- and octacyanides of
molybdenum, tungsten and iron. In addition, other examples include
macrocycles and chelating ligands of transition metals such as
cobalt, ruthenium and nickel, including Co(ethylenediamine).sub.3
and Ru(ethylenediamine).sub.3 and the trisbypyridyl 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.
[0146] Precise control and variations in the applied potential can
be via a potentiostat and a three electrode system (one reference,
one sample and one counter electrode). This allows matching of
applied potential to peak electron transfer potential of the system
which depends in part on the choice of electron acceptors attached
to the nucleic acid. High driving forces are achieved using
bisbipyridyl complexes of transition metals, for example, ruthenium
and rhenium bisbipyridyl complexes such as (Ru(bpy).sub.2im-) as
electron acceptors.
[0147] Alternatively, electrochemical initiation of electron
transfer may be used. The redox states of the electron donating and
accepting moieties attached to nucleic acid can be electrochemicaly
changed using water soluble chemical oxidants and reductants,
either with or without photo- or electrical activation. Such
compounds include numerous derivatives known in the art (T. Kuwana,
Electrochemical Studies of Biological Systems, (D. T. Sawyer Ed.)
ACS Symp. Series #38, (1977)) and include hexacyano iron complexes,
zinc-mercury amalgam, and trisphenanthroline complexes of ruthenium
and iron.
[0148] Electron transfer through nucleic acid can be detected in a
variety of ways. A variety of detection methods may be used,
including, but not limited to, optical detection, which includes
fluorescence, phosphorescence, and refractive index; and electronic
detection, including, but not limited to, amperommetry,
voltammetry, capacitance and impedence. These methods include time
or frequency dependent methods based on AC or DC currents, pulsed
methods, lock-in techniques, filtering (high pass, low pass, band
pass), and time-resolved techniques including time-resolved
fluorescence. In some embodiments, all that is required is electron
transfer detection; in others, the rate of electron transfer may be
determined.
[0149] In one embodiment, the efficient transfer of electrons from
one end of a nucleic acid double helix to the other results in
stereotyped changes in the redox state of both the electron donor
and acceptor. With many electron transfer moieties including the
complexes of ruthenium containing bipyridine, pyridine and
imidazole rings, these changes in redox state are associated with
changes in spectral properties ("light out"). Significant
differences in absorbance are observed between reduced and oxidized
states for these molecules. These differences can be monitored
using a spectrophotometer or simple photomultiplier tube
device.
[0150] In this embodiment, possible electron donors and acceptors
include all the derivatives listed above for photoactivation or
initiation. Preferred electron donors and acceptors have
characteristically large spectral changes upon oxidation and
reduction (large extinction coefficient "deltas") resulting in
highly sensitive monitoring of electron transfer. Such examples
include Ru(NH.sub.3).sub.4py and Ru(bpy).sub.2im as preferred
examples. It should be understood that only the donor or acceptor
that is being monitored by absorbance need have ideal spectral
characteristics. That is, the electron acceptor can be optically
invisible if only the electron donor is monitored for absorbance
changes.
[0151] In a preferred embodiment, the electron transfer is detected
fluorometrically. Numerous transition metal complexes, including
those of ruthenium, have distinct fluorescence properties.
Therefore, the change in redox state of the electron donors and
electron acceptors attached to the nucleic acid can be monitored
very sensitively using fluorescence. Highly efficient electron
transfer through double stranded nucleic acid can, for example,
result in the production of fluorescent
Ru(4,7-biphenyl.sub.2-phenanthroline).sub.3.sup.2+ at one end of a
nucleic acid probe when the electron transfer moiety on the other
end is excited. The production of this compound can be easily
measured using standard fluorescence assay techniques. For example,
laser induced fluorescence can be recorded in a standard single
cell fluorimeter, a flow through "on-line" fluorimeter (such as
those attached to a chromatography system) or a multi-sample
"plate-reader" similar to those marketed for 96-well immuno assays.
Alternatively, fluorescence can be measured using fiber optic
sensors with nucleic acid probes in solution or attached to the
fiber optic. Fluorescence is monitored using a photomultiplier tube
or other light detection instrument attached to the fiber optic.
The advantage of this system is the extremely small volumes of
sample that can be assayed.
[0152] In addition, scanning fluorescence detectors such as the
FluorImager sold by Molecular Dynamics are ideally suited to
monitoring the fluorescence of modified nucleic acid molecules
arrayed on solid surfaces. The advantage of this system is the
large number of electron transfer probes that can be scanned at
once using chips covered with thousands of distinct nucleic acid
probes.
[0153] Many transition metal complexes display fluorescence with
large Stokes shifts. Suitable examples include bis- and
trisphenanthroline complexes and 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 display efficient fluorescence (reasonably high quantum
yields) as well as low reorganization energies. These include
Ru(4,7-biphenyl.sub.2-phenanthroline).sub.3.sup.2+ and
Ru(4,4'-diphenyl-2,2'-bipyridine).sub.3.sup.2+.
[0154] Alternatively, a reduction in fluorescence associated with
hybridization can be measured using these systems. An electron
transfer "donor" molecule that fluoresces readily when on single
stranded nucleic acid (with an "acceptor" on the other end) will
undergo a reduction in fluorescent intensity when complementary
nucleic acid binds the probe allowing efficient transfer of the
excited state electron. This drop in fluorescence can be easily
monitored as an indicator of the presence of a target sequence
using the same methods as those above.
[0155] In a further embodiment, electrochemiluminescence is used as
the basis of the electron transfer detection. With some electron
transfer moieties such as Ru.sup.2+ (bpy).sub.3, direct
luminescence accompanies excited state decay. Changes in this
property are associated with nucleic acid hybridization and can be
monitored with a simple photomultiplier tube arrangement (see
Blackburn, G. F. Clin. Chem. 37: 1534-1539 (1991); and Juris et
al., supra.
[0156] In a preferred embodiment, electronic detection is used,
including amperommetry, voltammetry, capacitance, and impedence.
Suitable techniques include, but are not limited to,
electrogravimetry; coulometry (including controlled potential
coulometry and constant current coulometry); voltametry (cyclic
voltametry, pulse voltametry (normal pulse voltametry, square wave
voltametry, differential pulse voltametry, Osteryoung square wave
voltametry, and coulostatic pulse techniques); stripping analysis
(aniodic stripping analysis, cathiodic stripping analysis, square
wave stripping voltammetry); conductance measurements (electrolytic
conductance, direct analysis); time-dependent electrochemical
analyses (chronoamperometry, chronopotentiometry, cyclic
chronopotentiometry and amperometry, AC polography,
chronogalvametry, and chronocoulometry); AC impedance measurement;
capacitance measurement; and photoelectrochemistry.
[0157] In a preferred embodiment, monitoring electron transfer
through nucleic acid is via amperometric detection, either directly
using a covalently attached electrode, or indirectly using electron
transport "mediators" to shuttle electrons from the nucleic acid to
an electrode. Modes of attaching nucleic acids to electrodes and
possible mediators are described below. An amperometric detector
would resemble the numerous enzyme-based biosensors currently used
to monitor blood glucose, for example. This method of detection
involves applying a potential (as compared to a separate reference
electrode) between the nucleic acid-conjugated electrode and an
auxiliary (counter) electrode in the sample containing target genes
of interest. Electron transfer of differing efficiencies is induced
in samples in the presence or absence of target nucleic acid; that
is, the single stranded probe exhibits a different rate than the
probe hybridized to the target sequence. The differing efficiencies
of electron transfer result in differing currents being generated
in the electrode.
[0158] The device for measuring electron transfer amperometrically
involves sensitive (nanoamp to picoamp) current detection and
includes a means of controlling the voltage potential, usually a
potentiostat. This voltage is optimized with reference to the
potential of the electron donating complex on the nucleic acid.
Possible electron donating complexes include those previously
mentioned with complexes of ruthenium being preferred and complexes
of rhenium being most preferred.
[0159] In a preferred embodiment, alternative electron detection
modes are utilizes. For example, potentiometric (or voltammetric)
measurements involve non-faradaic (no net current flow) processes
and are utilized traditionally in pH and other ion detectors.
Similar sensors are used to monitor electron transfer through
nucleic acid. In addition, other properties of insulators (such as
resistance) and of conductors (such as conductivity, impedance and
capicitance) could be used to monitor electron transfer through
nucleic acid. Finally, any system that generates a current (such as
electron transfer) also generates a small magnetic field, which may
be monitored in some embodiments.
[0160] It should be understood that one benefit of the fast rates
of electron transfer observed in the compositions of the invention
is that time resolution can greatly enhance the signal-to-noise
results of monitors based on absorbance, fluorescence and
electronic current. The fast rates of electron transfer of the
present invention result both in high signals and stereotyped
delays between electron transfer initiation and completion. By
amplifying signals of particular delays, such as through the use of
pulsed initiation of electron transfer and "lock-in" amplifiers of
detection, between two and four orders of magnitude improvements in
signal-to-noise may be achieved. This is particularly true using AC
methodology, as is more fully described below.
[0161] In a preferred embodiment, electron transfer is initiated
and detected using alternating current (AC) methods, particularly
when one of the electron transfer moieties is an electrode; that
is, when the nucleic acid is attached to an electrode. This system
is particularly advantageous for a number of reasons. In general,
the use of AC techniques can result in good signals and low
background noise. Without being bound by theory, there are a number
of possible contributors to background noise, or "parasitic"
signals, i.e. detectable signals that are inherent to the system
but are not the result of the presence of the target sequence.
[0162] However, all of the contributors to parasitic noise will
generally give very fast signals; that is, the rate of electron
transfer through the double helix, i.e. the "n-way", is generally
significantly slower than the rate of electron transfer of the
parasitic components, such as the contribution of charge carriers
in solution, and other "short circuiting" mechanisms. As a result,
the parasitic components are generally all phase related; that is,
they exhibit a constant phase delay or phase shift that will scale
directly with frequency. The hybridization complex, in contrast,
exhibits a time delay between the input and output signals, which
is independent of frequency. Thus, for the hybridization signal,
the time it takes electrons to travel between the electron transfer
moieties will remain constant and relatively large as compared to
parasitic background. As a consequence, at different frequencies,
the phase of the system will change. This is very similar to the
time domain detection used in fluorescent systems.
[0163] This difference can be exploited in various methods to
decrease the signal to noise ratio. Accordingly, the preferred
detection methods comprise applying an AC input signal to a
hybridization complex comprising a first single stranded nucleic
acid containing an electrode and a second electron transfer moiety
and a target single stranded nucleic acid. The presence of the
target nucleic acid (hybridization complex) is detected via an
output signal characteristic of electron transfer through the
hybridization complex; that is, the output signal is characteristic
of the hybridization complex rather than the parasitic components
or single stranded nucleic acid. Thus, for example, the output
signal will exhibit a time delay dependent on the rate of electron
transfer between the two electron transfer moieties. It should be
noted that this time delay will also vary depending on the distance
between the electron transfer moieties; the farther apart the
electron transfer moieties are, the longer the time delay.
[0164] In a preferred embodiment, the input signals are applied at
a plurality of frequencies, since this again allows the distinction
between true signal and noise. "Plurality" in this context means at
least two, and preferably more, frequencies. In general, the AC
frequencies will range from about 0.1 Hz to about 10 mHz, with from
about 1 Hz to 100 KHz being preferred.
[0165] In a preferred embodiment, DNA is modified by the addition
of electron donor and electron acceptor moieties. In an alternative
embodiment, RNA is modified. In a further embodiment, a double
stranded nucleic acid for use as a bioconductor will contain some
deoxyribose nucleosides, some ribose nucleosides, and a mixture of
adenosine, thymidine, cytosine, guanine and uracil bases.
[0166] In accordance with a further aspect of the invention, the
preferred formulations for donors and acceptors will possess a
transition metal covalently attached to a series of ligands and
further covalently attached to an amine group as part of the ribose
ring (2' or 3' position) or to a nitrogen or sulfur atom as part of
a nucleoside dimer linked by a peptide bond, phosphoramidate bond,
phosphorothioate bond, phosphorodithioate bond or O-methyl
phosphoramidate bond.
[0167] In a preferred embodiment, an oligonucleotide containing at
least one electron transfer moiety is attached to an electrode,
which also serves as an electron transfer moiety, thus forming a
single stranded nucleic acid with both an electron donor moiety and
an electron acceptor moiety attached in the manner outlined above.
Preferably, the single stranded nucleic acid containing an electron
transfer moiety is attached covalently or in such a way that allows
the transfer of electrons from the electrode to the single stranded
nucleic acid in order to allow electron transfer between the
electron donor and acceptor. Preferably, the non-electrode electron
transfer moiety is attached at or near the terminus of the
oligonucleotide, such that the probe sequence to be hybridized to
the target sequence is between the donor and acceptor. The
electrode may be immersed in a sample containing the target
sequence such that the target sequence hybridizes to the probe and
electron transfer may be detected using the techniques outlined
above.
[0168] In an additional embodiment, two nucleic acids are utilized
as probes as described previously. For example, one nucleic acid is
covalently attached to a solid electrode which serves as an
electron transfer moiety, and the other, with a covalently attached
electron transfer moiety, is free in solution. Upon hybridization
of a target sequence, the two nucleic acids are aligned such that
electron transfer between the electron transfer moiety of the
hybridized nucleic acid and the electrode occurs. The electron
transfer is detected as outlined above, using techniques well known
in the art.
[0169] The following examples serve to more fully describe the
manner of using the above-described invention, as well as to set
forth the best modes contemplated for carrying out various aspects
of the invention. It is understood that these examples in no way
serve to limit the true scope of this invention, but rather are
presented for illustrative purposes. The references cited herein
are expressly incorporated by reference.
EXAMPLES
[0170] The amino-modified monomer units are prepared by variation
of published procedures and are incorporated into a growing
oligonucleotide by standard synthetic techniques. The procedure is
applicable to both DNA and RNA derivatives.
Example 1
Synthesis of an Oligonucleotide Duplex with Electron Transfer
Moieties at the 5' Termini
[0171] In this example an eight nucleotide double stranded nucleic
acid was produced, with each single strand having a single electron
transfer moiety covalently attached to the 5' terminal uridine
nucleotide at the 2' carbon of the ribose sugar.
[0172] Step 1: Synthesis of 5'-di(p-methoxyphenyl)methyl
ether-2'-(trifluoroacetamido)-2'-deoxyuridine
2'-(trifluoroacetamido)-2'-- deoxyuridine (2.0 g, 5.9 mmoles)
prepared by minor modification of published procedures (Imazawa,
supra) was repeatedly dissolved in a minimum of very dry CH.sub.3CN
and rotary evaporated to dryness and then transferred to inert
atmosphere vacuum line and further dried for a period of 1 hour.
The following procedure for the synthesis of the material was
adapted from Gait (supra): Under positive pressure argon, the
material was dissolved in freshly dried and distilled pyridine and
with stirring, 0.05 equivalents (wt.) of 4-dimethylaminopyridine
(DMAP), 1.5 equivalents of triethylamine (TEA) and 1.2 equivalents
of 4,4'-dimethoxytrityl chloride (DMTr-Cl) were added to the
reaction mixture. The progress of the reaction was monitored by
silica gel TLC (98:2 methylene chloride:methanol, mobile phase).
After 30 minutes, an additional 0.5 equivalents each of DMTr-Cl and
TEA were added and the reaction allowed to proceed for an
additional three hours. To this reaction mixture was added an equal
volume of water and the solution extracted several times with
diethyl ether. The ether layers were rotary evaporated to dryness,
redissolved in a minimum amount of methylene chloride and purified
by flash chromatography (99:1 methylene chloride:methanol, mobile
phase), to obtain the 5'-di(p-methoxyphenyl)met- hyl
ether-2'(trifluoroacetamido)-2'-deoxyuridine product.
[0173] Step 2: 5'-2'-aminouridine-GCTACGA and
5'-2'-aminouridine-CGTAGCA
[0174] 5'-di(p-methoxyphenyl)methyl
ether-2'-(trifluoroacetamido)-2'-deoxy- uridine was dried under
reduced pressure (glass) and dissolved in freshly dried and
distilled CH.sub.3CN and placed in a specially made conical vial
and placed on an ABI DNA synthesizer. The program for the
preparation of standard (i.e. unmodified) oligonucleotides was
altered during the final base (amino-modified) addition to a 15-30
minute coupling time. The oligonucleotide was cleaved from the
column by standard procedures and purified by C-18 reverse phase
HPLC. In this manner 5'-2'-aminouridine-GCTACGA and
5'-2'-aminouridine-CGTAGCA were prepared. In addition, unmodified
complementary strands to both products were made for use in the
electron transfer moiety synthesis below.
[0175] Step 3: 5'-2'-ruthenium
bisbipyridineimidazole-aminouridine-GCTACGA
[0176] 5'-2'-aminouridine GCTACGA produced in the previous step was
annealed to the complementary unmodified strand using standard
techniques. All manipulations of the annealed duplex, prior to the
addition of the transition metal complex were handled at 4.degree.
C. In order to insure that the DNA remained annealed during
modification, the reactions were performed in 1 M salt. The
5'-amino modified duplex DNA was dissolved in 0.2 M HEPES, 0.8 M
NaCl, pH 6.8 and repeatedly evacuated on a Schlenk line. Previously
prepared ruthenium bisbipyridine carbonate was dissolved in the
above buffer and oxygen was removed by repeated evacuation and
purging with argon via a Schlenk line. The ruthenium complex was
transferred to the DNA solution via cannulation (argon/vacuum) and
the reaction allowed to proceed under positive pressure argon with
stirring for 24 hours. To this reaction, 50 equivalents of
imidazole was added to the flask and the reaction allowed to
proceed for an additional 24 hours. The reaction mixture was
removed from the vacuum line and applied to a PD-10 gel filtration
column and eluted with water to remove excess ruthenium complex.
The volume of the collected fractions was reduced to dryness via a
speed vac and the solid taken up in 0.1 M triethylammonium acetate
(TEAC) pH 6.0. The duplex DNA was heated to 60.degree. C. for 15
minutes with 50% formamide to denature the duplex. The single
stranded DNA was purified using a C-18 reverse phase HPLC column
equipped with a diode array detector and employing a gradient from
3% to 35% acetonitrile in 0.1 M TEAC, pH 6.0.
[0177] Step 4: 5'-2'-ruthenium
tetraminepyridine-aminouridine-CGTAGCA
[0178] 5'-aminouridine-CGTAGCA (0.3 .mu.m) was dissolved in 0.2 M
HEPES, 0.8 M NaCl buffer, pH 6.8 and degassed on the vacuum line.
To a 10 ml conical shaped flask equipped with a stirring bar and
septum was slurried Ru(III) tetraaminepyridine chloride (10 .mu.m),
in the same buffer. In a separate flask, Zn/Hg amalgam was prepared
and dried under reduced pressure and the ruthenium(III) solution
transferred (via cannulation) to the Zn/Hg amalgam. The immediate
formation of a clear yellow solution (.lambda..sub.max=406 nm)
indicated that the reduced form of the ruthenium had been achieved
and the reaction allowed to proceed for 30 minutes. This solution
was transferred to the flask containing the amino-modified DNA and
the reaction allowed to proceed at room temperature for 24 hours
under argon. The reaction mixture was removed from the vacuum line
and a 50 fold excess of cobalt EDTA (Kirschner, Inorganic Synthesis
(1957), pp 186) added to the solution. The solution was applied to
Sephadex G-25 gel filtration column to remove excess ruthenium
complex and further purified by reverse phase HPLC as described
above. The two ruthenium modified nucleotides were annealed by
standard techniques and characterized (see Example 5).
Example 2
Synthesis of Long DNA Duplexes with Electron Transfer Moieties at
the 5' Termini
[0179] In this example, an in vitro DNA amplification technique,
PCR (reviewed in Abramson et al., Curr. Op. in Biotech. 4:41-47
(1993)) is used to generate modified duplex DNA by polymerization
of nucleotides off modified primer strands (Saiki et al., Science
239:487 (1988)). Two oligonucleotides 18 bases in length and not
complementary to each other are synthesized with amino-modification
to the 2'-ribose position of the 5' nucleotides, as in example
1.
[0180] A series of oligonucleotides of increasing lengths starting
at 40 bases are chemically synthesized using standard chemistry.
Each of the PCR templates shares a 5' sequence identical to one
modified 18mer. The 3' end of the template oligonucleotide shares a
sequence complementary to the other 18mer.
[0181] PCR rapidly generates modified duplex DNA by the catalysis
of 5'-3' DNA synthesis off of each of the modified 18mers using the
unmodified strand as a template. One hundred nanomoles of each of
the two modified 18mers are mixed in 1 ml of an aqueous solution
containing 2,000 units of Taq polymerase, deoxyribonucleoside
triphosphates at 0.2 M each, 50 mM KCl, 10 mM Tris-Cl, pH 8.8, 1.5
mM MgCl.sub.2, 3 mM dithiothreitol and 0.1 mg/ml bovine serum
albumin. One femtomole of the template strand 40 bases in length is
added to the mixture. The sample is heated at 94.degree. C. for one
minute for denaturation, two minutes at 55.degree. C. for annealing
and three minutes at 72.degree. C. for extension. This cycle is
repeated 30 times using an automated thermal cycler.
[0182] The amplified template sequences with transition metal
complexes on both 5' termini are purified by agarose gel
electrophoresis and used directly in electron transfer
applications.
Example 3
Synthesis of Covalently Bound Electron Transfer Moieties at
Internucleotide Linkages of Duplex DNA
[0183] In this example, alternative backbones to phophodiester
linkages of oligonucleotides are employed. Functional groups
incorporated into these internucleotide linkages serve as the site
for covalent attachment of the electron transfer moieties. These
alternate internucleotide linkages include, but are not limited to,
peptide bonds, phosphoramidate bonds, phosphorothioate bonds,
phosphorodithioate bonds and O-methylphosphoramidate bonds.
[0184] The preparation of peptide nucleic acid (PNA) follows
literature procedures (See Engholm, supra), with the synthesis of
Boc-protected pentaflurophenyl ester of the chosen base
(thymidine). The resulting PNA may be prepared employing
Merrifield's solid-phase approach (Merrifield, Science, 232:341
(1986)), using a single coupling protocol with 0.1 M of the
thiminyl monomer in 30% (v/v) DMF in CH.sub.2Cl.sub.2. The progress
of the reaction is followed by quantiative ninhydrin analysis
(Sarin, Anal. Biochem., 117:147 (1981)). The resulting PNA may be
modified with an appropriate transition metal complex as outlined
in example 1.
[0185] The synthesis of phosphoramidate (Beaucage, supra,
Letsinger, supra, Sawai, supra) and N-alkylphosphoramidates (Jager,
supra) internucleotide linkages follows standard literature
procedures with only slight modification (the procedures are halted
after the addition of a single base to the solid support and then
cleaved to obtain a dinucleotide phosphoramidate). A typical
example is the preparation of the phenyl ester of
5'O-isobutyloxycarbonylthymidyl-(3'-5')-5'-amino-5'-d-
eoxythymidine (Letsinger, J. Org. Chem., supra). The dimer units
are substituted for standard oligonucleotides at chosen intervals
during the preparation of DNA using established automated
techniques. Transition metal modification of the modified linkages
takes place as described in Example 1.
[0186] The synthesis of phosphorothioate and phosphorodithioate
(Eckstein, supra, and references within) internucleotide linkages
is well documented. A published protocol utilizes an Applied
Biosystems DNA synthesizer using a modified
.beta.-cyanoethylphosphoramidite cycle that caps after
sulphurization with tetraethylthiuram disulfide (TETD) (Iyer, J.
Org. Chem. 55:4693 (1990)). The phosphorothioate and
phosphorodithioate analogs are prepared as dimers and cleaved from
the solid support and purified by HPLC
(acetonitrile/triethylammonium acetate mobile phase).
Example 4
Synthesis of Two Oligonucleotides Each with an Electron Transfer
Moiety at the 5' Terminus
[0187] In this example, two oligonucleotides are made which
hybridize to a single target sequence, without intervening
sequences. One oligonucleotide has an electron donor moiety
covalently attached to the 5' terminus, and the other has an
electron acceptor moiety covalently attached to the 5' terminus. In
this example, the electron transfer species are attached via a
uradine nucleotide, but one skilled in the art will understand the
present methods can be used to modify any of the nucleotides. In
addition, one skilled in the art will recognize that the procedure
is not limited to the generation of 8-mers, but is useful in the
generation of oligonucleotide probes of varying lengths.
[0188] The procedure is exactly as in Example 1, except that the
8-mers generated are not complementary to each other, and instead
are complementary to a target sequence of 16 nucleotides. Thus the
final annealing step of step 4 of Example 1 is not done. Instead,
the two modified oligonucleotides are annealed to the target
sequence, and the resulting complex is characterized as in Example
5.
Example 5
Characterization of Modified Nucleic Acids
[0189] Enzymatic Digestion
[0190] The modified oligonucleotides of example 1 were subjected to
enzymatic digestion using established protocols and converted to
their constituent nucleosides by sequential reaction with
phosphodiesterase and alkaline phosphatase. By comparison of the
experimentally obtained integrated HPLC profiles and UV-vis spectra
of the digested oligonucleotides to standards (including
2'-aminouridine and 2'-aminoadenine), the presence of the
amino-modified base at the predicted retention time and
characteristic UV-vis spectra was confirmed. An identical procedure
was carried out on the transition metal modified duplex DNA and
assignments of constituent nucleosides demonstrated single-site
modification at the predicted site.
[0191] Fluorescent Labeled Amino-Modified Oligonucleotides
[0192] It has been demonstrated that the fluorochrome, fluorescein
isothiocyanate (FITC) is specific for labeling primary amines on
modified oligonucleotides while not bonding to amines or amides
present on nucleotide bases (Haugland, Handbook of Fluorescent
Probes and Research Chemicals, 5th Edition, (1992)). This reaction
was carried out using the amino-oligonucleotide synthesized as
described in example 1 and on an identical bases sequence without
the 2'-amino-ribose group present. Fluorescence spectroscopic
measurements were acquired on both these oligonucleotides and the
results confirm the presence of the amine on the 5'-terminal ribose
ring.
[0193] Thermodynamic Melting Curves of Modified Duplex DNA
[0194] A well established technique for measuring thermodynamic
parameters of duplex DNA is the acquisition of DNA melting curves.
A series of melting curves as a function of concentration of the
modified duplex DNA was measured via temperature controlled UV-vis
(Hewlett-Packard), using techniques well known in the art. These
results confirm that hybridization of the amino-modified and
transition metal modified DNA had taken place. In addition, the
results indicate that the modified DNA form a stable duplex
comparable to the stability of unmodified oligonucleotide
standards.
[0195] Two Dimensional Nuclear Magnetic Resonance (NMR)
Spectroscopy
[0196] The amino-modified oligonucleotides synthesized as a part of
this work were prepared in sufficient quantities (6 micromoles) to
permit the assignment of the .sup.1H proton NMR spectra using a 600
MHz Varian NMR spectrometer.
[0197] Measurement of the Rate of Electron Transfer
[0198] An excellent review of the measurement techniques is found
in Winkler et al., Chem. Rev. 92:369-379 (1992). The donor is
Ru(bpy).sub.2(NHuridine)im, E.sup.0.about.1 V, and the acceptor is
Ru(NH.sub.3).sub.4py(NHuridine)im, E.sup.0.about.330 mV. The
purified transition metal modified oligonucleotides
(U.sub.NHRu(bpy)2imGCATCGA and U.sub.NHRu(NH3)4(py)imCGATGCA were
annealed by heating an equal molar mixture of the oligonucleotides
(30 .mu.molar: 60 nmoles of DNA in 2 ml buffer) in pH 6.8 (100 mM
NaPi, 900 mM NaCl) to 60.degree. C. for 10 minutes and slowly
cooling to room temperature over a period of 4 hours. The solution
was transferred to an inert atmosphere cuvette equipped with
adapters for attachment to a vacuum line and a magnetic stirring
bar. The solution was degassed several times and the sealed
apparatus refilled repeatedly with Ar gas.
[0199] The entire apparatus was inserted into a cuvette holder as
part of the set-up using the XeCl excimer-pumped dye laser and data
acquired at several wavelengths including 360, 410, 460 and 480 nm.
The photoinduced electron transfer rate is 1.6.times.10.sup.6
s.sup.-1 over a distance of 28 .ANG..
Example 6
Synthesis of a Single Stranded Nucleic Acid Labeled with Two
Electron Transfer Moieties
[0200] This example uses the basic procedures described earlier to
generate two modified oligonucleotides each with an electron
transfer moiety attached. Ligation of the two modified strands to
each other produces a doubly labeled nucleic acid with any of four
configurations: 5' and 3' labeled termini, 5' labeled terminus and
internal nucleotide label, 3' labeled terminus and internal
nucleotide label, and double internal nucleotide labels.
Specifically, the synthesis of an oligonucleotide 24 bases in
length with an electron transfer donor moiety on the 5' end and an
internal electron transfer moiety is described.
[0201] Five hundred nanomoles of each of two 5'-labeled
oligonucleotides 12 bases in length are synthesized as detailed
above with ruthenium (II) bisbipyridine imidazole on one
oligonucleotide, "D" and ruthenium (III) tetraamine pyridine on a
second oligonucleotide, "A".
[0202] An unmodified oligonucleotide 24 bases in length and
complementary to the juxtaposition of oligonucleotide "D" followed
in the 5' to 3' direction by oligonucleotide "A" is produced by
standard synthetic techniques. Five hundred nanomoles of this
hybridization template is added to a mixture of oligonucleotides
"A" and "D" in 5 ml of an aqueous solution containing 500 mM
Tris-Cl, pH 7.5, 50 mM MgCl.sub.2, 50 mM dithiothreitol and 5 mg/ml
gelatin. To promote maximal hybridization of labeled
oligonucleotides to the complementary strand, the mixture is
incubated at 60.degree. C. for 10 minutes then cooled slowly at a
rate of approximately 10.degree. C. per hour to a final temperature
of 12.degree. C. The enzymatic ligation of the two labeled strands
is achieved with T4 DNA ligase at 12.degree. C. to prevent the
ligation and oligomerization of the duplexed DNA to other duplexes
(blunt end ligation). Alternatively, E. coli DNA ligase can be used
as it does not catalyze blunt end ligation.
[0203] One hundred Weiss units of T4 DNA ligase is added to the
annealed DNA and adenosine triphosphate is added to a final
concentration of 0.5 mM. The reaction which catalyzes the formation
of a phosphodiester linkage between the 5' terminal phosphate of
oligonucleotide "A" and the 3' terminal hydroxyl group of
oligonucleotide "D" is allowed to proceed for 18 hours at
12.degree. C. The reaction is terminated by heat inactivation of
the enzyme at 75.degree. C. for 10 minutes. The doubly labeled
oligonucleotide is separated from the singly labeled
oligonucleotides and the complementary unlabeled oligonucleotide by
HPLC in the presence of urea as in the previous examples. The
doubly labeled oligonucleotide of this example is ideally suited
for use as a photoactive gene probe as detailed below.
Example 7
[0204] Use of a Doubly Modified Oligonucleotide with Electron
Transfer Moieties as a Photoactive Probe for Homologous Nucleic
Acid Sequence Detection
[0205] This example utilizes the oligonucleotide 24mer of example 6
in a unique type of gene-probe assay in which removal of
unhybridized probe prior to signal detection is not required. In
the assay procedure, a region of the gag gene of human
immunodeficiency virus type I (HIV-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 different
clinical isolates.
[0206] The amplified target DNA versus controls lacking in HIV-I
DNA are added to a hybridization solution of 6.times.SSC (0.9 M
NaCl, 0.09 M Na citrate, pH 7.2) containing 50 nanomoles of doubly
labeled 24mer probe of example 6. Hybridization is allowed to
proceed at 60.degree. C. for 10 minutes with gentle agitation.
Detection of electron transfer following laser excitation is
carried out as in example 5. Control samples which lack the
hybridized probe show negligible electron transfer rates. Probes
hybridized to the gag sequence show efficient and rapid electron
transfer through the DNA double helix, providing a highly specific,
homogeneous and automatable HIV-I detection assay.
[0207] A similar homogeneous gene probe assay involves the use of
two probes, one an electron donor and the other an electron
acceptor, which hybridize with the gag region of HIV-I in a tandem
configuration, one probe abutting the other. In this assay,
electronic coupling between the two electron transfer moieties
depends entirely on hybridization with the target DNA. If
appropriate, the electron transfer from one probe to the other is
enhanced by the ligation of the juxtaposed ends using T4 DNA ligase
as in example 6.
Example 8
[0208] Preparation of a Hydroxythiol for Attachment to a Gold
Electrode
[0209] OH(CH.sub.2).sub.16OH was purchased from Aldrich and the
monoacetate form prepared by slurring the material in dry
CH.sub.2Cl.sub.2. 0.5 equiv. of dimethylaminopyridine was added
along with 1.4 equivalents of triethylamine and 1 equivalent of
acetic anhydride. The reaction was allowed to proceed for 2 hours
and purified by flash chromatography (80:20 hexane:diethyl
ether.
[0210] The monoacetate compound was converted to the
monotosylate-monoacetate using p-TSOCl by literature procedures and
then treated with triphenyl methylmercaptan. To remove the
monoacetate, the product was dissolved in MeOH (1 mmol, 9 ml),
cooled to 0.degree. C., and aqueous solution of NaOH (1 mmol, in 2
ml water) added. The temperature was allowed to rise to room
temperature slowly, and the reaction followed by TLC (5%
MeOH/CH.sub.2Cl.sub.2). When the ester was gone the mixture was
recooled to 0.degree. C., and acidified with KHSO.sub.4 to pH 5-6
using pH paper. The MeOH was evaporated, and the residue was
extracted with CH.sub.2Cl.sub.2 (200 ml), dried (Na.sub.2SO.sub.4),
evaporated and checked via TLC. The material was phosphoroamidited
by standard procedures. This material was inserted into the DNA
synthesizer and an modified oligonucleotide produced. The
phosphoramidited oligonucleotide was modified with a ruthenium
complex by adding Ru(bpy).sub.2CO.sub.3 followed by imidazole to
yield a Ru(bpy).sub.2im oligonucleotide. The trityl protecting
group was removed by dissolving the nucleotide in 200 .mu.l of 0.1
M triethylammonium acetate (TEAA) buffer, pH 7.5. 30 .mu.l of 1 M
silver nitrate solution was added and the mixture vortexed and
incubated at room temperature for 30 minutes. 50.mu. of 1 M
dithiothritol (DTT) was added, the mixture vortexed and incubated
for 15 minutes, at which point it was microcentrifuged for 15
minutes to remove precipitated Ag+DTT. The supernatant was
collected and the pellet was washed with 100 .mu.l of TEAA buffer
and the solutions pooled. The resulting oligonucleotide was then
attached to the gold surface by standard techniques.
Example 9
[0211] Synthesis of a Single Stranded Nucleic Acid containing both
an Electron Acceptor and an Electron Donor Moiety
[0212] In order to evaluate the path dependent nature of the
electron transfer process through duplex DNA, an oligonucleotide
was prepared with an electron donor at the 3' end and an electron
acceptor at the 5' end. This multiply-modified oligonucleotide was
prepared by synthesizing a derivative with an amine at the
2'-position of the terminal ribose of both ends.
[0213] Synthesis of Bis-3',5'-2'-deoxyuridine Oligonucleotides
[0214] A DMT-2'-N-trifluoroacetyl-protected phosphoroamidite of
2'-amino-2'-deoxyuridine (UNH2) was prepared as described earlier
and reacted with succinic anhydride. This material was reacted with
p-nitrophenol to produce the precursor for the attachment to the
controller pore glass (GPG) resin as in FIG. 6A. The modified
oligonucleotide were assembled by standard solid phase automated
DNA synthesis techniques and the
bis-3',5',-2'-amino-2'-deoxyuridine oligonucleotide isolated and
characterized by mass spectrometry and HPLC digestion analysis. In
addition, the aminoribose oligomers and their complements were
reacted with FITC under conditions that favor 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 an
example, a 11 base pair sequence was prepared (calc. for
U.sub.NH2.gamma.TCCTACACU.sub.NH2-3229; found 3229.1) and the
subsequent digestion map was consistent with the proposed
structure. The metal modification of the bis-amino modified
oligonucleotide was performed in a similar manner. The new
metal-modified oligonucleotides were characterized by fluorescent
labelling, enzymatic digestion, and duplex-melting temperature
studies.
[0215] Thermal denaturing and annealing experiments display similar
melting temperatures for both ruthenium and aminoribose oligomers.
In addition, the amino-modified duplex DNA has been characterized
by 2D NMR. These data confirm that the donors and acceptors are
covalently attached to the 2'-amino-2'-deoxyribose position and
indicate that the DNA structure is unperturbed by the presence of
the ruthenium complexes.
Sequence CWU 1
1
4 1 10 DNA Artificial synthetic 1 cacatcctcu 10 2 11 DNA Artificial
synthetic 2 ucacatcctc u 11 3 11 DNA Artificial synthetic 3
uctcctacac u 11 4 11 DNA Artificial synthetic 4 agtgtaggag a 11
* * * * *