U.S. patent application number 09/866067 was filed with the patent office on 2001-10-25 for nucleic acid mediated electron transfer.
This patent application is currently assigned to California Institute of Technology. Invention is credited to Fraser, Scott E., Kayyem, Jon Faiz, Meade, Thomas J..
Application Number | 20010034033 09/866067 |
Document ID | / |
Family ID | 22601539 |
Filed Date | 2001-10-25 |
United States Patent
Application |
20010034033 |
Kind Code |
A1 |
Meade, Thomas J. ; et
al. |
October 25, 2001 |
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.; (Altadena,
CA) ; Kayyem, Jon Faiz; (Pasadena, CA) ;
Fraser, Scott E.; (Newport Beach, CA) |
Correspondence
Address: |
Robin M. Silva
FLEHR HOHBACH TEST ALBRITTON & HERBERT LLP
Suite 3400
Four Embarcadero Center
San Francisco
CA
94111-4187
US
|
Assignee: |
California Institute of
Technology
|
Family ID: |
22601539 |
Appl. No.: |
09/866067 |
Filed: |
May 23, 2001 |
Related U.S. Patent Documents
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09866067 |
May 23, 2001 |
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09454498 |
Dec 6, 1999 |
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6268149 |
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09454498 |
Dec 6, 1999 |
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08946679 |
Oct 8, 1997 |
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6087100 |
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08946679 |
Oct 8, 1997 |
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08709263 |
Sep 6, 1996 |
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5780234 |
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08709263 |
Sep 6, 1996 |
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08166036 |
Dec 10, 1993 |
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5591578 |
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Current U.S.
Class: |
435/6.11 ;
435/287.2; 435/91.1; 435/91.5; 536/23.1 |
Current CPC
Class: |
C12Q 1/6825 20130101;
C12Q 1/6818 20130101; C12Q 1/6825 20130101; C12Q 1/6818 20130101;
C12Q 1/6827 20130101; C07H 23/00 20130101; C12Q 2563/113 20130101;
C12Q 2563/113 20130101; C12Q 2563/113 20130101; C12Q 2563/137
20130101; C12Q 2563/113 20130101; C12Q 2565/1015 20130101; C12Q
2563/113 20130101; C12Q 2565/101 20130101; C12Q 2563/137 20130101;
C12Q 2565/101 20130101; C12Q 2563/113 20130101; C12Q 2537/101
20130101; C12Q 2565/101 20130101; C12Q 2565/101 20130101; C12Q
2565/101 20130101; C12Q 1/6825 20130101; C12Q 2565/301 20130101;
C12Q 1/6825 20130101; C12Q 1/6825 20130101; C12Q 1/6827 20130101;
C12Q 1/6818 20130101; C12Q 1/6818 20130101; C07H 21/00
20130101 |
Class at
Publication: |
435/6 ; 435/91.1;
435/91.5; 536/23.1 |
International
Class: |
C12Q 001/68; C07H
021/02; C07H 021/04; C12P 019/34 |
Claims
What is claimed is:
1. A single-stranded nucleic acid containing at least one electron
donor moiety and at least one electron acceptor moiety, said
electron donor moiety and said electron acceptor moiety being
covalently attached to said nucleic acid.
2. A single-stranded nucleic acid according to claim 1 wherein said
covalent attachment is to the ribose-phosphate backbone of said
nucleic acid.
3. A single stranded nucleic acid according to claim 1, wherein
said single stranded nucleic acid is capable of hybridizing to a
complementary target sequence in a second single stranded nucleic
acid to form a hybridization complex.
4. A single stranded nucleic acid according to claim 3, wherein
said hybridization complex is capable of transferring at least one
electron between said electron donor moiety and said electron
acceptor moiety.
5. A composition comprising a first single stranded nucleic acid
containing at least one electron donor moiety and a second single
stranded nucleic acid containing at least one electron acceptor
moiety, wherein said electron donor moiety and electron acceptor
moiety are covalently linked to the ribose-phosphate backbone of
said first and second single stranded nucleic acids.
6. A composition according to claim 5 wherein said first single
stranded nucleic acid is capable of hybridizing to said second
single stranded nucleic acid to form a double stranded nucleic
acid.
7. A double stranded nucleic acid composition according to claim 6
wherein said first single stranded nucleic acid is hybridized to
said second single stranded nucleic acid.
8. A composition according to claim 7 wherein said composition is
capable of transferring at least one electron between said electron
donor moiety and said electron acceptor moiety.
9. A composition according to claim 5 wherein said first and second
single stranded nucleic acids are capable of hybridizing to a
target sequence stranded nucleic acid, comprising at least a first
target domain and a second target domain, wherein said first
nucleic acid is capable of hybridizing to said first target domain
and said second nucleic acid is capable of hybridizing to said
second target domain to form a hybridization complex.
10. A composition according to claim 9 wherein said first target
domain and said second target domain are adjacent to one
another.
11. A composition according to claim 10 wherein said first nucleic
acid and said second nucleic acid in said hybridization complex are
ligated together.
12. A composition according to claim 10 wherein said hybridization
complex is capable of transferring at least one electron between
said electron donor moiety and said electron acceptor moiety.
13. A composition according to claim 10 wherein said target
sequence further comprises an intervening target domain between
said first and said second target domain.
14. A composition according to claim 13 further comprising an
intervening single stranded nucleic acid sequence capable of
hybridizing to said intervening target domain to form a
hybridization complex.
15. A composition according to claim 14 wherein said hybridization
complex is capable of transferring at least one electron between
said electron donor moiety and said electron acceptor moiety.
16. A method for making a single stranded nucleic acid containing
an electron transfer moiety at the 5' terminus, comprising a)
incorporating a modified nucleotide into a growing nucleic acid at
the 5' position to form a modified single stranded nucleic acid; b)
hybridizing said modified single stranded nucleic acid with a
complementary single stranded nucleic acid to form a double
stranded nucleic acid; c) reacting said double stranded nucleic
acid with an electron transfer moiety such that said moiety is
covalently attached to said modified single stranded nucleic acid;
and d) separating said complementary single stranded nucleic acid
from said modified single stranded nucleic acid containing said
electron transfer moiety.
17. A method for making a single stranded nucleic acid containing
an electron transfer moiety covalently attached to an internal
nucleotide, comprising a) incorporating a modified nucleotide dimer
into a growing nucleic acid to form a modified single stranded
nucleic acid; b) hybridizing said modified single stranded nucleic
acid with a complementary single stranded nucleic acid to form a
double stranded nucleic acid; c) reacting said double stranded
nucleic acid with an electron transfer moiety such that said moiety
is covalently attached to said modified single stranded nucleic
acid; and d) separating said complementary single stranded nucleic
acid from the modified single stranded nucleic acid containing said
electron transfer moiety.
18. A method for making a single stranded nucleic acid containing
an electron transfer moiety covalently attached to the 3' terminal
nucleotide, comprising a) incorporating a modified nucleotide via
enzymatic addition or replacement into a nucleic acid; b)
hybridizing said modified single stranded nucleic acid with a
complementary single stranded nucleic acid to form a double
stranded nucleic acid; c) reacting said double stranded nucleic
acid with an electron transfer moiety such that said moiety is
covalently attached through said phosphoramide bond of said
modified single stranded nucleic acid; and d) separating said
complementary single stranded nucleic acid from the modified single
stranded nucleic acid containing said electron transfer moiety.
19. A method of detecting a target sequence in a nucleic acid
sample comprising a) hybridizing a single stranded nucleic acid
containing at least one covalently attached electron donor moiety
and at least one covalently attached electron acceptor moiety to
said target sequence to form a hybridization complex; b)
determining the electron transfer rate between said electron donor
moiety and said electron acceptor moiety in the hybridization
complex; and c) comparing said electron transfer rate with the
electron transfer rate in the absence of the target sequence as an
indicator of the presence or absence of said target sequence.
20. A method of detecting a target sequence in a nucleic acid
wherein said target sequence comprises a first target domain and a
second target domain adjacent to said first target domain, wherein
said method comprises: a) hybridizing a first nucleic acid
containing at least one electron donor moiety to said first target
domain; b) hybridizing a second nucleic acid containing at least
one electron acceptor moiety to said second target domain; c)
determining the electron transfer rate between said electron donor
moiety and said electron acceptor moiety while said first and
second nucleic acids are hybridized to said first and second target
domains; and d) comparing said electron transfer rate with the
electron transfer rate in the absence of the target sequence as an
indicator of the presence or absence of said target sequence in
said nucleic acid sample.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to electron transfer via
nucleic acids. More particularly, the invention is directed to the
site-selective modification of nucleic acids with electron transfer
moieties such as redox active transition metal complexes to produce
a new series of biomaterials and to methods of making and using
them. The novel biomaterials of the present invention may be used
as bioconductors and diagnostic probes.
BACKGROUND OF THE INVENTION
[0002] The present invention, in part, relates to methods for the
site-selective modification of nucleic acids with redox active
moieties such as transition metal, complexes, the modified nucleic
acids themselves, and their uses. Such modified nucleic acids are
particularly useful as bioconductors and photoactive nucleic acid
probes.
[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 labelled 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 non-separation 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 labelled oligonucleotides for traditional gene probe
assays (Oligonucleotide Synthesis: A Practical Approach. Gait et
al., Ed., IRL Press: Oxford, UK, 1984; Oligonucleotides and
Analogues: A Practical Approach. Ed. F. Eckstein, Oxford University
Press, 1991). As a result, several techniques currently exist for
the synthesis of tailored nucleic acid molecules. Since nucleic
acids do not naturally contain functional groups to which molecules
of interest may easily be attached covalently, methods have been
developed which allow chemical modification at either of the
terminal phosphates or at the heterocyclic bases (Dreyer et al.
Proc. Natl. Acad. Sci. USA, 1985, 82:968).
[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 labelled to serve as a probe by
altering a nucleotide which then serves as a replacement analogue
in the nick translational resynthesis of double stranded DNA. The
chemically altered nucleotides may then provide reactive sites for
the attachment of immunological or other labels such as 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 selective
modification of nucleic acids at specific sites with redox active
moieties such as transition metal complexes. An electron donor
and/or electron acceptor moiety are covalently bound preferably
along the ribose-phosphate backbone of the nucleic acid at
predetermined positions. The resulting complexes represent a 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 a
single stranded nucleic acid which has both an electron donor
moiety and an electron acceptor moiety covalently attached thereto.
These moieties are attached through the ribose phosphate or
analogous backbone of the nucleic acid. The single stranded nucleic
acid is capable of hybridizing to a complementary target sequence
in a single stranded nucleic acid, and transferring electrons
between the donor and acceptor.
[0031] It is a further object of the present invention to provide
for a nucleic acid probe which can detect basepair mismatches. In
this embodiment, the single stranded nucleic acid with a covalently
attached electron donor and electron acceptor moiety is hybridized
to a complementary target sequence in a single stranded nucleic
acid. When the region of hybridization contains at least one base
pair mismatch, the rate of electron transfer between the donor
moiety and the acceptor moiety is decreased or eliminated, as
compared to when there is perfect complementarity between the probe
and target sequence.
[0032] It is an additional object of the present invention to
provide a complex which contains a first single stranded nucleic
acid with at least one electron donor moiety and a second single
stranded nucleic acid with at least one electron acceptor moiety.
As with the other embodiments of the present invention, the
moieties are covalently linked to the ribose-phosphate backbone of
the nucleic acids.
[0033] In one aspect of the present invention, the first and second
single stranded nucleic acids are capable of hybridizing to each
other to form a double stranded nucleic acid, and of transferring
electrons between the electron donor moiety and the electron
acceptor moiety.
[0034] In another aspect of the present invention, a target
sequence in a single stranded nucleic acid comprises at least first
and second target domains, which are directly adjacent to one
another. The first single stranded nucleic acid hybridizes to the
first target domain and the second single stranded nucleic acid
hybridizes to the second target domain, such that the first and
second single stranded nucleic acids are adjacent to each other.
This resulting hybridization complex is capable of transferring
electrons between the electron donor moiety and the electron
acceptor moiety on the first and second nucleic acids.
[0035] In another aspect of the present invention, a target
sequence in a single stranded nucleic acid comprises a first target
domain, an intervening target domain, and a second target domain.
The intervening target domain comprises one or more nucleotides.
The first and second single stranded nucleic acids hybridize to the
first and second target domains. An intervening nucleic acid
comprising one or more nucleotides hybridizes to the target
intervening domain such that electrons are capable of being
transferred between the electron donor moiety and the electron
acceptor moiety on the first and second nucleic acids.
[0036] The invention also provides for a method of making a single
stranded nucleic acid containing an electron transfer moiety
covalently attached to the 5' terminus of the nucleic acid. The
method comprises incorporating a modified nucleotide into a growing
nucleic acid at the 5' position to form a modified single stranded
nucleic acid. The modified single stranded nucleic acid is then
hybridized with a complementary single stranded nucleic acid to
form a double stranded nucleic acid. The double stranded nucleic
acid is reacted with an electron transfer moiety such that the
moiety is covalently attached to the modified single stranded
nucleic acid. The modified single stranded nucleic acid containing
the electron transfer moiety is separated from the complementary
unmodified single stranded nucleic acid.
[0037] The present invention also provides a method for making a
single stranded nucleic acid containing an electron transfer moiety
covalently attached to an internal nucleotide. The method comprises
creating a nucleotide dimer joined by a phosphoramide bond and
incorporating said nucleotide dimer into a growing nucleic acid to
form a modified single stranded nucleic acid. The modified single
stranded nucleic acid is then hybridized with a complementary
single stranded nucleic acid to form a double stranded nucleic
acid. The double stranded nucleic acid is reacted with an electron
transfer moiety such that the moiety is covalently attached to the
modified single stranded nucleic acid. The modified single stranded
nucleic acid containing the electron transfer moiety is separated
from the complementary unmodified single stranded nucleic acid.
[0038] Another aspect of the present invention provides a method of
detecting a target sequence. The method comprises creating a single
stranded nucleic acid with an electron donor moiety and an electron
acceptor moiety covalently attached. The single stranded nucleic
acid containing the electron transfer moieties is then hybridized
to the target sequence, and an electron transfer rate determined
between the electron donor and the electron acceptor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 illustrates possible orientations of electron donor
(EDM) and electron acceptor (EAM) moieties on a single stranded
nucleic acid.
[0040] FIG. 2 illustrates 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.
[0041] FIG. 3 illustrates a series of amino-modified nucleoside
precursors prior to, incorporation into an oligonucleotide.
[0042] FIGS. 4A and 4B depict the structure of electron transfer
moieties.
[0043] FIG. 4A depicts the general formula of a representative
class of electron donors and acceptors.
[0044] FIG. 4B depicts a specific example of a ruthenium electron
transfer moiety using bisbipyridine and imidazole as the
ligands.
DETAILED DESCRIPTION
[0045] 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 may have
an analogous backbone, comprising, for example, phosphoramide
(Beaucage et al., Tetrahedron 49(10):1925 (1993) and references
therein; Letsinger, J. Org. Chem. 35:3800 (1970)),
phosphorothioate, phosphorodithioate, O-methylphophoroamidite
linkages (see Eckstein, Oligonucleotides and Analogues: A Practical
Approach, Oxford University Press), or peptide nucleic acid
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)). The nucleic acids may be single stranded or double
stranded, as specified. The nucleic acid may be DNA, RNA or a
hybrid, where the nucleic acid contains any combination of
deoxyribo- and ribo-nucleotides, and any combination of uracil,
adenine, thymine, cytosine and guanine. In some instances, e.g. in
the case of an "intervening nucleic acid", the term nucleic acid
refers to one or more nucleotides.
[0046] 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. Generally, electron transfer moieties contain
transition metals as components, but not always.
[0047] 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, mRNA, or others. It may be any length, with
the understanding that longer sequences are more specific.
Generally speaking, this term will be understood by those skilled
in the art.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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. The latter confers 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.
[0052] In one embodiment, the present invention provides for novel
gene probes, which are useful in molecular biology and diagnostic
medicine. In this embodiment, single stranded nucleic acids having
a predetermined sequence and covalently attached electron donor and
electron acceptor moieties are synthesized. The sequence is
selected based upon a known target sequence, such that if
hybridization to a complementary target sequence occurs in the
region between the electron donor and the electron acceptor,
electron transfer proceeds at an appreciable and detectable rate.
Thus, the present invention has broad general use, as a new form of
labelled gene probe. In addition, since detectable electron
transfer in 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.
[0053] 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
nucleotides will measurably affect the electron transfer rate. This
is the case if the mutation is a substitution, insertion or
deletion. Accordingly, the present invention provides for the
detection of mutations in target sequences.
[0054] 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.
[0055] 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). 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,.
[0056] 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.
[0057] 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.
[0058] 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 nucleotide.
[0059] In one embodiment, the, electron donor and acceptor moieties
are added to the 3' and/or 5' termini of the nucleic acid. In
alternative embodiments, the electron donor and acceptor moieties
are added to the backbone of one or more internal nucleotides, that
is, any nucleotide which is not the 3' or 5' terminal nucleotide.
In a further embodiment, the electron donor and acceptor moieties
are added to the backbone of both internal and terminal
nucleotides.
[0060] In a preferred embodiment, the transition metal electron
transfer moieties are added through a procedure which utilizes
modified nucleotides, preferably amino-modified nucleotides. In
this embodiment, the electron transfer moieties are added to the
sugar phosphate backbone through the nitrogen group in
phosphoramide linkages. The modified nucleotides are then used to
site-specifically add a transition metal electron transfer moiety,
either to the 3' or 5' termini of the nucleic acid, or to any
internal nucleotide.
[0061] Molecular mechanics calculations indicate that perturbations
due to the modification of the terminal nucleotides of nucleic
acids are minimal and Watson-Crick base pairing is not disrupted
(unpublished data using Biograf from Molecular Simulations Inc.,
San Diego, Calif.). Accordingly, in one embodiment, modified
nucleotides are used to add an electron transfer moiety to the 5'
terminus of a nucleic acid. In this embodiment, 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.
In a preferred embodiment, an amino group is added to the 2' 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)).
[0062] Once the modified nucleotides 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) as the
5' terminal nucleotide, This method therefore allows the addition
of a transition metal electron transfer moiety to the 5' terminus
of a nucleic acid.
[0063] In an alternative embodiment, the 3' terminal nucleoside is
modified in order to add a transition metal electron transfer
moiety. In this embodiment, the 3' nucleoside is modified at either
the 2' or 3' carbon of the ribose sugar. In a preferred embodiment,
an amino group is added to the 2' or 3' carbon of the sugar using
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)).
[0064] The above procedures are applicable to both DNA and RNA
derivatives as shown in FIG. 3.
[0065] The amino-modified nucleotides 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)). One or more modified nucleosides
are then attached at the 3' end using standard molecular biology
techniques such as with the use of the enzyme DNA polymerase I or
terminal deoxynucleotidyltransferase (Ratliff, Terminal
deoxynucleotidyltransferase. In The Enzymes, Vol 14A. P. D. Boyer
ed. pp 105-118. Academic Press, San Diego, Calif. 1981).
[0066] In other embodiments, the transition metal electron transfer
moiety or moieties are added to the middle of the nucleic acid,
i.e. to an internal nucleotide. This may be accomplished in three
ways.
[0067] In a preferred embodiment, an oligonucleotide is
amino-modified at the 5' terminus as described above. In this
embodiment, oligonucleotide synthesis simply extends the 5' end
from the amino-modified nucleotide using standard techniques. This
results in an internally amino modified oligonucleotide.
[0068] In an alternate embodiment, electron transfer moieties are
added to the backbone at a site other than ribose. For example,
phosphoramide rather than phosphodiester linkages can be used 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.
[0069] 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 nucleotide, thus eliminating the need for
the extra steps required to produce the 3' terminally labelled
nucleotide.
[0070] 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' nucleotide
modification. The modified nucleosides are inserted internally into
nucleic acid using standard molecular biological techniques for
labelling DNA and RNA. Enzymes used for said labelling include DNA
polymerases such as polymerase I, T4 DNA polymerase, T7 DNA
polymerase, Taq DNA polymerase, reverse transcriptase and RNA
polymerases such as E. coli RNA polymerase or the RNA polymerases
from phages SP6, T7 or T3 (Short Protocols in Molecular Biology,
1992. Ausubel et al. Ed. pp 3.11-3.30).
[0071] In a preferred embodiment, the electron donor and acceptor
moieties are attached to the modified nucleotide 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. 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 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)). 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.
[0072] 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 nucleotides, or one to an internal nucleotide
and one to a terminal nucleotide. 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
nucleotides may be utilized in this embodiment.
[0073] 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 nucleotide or nucleotides,
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.
[0074] 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 nucleotides. 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.
[0075] It is to be understood that the rate of electron transfer
through a double stranded nucleic acid helix depends on the
nucleotide distance between the electron donor and acceptor
moieties. Longer distances will have slower rates, and
consideration of the rates will be a parameter in the design of
probes and bioconductors. Thus, while it is possible to measure
rates for distances in excess of 100 nucleotides, a preferred
embodiment has the electron donor moiety and the electron acceptor
moiety separated by at least 3 and no more than 100 nucleotides.
More preferably the moieties are separated by 8 to 64 nucleotides,
with 15 being the most preferred distance.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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 nucleotide.
[0080] 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.
[0081] 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.
[0082] In the design of probes comprised of two single stranded
nucleic acids which hybridize to adjacent first and second target
sequences, several factors should be considered. These factors
include the distance between the electron donor moiety and the
electron acceptor moiety in the hybridized form, and the length of
the individual single stranded probes. For example, it may be
desirable to synthesize only 5' terminally labelled probes. In this
case, the single stranded nucleic acid which hybridizes to the
first sequence may be relatively short, such that the desirable
distance between the probes may be accomplished. For example, if
the optimal distance between the electron transfer moieties is 15
nucleotides, then the first probe may be 15 nucleotides long.
[0083] 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.
[0084] 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 nucleotide. 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] In one bioconductor embodiment, the double stranded nucleic
acid has one single strand nucleic acid which carries all of the
electron transfer moieties. In another embodiment, the electron
transfer moieties may be carried on either strand, and in any
orientation. For example, one strand may carry only electron
donors, and the other only electron acceptors or both strands may
carry both.
[0089] 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.
[0090] 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.
[0091] 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:
[0092] D-A+h.nu..fwdarw.D-A*
[0093] D-A'+Q.fwdarw.D-A.sup.++Q.sup.-
[0094] D-A.sup.+.fwdarw.D.sup.+-A
[0095] D.sup.+-A+Q.sup.-.fwdarw.D-A+Q
[0096] The upper limit of measurable intramolecular electron
transfer rates using this method is about 10.sup.4 per second.
Alternative embodiments use the pulse-radiolytic generation of
reducing or oxidizing radicals, which inject electrons into a donor
or remove electrons from a donor, as reviewed in Winkler et al.,
supra.
[0097] Electron transfer will be initiated using electrical,
electrochemical, photon (including laser) or chemical activation of
the electron transfer moieties. These events are detected by
changes in transient absorption or by fluorescence or
phosphorescence or chemiluminescence of the electron transfer
moieties.
[0098] 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.
[0099] 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 nucleotides, some ribose nucleotides, and a mixture of
adenosine, thymidine, cytosine, guanine and uracil bases.
[0100] 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 nucleotide dimer linked by a peptide bond, phosphoramidate bond,
phosphorothioate bond, phosphorodithioate bond or 0-methyl
phosphoramidate bond.
[0101] A general formula is representative of a class of donors and
acceptors that may be employed is shown in FIG. 4A. In this figure,
M may be Cd, Mg, Cu, Co, Pd, Zn, Fe, Ru with the most preferred
being ruthenium. 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 ligands such
as NH.sub.3, pyridine, isonicotinamide, imidazole, bipyridine, and
substituted derivative of bipyridine, phenanthrolines and
substituted derivatives of phenanthrolines, porphyrins and
substituted derivatives of the porphyrin family. 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 Where: Ru = ruthenium bpy =
bisbipyridine im = imidazole py = pyridine
[0102] 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.
[0103] In an alternate embodiment, one of the electron transfer
moieties may be in the form of a solid support such as an
electrode. When the other electron transfer moiety is in solution
the system is referred to as a heterogenous system as compared to a
homogenous system where both electron donor and electron transfer
moities are in the same phase.
[0104] The techniques used in this embodiment are analogos to the
wiring of proteins to an electrode except that the nucleic acids of
the present invention are used rather than a redox protein (see for
example Gregg et al., J. Phys. Chem. 95:5970 (1991); Heller et al.,
Sensors and Actuators R., 13-14:180 (1993); and Pishko et al.,
Anal. Chem., 63:2268 (1991)). In this embodiment, it is preferred
that a redox polymer such as a poly-(vinylpyridine) complex of
Os(bpy).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.
[0105] In this embodiment, a single stranded nucleic acid probe
containing at least one electron transfer moiety is attached via
this redox hydrogel to the surface of an electrode. Hybridization
of a target sequence can then be measured as a function of
conductivity between the electron transfer moiety covalently
attached to one end of the nucleic acid and the electrode at the
other end. This may be done using equipment and techniques well
known in the art, such as those described in the references cited
above.
[0106] In similar embodiments, two nucleic acids are utilized as
probes as described previously. For example, one nucleic acid is
attached to a solid electrode, 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, or by use of
amperometric, potentiometric or conductometric electrochemical
sensors using techniques well known in the art.
[0107] 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.
EXAMPLES
[0108] 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
[0109] 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.
[0110] Step 1: Synthesis of 5'-di(p-methoxyphenyl)methyl
ether-2'-(trifluoroacetamido)-2'-deoxyuridine
[0111] 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-
hylether-2'-(trifluoroacetamido)-2'-deoxyuridine product.
[0112] Step 2: 5'-2'-aminouridine-GCTACGA and
5'-2'-aminouridine-CGTAGCA
[0113] 5'-di (p-methoxyphenyl)methyl ether-2
'-(trifluoroacetamido)-2'-deo- xyuridine 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.
[0114] Step 3: 5'-2'-ruthenium
bisbipyridineimidazole-aminouridine-GCTACGA
[0115] 5'1-2 '-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 1M 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 equiped with a
diode array detector and employing a gradient from 3% to 35%
acetonitrile in 0.1 M TEAC, pH 6.0.
[0116] Step 4: 51-2'-ruthenium
tetraminepyridine-aminouridine-CGTAGCA
[0117] 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
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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
,oiety at the 5' Terminus
[0126] 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.
[0127] 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
[0128] Enzymatic Digestion
[0129] 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 WV-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.
[0130] Fluorescent Labeled Amino-Modified Oligonucleotides
[0131] It has been demonstrated that the fluorochrome, fluorescein
isothiocyanate (FITC) is specific for labeling primary amines on
modified oligonucleotides while not bonding to amines or amides
present on nucleotide bases (Haugland, Handbood of Fluorescent
Probes and Research Chemicals, 5th Edition, (1992)). This reaction
was carried out using the amino-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.
[0132] Thermodynamic Melting Curves of Modified Duplex DNA
[0133] A well established technique for measuring thermodynamic
parameters of duplex DNA is the acquisition of DNA melting curves.
A series of melting curves as a function of concentration of the
modified duplex DNA was measured via temperature controlled UV-vis
(Hewlett-Packard), using techniques well known in the art. These
results confirm that hybridization of the amino-modified and
transition metal modified DNA had taken place. In addition, the
results indicate that the modified DNA form a stable duplex
comparable to the stability of unmodified oligonucleotide
standards.
Two Dimensional Nuclear Magnetic Resonance (NMR) Spectroscopy
[0134] 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.
Measurement of the Rate of Electron Transfer
[0135] 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.
[0136] 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
[0137] 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.
[0138] 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".
[0139] 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.
[0140] 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
Use of a Doubly Modified Oligonucleotide with Electron Transfer
Moieties as a Photoactive Probe for Detection of Complementary
Nucleic Acid Sequence
[0141] 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.
[0142] 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.
[0143] 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.
* * * * *