U.S. patent application number 09/753362 was filed with the patent office on 2002-05-09 for electrochemical sensor using intercalative, redox-active moieties.
Invention is credited to Barton, Jacqueline K., Hill, Michael G., Kelley, Shana O..
Application Number | 20020055103 09/753362 |
Document ID | / |
Family ID | 22007859 |
Filed Date | 2002-05-09 |
United States Patent
Application |
20020055103 |
Kind Code |
A1 |
Barton, Jacqueline K. ; et
al. |
May 9, 2002 |
Electrochemical sensor using intercalative, redox-active
moieties
Abstract
Compositions and methods for electrochemical detection and
localization of genetic point mutations and other base-stacking
perturbations within oligonucleotide duplexes adsorbed onto
electrodes and their use in biosensing technologies are described.
An intercalative, redox-active moiety (such as an intercalator or
nucleic acid-binding protein) is adhered and/or crosslinked to
immobilized DNA duplexes at different separations from an electrode
and probed electrochemically in the presence or absence of a
non-intercalative, redox-active moiety. Interruptions in
DNA-mediated electron-transfer caused by base-stacking
perturbations, such as mutations or binding of a protein to its
recognition site are reflected in a difference in electrical
current, charge and/or potential.
Inventors: |
Barton, Jacqueline K.; (San
Marino, CA) ; Hill, Michael G.; (Pasadena, CA)
; Kelley, Shana O.; (Boston, MA) |
Correspondence
Address: |
LISA A. HAILE, PH.D.
GRAY CARY WARE & FREIDENRICH LLP
Suite 1600
4365 Executive Drive
San Diego
CA
92121
US
|
Family ID: |
22007859 |
Appl. No.: |
09/753362 |
Filed: |
December 29, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09753362 |
Dec 29, 2000 |
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09056995 |
Apr 8, 1998 |
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6221586 |
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60043146 |
Apr 9, 1997 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
B82Y 30/00 20130101;
C12Q 1/6827 20130101; C12Q 2563/113 20130101; C12Q 2563/173
20130101; C12Q 2565/607 20130101; C12Q 2563/113 20130101; C12Q
2563/113 20130101; C12Q 2563/173 20130101; C12Q 2565/607 20130101;
C12Q 1/6825 20130101; C12Q 2563/173 20130101; C12Q 1/6827 20130101;
C12Q 1/6837 20130101; C12Q 1/6825 20130101; B82Y 5/00 20130101;
C12Q 1/6825 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Goverment Interests
[0002] The U.S. Government has certain rights in this invention
pursuant to Grant No. GM 49216 awarded by the National Institute of
Health.
Claims
What is claimed is:
1. A method of detecting one or more base-stacking perturbations in
a target sequence comprising: (a) hybridizing a first single
stranded nucleic acid to a second single stranded nucleic acid to
form a first complex; (b) depositing said first complex onto an
electrode or an addressable multielectorde array; (c) adding an
intercalative, redox-active moiety to said first complex to form a
second complex; and (d) measuring an electron transfer event
between said electrode or addressable multielectrode array and said
intercalative, redox-active moiety as an indication for the
presence or absence of said base-stacking perturbations.
2. A method according to claim 1, wherein said base-stacking
perturbations are point mutations, protein-DNA adducts, adducts
between any chemical entity and said target sequence, or
combinations thereof.
3. A method according to claim 1, wherein said intercalative,
redox-active moiety is either noncovalently adsorbed or crosslinked
to said first complex.
4. A method according to claim 1, wherein said intercalative,
redox-active moiety is an intercalator.
5. A method according to claim 1, wherein said intercalative,
redox-active moiety is an intercalator selected from the group
consisting of phenanthridines, phenothiazines, phenazines,
acridines, and anthraquinones.
6. A method according to claim 1, wherein said intercalative,
redox-active moiety is daunomycin.
7. A method according to claim 1, wherein said intercalative,
redox-active moiety is a part of a protein.
8. A method according to claim 1, wherein said intercalative,
redox-active moiety is mut Y.
9. A method according to claim 1, wherein said electrode or
addressable multielectrode array is gold.
10. A method according to claim 1, wherein said electrode or
addressable multielectrode array is carbon.
11. A method according to claim 1, wherein one of said
single-stranded nucleic acids is deriatized with a functionalized
linker.
12. A method according to claim 11, wherein said functionalized
linker is comprised of 5 to 20 .sigma. bonds.
13. A method according to claim 11, wherein said functionalized
linker is thiol-terminated.
14. A method according to claim 11, wherein said functionalized
linker is amine-terminated.
15. A method according to claim 1, wherein said addressable
multielectrode array is comprised of a monolayer of oligonucleotide
duplexes of 5 to 10 base-pairs in length deposited onto said array,
wherein each of said oligonucleotide duplexes is derivatized on one
end with a functionalized linker and on the opposite end with a
first single-stranded overhang of known sequence composition, and
wherein one of said single-stranded nucleic acids contains a second
single-stranded overhang complementary to said first
single-stranded overhang on said electrode or addressable
multielectrode array.
16. A method of detecting one or more base-stacking perturbations
in a target sequence comprising: (a) hybridizing a first single
stranded nucleic acid to a second single stranded nucleic acid to
form a first complex, wherein said nucleic acids are comprised of
12 to 25 nucleotides, and wherein one of said single-stranded
nucleic acids is derivatized with a thiol-terminated linker
comprised of 5 to 20 .sigma. bonds; (b) depositing said first
complex onto an addressable gold multielectrode array; (c) adding
daumomycin to said electrode-bound first complex to form a second
complex; and (d) measuring an electron transfer event between said
addressable gold multielectrode array and daunomycin as an
indication for the presence or absence of said base-stacking
perturbations.
17. A method according to claim 16, wherein said base-stacking
perturbations are point mutations, protein-DNA adducts, adducts
between any chemical entity and said target sequence, or
combinations thereof.
18. A method of detecting one or more base-stacking perturbations
in a target sequence comprising: (a) hybridizing a first single
stranded nucleic acid to a second single stranded nucleic acid to
form a first complex, wherein said nucleic acids are comprised of
12 to 25 nucleotides, and wherein one of said single-stranded
nucleic acids is derivatized with a amine-terminated linker
comprised of 5 to 20 .sigma. bonds; (b) depositing said first
complex onto an addressable carbon multielectrode array; (c) adding
daunomycin to said electrode-bound first complex to form a second
complex; and (d) measuring an electron transfer event between said
addressable carbon multielectrode array and daumonycin as an
indication for the presence or absence of said base-stacking
perturbations.
19. A method according to claim 18, wherein said base-stacking
perturbations are point mutations, protein-DNA adducts, adducts
between any chemical entity and said target sequence, or
combinations thereof.
20. A method of detecting one or more base-stacking perturbations
in a target sequence comprising: (a) hybridizing a first
single-stranded nucleic acid to a second single-stranded nucleic
acid to form a first complex of 12 to 25 nucleotides in length,
wherein said first complex contains a first single-stranded
overhang of known sequence composition, and wherein said first
single-stranded overhang can be the same or different; (b)
depositing said first complex onto an addressable multielectrode
array, wherein said addressable multielectrode array is comprised
of a monolayer of oligonucleotide duplexes of 5 to 10 base-pairs in
length deposited onto said array, wherein each of said
oligonucleotide duplexes is derivatized on one end with a
functionalized linker and on the opposite end with a second
single-stranded overhang complementary to said first
single-stranded overhang; (c) adding daumomycin to said
electrode-bound first complex to form a second complex; and (d)
measuring an electron transfer event between said addressable
multielectrode array and daunomycin as an indication for the
presence or absence of said base-stacking perturbations.
21. A method according to claim 20, wherein said base-stacking
perturbations are point mutations, protein-DNA adducts, adducts
between any chemical entity and said target sequence, or
combinations thereof.
22. A method of detecting one or more base-stacking perturbations
electrocatalytically in a target sequence comprising: (a)
hybridizing a first single stranded nucleic acid to a second single
stranded nucleic acid to form a first complex; (b) depositing said
first complex onto an electrode or an addressable multielectrode
array to form a second complex; (c) immersing said second complex
in a solution comprising an intercalative, redox-active species and
a non-intercalative, redox-active species; and (d) measuring an
electron transfer event as an indication for the presence or
absence of said base-stacking perturbations.
23. A method according to claim 22, wherein said base-stacking
perturbations are point mutations, protein-DNA adducts, adducts
between any chemical entity and said target sequence, or
combinations thereof.
24. A method according to claim 22, wherein said intercalative,
redox-active moiety is an intercalator.
25. A method according to claim 22, wherein said intercalative,
redox-active moiety is an intercalator selected from the group
consisting of phenanthridines, phenothiazines, phenazines,
acridines, and anthraquinones.
26. A method according to claim 22, wherein said non-intercalative,
redox-active moiety is selected from the group consisting of
ferricyanide, ferrocenes, hexacyanoruthenate, and
hexacyanoosmate.
27. A method according to claim 22, wherein said intercalative,
redox-active moiety is a protein.
28. A method according to claim 22, wherein said intercalative,
redox-active moiety is methylene blue, and wherein said
non-intercalative, redox-active moiety is ferricyanide.
29. A method according to claim 22, wherein said electrode or
addressable multielectrode array is gold.
30. A method according to claim 22, wherein said electrode or
addressable multielectrode array is carbon.
31. A method according to claim 22, wherein one of said
single-stranded nucleic acids is derivatized with a functionalized
linker.
32. A method according to claim 31, wherein said functionalized
linker is comprised of 5 to 20 .sigma. bonds.
33. A method according to claim 31, wherein said functionalized
linker is thiol-terminated.
34. A method according to claim 31, wherein said functionalized
linker is amine-terminated.
35. A method according to claim 22, wherein said addressable
multielectrode array is comprised of a monolayer of oligonucleotide
duplexes of 5 to 10 base-pairs in length deposited onto said array,
wherein each of said oligonucleotide duplexes are derivatized on
one end with a functionalized linker and on the opposite end with a
first single-stranded overhang of distinct sequence composition,
and wherein one of said single-stranded nucleic acids contains a
second single-stranded overhang complementary to said first
single-stranded overhang on said electrode or addressable
multielectrode array.
36. A method of detecting one or more point mutations
electrocatalytically within the p53 gene, comprising: (a) forming a
set of oligonucleotide duplexes of approximately 20 base-pairs in
length corresponding to the approximately 600 base pair long region
within exons 5 through 8 of the p53 gene, wherein said
oligonucleotide duplexes are derivatized with a thiol-terminated
linker comprised of 5 to 20 .sigma. bonds; (b) depositing said
oligonucleotide duplexes onto an addressable gold multielectrode
array; (c) denaturing said oligonucleotide duplexes by immersing
them in aqueous solution at elevated temperatures for 1 minute and
removing the complementary strands to form a single-stranded
monolayer; (d) exposing said single-stranded monolayer to a first
samnple comprising PCR-amplified and fragmented p53 gene DNA under
hybridizing conditions to form a first complex; (e) rinsing said
electrode-bound first complex to remove any unhybridized material;
(f) immersing said electrode-bound first complex into a dilute
solution comprised of 1.0 .mu.M methylene blue and 1.0 mM
ferricyanide; (g) measuring an electron transfer event as an
indication for the presence or absence of said point mutations; (h)
denaturing said electrode-bound first complex by immersing it in an
aqueous solution at elevated temperatures for 1 minute, and
regenerating said single-stranded monolayer; (i) exposing said
single-stranded monolayer to a second sample containing
PCR-amplified and fragmented p53 gene DNA under hybridizing
conditions to form a second complex; and (k) repeating steps (e)
through (h) using several sample solutions.
Description
2. RELATED APPLICATION
[0001] This application is a continuation-in part of Ser. No.
60/043,146, filed Apr. 9, 1997.
3. FIELD OF THE INVENTION
[0003] The present invention relates to the detection and
localization of base-pair mismatches and other perturbations in
base-stacking within an oligonucleotide duplex.
4. DESCRIPTION OF RELATED ART
[0004] It is now well known that mutations in DNA can lead to
severe consequences in metabolic functions (e.g., regulation of
gene expression, modulation of protein production) which ultimately
are expressed in a variety of diseases. For example, a significant
number of human cancers are characterized by a single base mutation
in one of the three ras genes (Bos, 1989). In order to unravel the
genetic components of such diseases, it is of utmost importance to
develop DNA sensors that are capable of detecting single-base
mismatches rapidly and efficiently and to establish routine
screening of disease-related genetic mutations based on such
sensors (Skogerboe, 1993; Southern, 1996; Chee, 1996; Eng,
1997).
[0005] Various methods that have been developed for the detection
of differences between DNA sequences rely on hybridization events
to differentiate native versus mutated sequences and are limited by
the small differences in base-pairing energies caused by point
mutations within extended polynucleotides (Millan, 1993; Hashimoto,
1994; Xu, 1995; Wang, 1996; Lockhart, 1996; Alivisatos, 1996;
Korriyoussoufi, 1997; Elghanian, 1997; Lin, 1997; Herne, 1997).
Typically, a nucleic acid hybridization assay to determine the
presence of a particular nucleotide sequence (i.e. the "target
sequence") in either RNA or DNA comprises a multitude of steps.
First, an oligonucleotide probe having a nucleotide sequence
complementary to at least a portion of the target sequence is
labeled with a readily detectable atom or group. When the labeled
probe is exposed to a test sample suspected of containing the
target nucleotide sequence, under hybridizing conditions, the
target will hybridize with the probe. The presence of the target
sequence in the sample can be determined qualitatively or
quantitatively in a variety of ways, usually by separating the
hybridized and non-hybridized probe, and then determining the
amount of labeled probe which is hybridized, either by determining
the presence of label in probe hybrids or by determining the
quantity of label in the non-hybridized probes. Suitable labels may
provide signals detectable by luminescence, radioactivity,
colorimetry, x-ray diffraction or absorption, magnetism or
enzymatic activity, and may include, for example, fluorophores,
chromophores, radioactive isotopes, enzymes, and ligands having
specific binding partners. However, the specific labeling method
chosen depends on a multitude of factors, such as ease of
attachment of the label, its sensitivity and stability over time,
rapid and easy detection and quantification, as well as cost and
safety issues. Thus, despite the abundance of labeling techniques,
the usefulness, versatility and diagnostic value of a particular
system for detecting a material of interest is often limited.
[0006] Some of the currently used methods of mismatch detection
include single-strand conformation polymorphism (SSCP) (Thigpen,
1992; Orita, 1989), denaturing gradient gel electrophoresis (DGGE)
(Finke, 1996; Wartell, 1990; Sheffield, 1989), RNase protection
assays (Peltonen and Pulkkinen, 1986; Osborne, 1991),
allele-specific oligonucleotides (Wu, 1989), allele-specific PCR
(Finke, 1996), and the use of proteins which recognize nucleotide
mismatches, such as the E. coli mutS protein (Modrich, 1991).
[0007] In the first three methods, the appearance of a new
electrophoretic band is observed by polyacrylamide gel
electrophoresis. SSCP detects the differences in speed of migration
of single-stranded DNA sequences in polacrylamide gel
electrophoresis under different conditions such as changes in pH,
temperature, etc. A variation in the nucleotide base sequence of
single-stranded DNA segments (due to mutation or polymorphism) may
lead to a difference in spatial arrangement and thus in mobility.
DGGE exploits differences in the stability of DNA segments in the
presence or absence of a mutation. Introduction of a mutation into
double-stranded sequences creates a mismatch at the mutated site
that destabilizes the DNA duplex. Using a gel with an increasing
gradient of formamide (denaturation gradient gel), the mutant and
wild-type DNA can be differentiated by their altered migration
distances. The basis for the RNase protection assay is that the
RNase A enzyme cleaves mRNA that is not fully hybridized with its
complementary strand, whereas a completely hybridized duplex is
protected from RNase A digestion. The presence of a mismatch
results in incomplete hybridization and thus cleavage by RNase A at
the mutation site. Formation of these smaller fragments upon
cleavage can be detected by polyacrylamide gel electrophoresis.
Techniques based on mismatch detection are generally being used to
detect point mutations in a gene or its mRNA product. While these
techniques are less sensitive than sequencing, they are simpler to
perform on a large number of tumor samples. In addition to the
RNase A protection assay, there are other DNA probes that can be
used to detect mismatches, through enzymatic or chemical cleavage.
See, e.g., Smooker and Cotton, 1993; Cotton, 1988; Shenk, 1975.
Other enzymatic methods include for example the use of DNA ligase
which covalently joins two adjacent oligonucleotides which are
hybridized on a complementary target nucleic acid, see, for example
Landegren (1988). The mismatch must occur at the site of
ligation.
[0008] Alternatively, mismatches can also be detected by shifts in
the electrophoretic mobility of mismatched duplexes relative to
matched duplexes (Cariello, 1988). With either riboprobes or DNA
probes, the cellular mRNA or DNA which may contain a mutation can
be amplified using polymerase chain reaction (PCR) prior to
hybridization. Changes in DNA of the gene itself can also be
detected using Southern hybridization, especially if the changes
are gross rearrangements, such as deletions and insertions.
[0009] DNA sequences of the specified gene which have been
amplified by use of PCR may also be screened using allele-specific
oligonucleotide probes. These probes are nucleic acid oligomers,
each of which is complementary to a corresponding segment of the
investigated gene and may or may not contain a known mutation. The
assay is performed by detecting the presence or absence of a
hybridization signal for the specific sequence. In the case of
allele-specific PCR, the PCR technique uses unique primers which
selectively hybridize at their 3'-ends to a particular mutated
sequence. If the particular mutation is not present, no
amplification product is observed.
[0010] In addition, restriction fragment length polymorphism (RFLP)
probes for the gene or surrounding marker genes can be used to
score alteration of an allele or an insertion in a polymorphic
fragment. However, since the recognition site of restriction
endonucleases ranges in general between 4 to 10 base pairs, only a
small portion of the genome is monitored by any one enzyme.
[0011] Another means for identifying base substitution is direct
sequencing of a nucleic acid fragment. The traditional methods are
based on preparing a mixture of randomly-terminated, differentially
labeled DNA fragments by degradation at specific nucleotides, or by
dideoxy chain termination of replicating strands (Maxam &
Gilbert, 1980; Sanger, 1977). Resulting DNA fragments in the range
of 1 to 500 basepairs are then separated on a gel to produce a
ladder of bands wherein the adjacent samples differ in length by
one nucleotide. The other method for sequencing nucleic acids is
sequencing by hybridization (SBH, Drmanac, 1993). Using mismatch
discriminative hybridization of short n-nucleotide oligomers
(n-mers), lists of constitutent n-mers may be determined for target
DNA. The DNA sequence for the target DNA may be assembled by
uniquely overlapping scored oligonucleotides. Yet another approach
relies on hybridization to high-density arrays of oligonucleotides
to determine genetic variation. Using a two-color labeling scheme
simultaneous comparison of a polymorphic target to a reference DNA
or RNA can be achieved (Lipshutz, 1995; Chee, 1996; Hacia,
1996).
[0012] Each of these known prior art methods for detecting base
pair mismatches has limitations that affect adequate sensitivity,
specificity and ease of automation of the assay. In particular,
these methods are unable to detect mismatches independent of
sequence composition and require carefully controlled conditions,
and most methods detect multiple mismatches only. Additional
shortcomings that limit these methods include high background
signal, poor enzyme specificity, and/or contamination.
[0013] Over the last decade, attention has also focused on DNA as a
medium of charge transfer in photoinduced electron transfer
reactions and its role in mutagenesis and carcinogenesis. For
example, studies were performed using various octahedral metal
complexes (which bind tightly to DNA by intercalation) as donors
and acceptors for photoinduced electron transfer. Dppz complexes of
ruthenium, osmium, cobalt, nickel, and rhenium showed tight
intercalative binding and unique photophysical and electrochemical
properties. No photoluminesence was observed upon irradiation of
the metal complexes in aqueous solution in absence of DNA (as a
result of quenching by proton transfer from the solvent), whereas
in the presence of DNA excitation of the complex afforded
significant, long-wavelength emission (because now the intercalated
complex was protected from quenching). Studies using rhodium
intercalators containing phenanthrenequinone-diimine (phi) ligands
displayed tight DNA binding by preferential intercalation, some
with affinities and specifities approaching DNA-binding
proteins.
[0014] Photoinduced electron transfer using DNA as a molecular
bridge has been established in various systems. Using metal
complexes intercalated into the base stack of DNA as donor and
acceptor it has been proposed that the DNA .pi.-stack could promote
electron transfer at long range. Additionally, the products of
redox-triggered reactions of DNA bases have been detected at sites
remote from intercalating oxidants (Hall, 1996; Dandliker, 1997;
Hall, 1997; Arkin, 1997). For example, it has been shown that a
metallointercalator can promote oxidative DNA damage through
long-range hole migration from a remote site. Oligomeric DNA
duplexes were prepared with a rhodium intercalator covalently
attached to one end and separated spatially from 5'-GG-3' doublet
sites of oxidation. Rhodium-induced photooxidation occurred
specifically at the 5'-G in the 5'-GG-3' doublets and was observed
up to 37 .ANG. away from the site of rhodium intercalation. In
addition it was found that rhodium intercalators excited with 400
nm light, initiated the repair of a thymine dimer incorporated
site-specifically in the center of a synthetic 16-mer
oligonucleotide duplex. The repair mechanism was thought to proceed
via oxidation of the dimer by the intraligand excited state of the
rhodium complex, in which an electron deficiency (hole) is
localized on the intercalated phi ligand. Like electron transfer
between metallointercalators, the efficiencies of long-range
oxidative processes were found to be remarkably sensitive to the
coupling of the reactants into the base stack (Holmlin, 1997) and
depended upon the integrity of the base stack itself (Kelley,
1997c, 1997d; Hall, 1997; Arkin, 1997) as well as on the oxidation
potential. Perturbations caused by mismatches or bulges greatly
diminished the yields of DNA-mediated charge transport.
[0015] Other studies have reported electron transfer through DNA
using nonintercalating ruthenium complexes coordinated directly to
amino-modified sugars at the terminal position of oligonucleotides
(Meade, 1995). In this system it was suggested that electron
transfer is protein-like. In proteins, where the energetic
differences in coupling depend upon .sigma.-bonded interactions,
small energetic differences between systems do not cause large
differences in electronic coupling. In the DNA double helix
however, .pi.-stacking can contribute to electronic coupling such
that small energetic differences could lead to large differences in
coupling efficiency. Most recently, Lewis and coworkers measured
rates of photo-oxidation of a guanine base in a DNA hairpin by an
associated stilbene bound at the top of the hairpin (Lewis, 1997).
By systematically varying the position of the guanine base within
the hairpin and measuring the rate of electron transfer, a value
for .beta., the electronic coupling parameter, could be made. Here,
.beta. was found to be intermediate between that seen in proteins,
with .sigma. bonded arrays, and that found for a highly coupled
.pi.-bonded array.
[0016] Electrochemical studies of small molecule/DNA complexes have
focused primarily on solution-phase phenomena, in which DNA-induced
changes in redox potentials and/or diffusion constants of organic
and inorganic species have been analyzed to yield association
constants (Carter, 1989, 1990; Rodriguez, 1990; Welch, 1995; Kelly,
1986; Molinier-Jumel, 1978; Berg, 1981; Plambeck, 1984). In
addition, rates of guanine oxidation catalyzed by electrochemically
oxidized transition-metal complexes have been used to evaluate the
solvent accessibility of bases for the detection of mismatches in
solution (Johnston, 1995). Electrochemical signals triggered by the
association of small molecules with DNA have also been applied in
the design of other novel biosensors. Toward this end,
oligonucleotides have been immobilized on electrode surfaces by a
variety of linkages for use in hybridization assays. These include
thiols on gold (Hashimoto, 1994a, 1994b; Okahata, 1992),
carbodiimide coupling of guanine residues on glassy carbon (Millan,
1993), and alkane bisphosphonate films on Al.sup.3+-treated gold
(Xu, 1994, 1995). Both direct changes in mass (measured at a quartz
crystal microbalance) (Okahata, 1992) and changes in current
(Hashimoto, 1994a, 1994b; Millan, 1993) or electrogenerated
chemiluminesence (Xu, 1994, 1995) due to duplex-binding molecules
have been used as reporters for double stranded DNA. Gold surfaces
modified with thiolated polynucleotides have also been used for the
detection of metal ions and DNA-binding drugs (Maeda, 1992,
1994).
[0017] Other known electrochemical sensors used in an increasing
number of clinical, environmental, agricultural and
biotechnological applications include enzyme based biosensors.
Amperometric enzyme electrodes typically require some form of
electrical communication between the electrode and the active site
of the redox enzyme that is reduced or oxidized by the substrate.
In one type of enzyme electrode, a non-natural redox couple
mediates electron transfer from the substrate-reduced enzyme to the
electrode. In this scheme, the enzyme is reduced by its natural
substrate at a given rate; the reduced enzyme is in turn, rapidly
oxidized by a non-natural oxidizing component of a redox couple
that diffuses into the enzyme, is reduced, diffuses out and
eventually diffuses to an electrode where it is oxidized.
[0018] Electrons from a substrate-reduced enzyme will be
transferred either to the enzyme's natural re-oxidizer or, via the
redox-centers of the polymer to the electrode. Only the latter
process contributes to the current. Thus, it is desirable to make
the latter process fast relative to the first. This can be
accomplished by (a) increasing the concentration of the redox
centers, or (b) assuring that these centers are fast, i.e. that
they are rapidly oxidized and reduced.
[0019] Most natural enzymes are not directly oxidized at electrodes
without being destroyed, even if the latter are maintained at
strongly oxidizing potentials. Also they are not reduced at
strongly reducing potentials without being decomposed. It has,
however, been shown that enzymes can be chemically modified by
binding to their proteins redox couples, whereupon, if in the
reduced state, they transfer electrons to an electrode. It has also
been shown that when redox polycations in solution
electrostatically complex polyanionic enzymes, electrons will flow
in these complexes from the substrate to the enzyme, and from the
enzyme through the redox polymer, to an electrode. In addition,
systems have been developed where a redox-active polymer, such as
poly(vinyl-pyridine), has been introduced which electrically
connects the enzyme to the electrode. In this case, the
polycationic redox polymer forms electrostatic complexes with the
polyanionic glucose oxidase in a manner mimicking the natural
attraction of some redox proteins for enzymes, e.g., cytochrome c
for cytochrome c oxidase.
[0020] The present invention provides a new approach for the
detection of mismatches based on charge transduction through DNA.
This electrochemical method is based on DNA-mediated electron
transfer using intercalative, redox-active species and detects
differences in electrical current or charge generated with fully
base-paired duplexes versus duplexes containing a base-stacking
perturbation, such as a mismatch. Carried out at an addressable
multielectrode array, this method allows the processing of multiple
sequences in the course of a single measurement, thus significantly
improving the efficiency of screening for multiple genetic defects.
Most importantly, the assay reports directly on the structural
difference in base pair stacking within the hybridized duplex,
rather than on a thermodynamic difference based on the
condition-dependent hybridization event itself. Consequently,
mismatch detection becomes independent of the sequence composition
and sensors based on this approach offer fundamental advantages in
both scope and sensitivity over any other existing methods.
5. SUMMARY OF THE INVENTION
[0021] The present invention provides a highly sensitive and
accurate method for the detection of genetic point mutations in
nucleic acid sequences and its application as a biosensor. In
particular, the invention relates to electrodes that are prepared
by modifying their surfaces with oligonucleotide duplexes combined
with an intercalative, redox-active species and their use as
sensors based on an electrochemical process in which electrons are
transferred between the electrode and the redox-active species.
[0022] One aspect of the invention relates to methods for
determining the presence of point mutations sequentially in a
series of oligonucleotide duplexes using an intercalative,
redox-active moiety. A preferred method comprises the steps of: (a)
contacting at least one strand of a first nucleic acid molecule
with a strand of a second nucleic acid molecule under hybridizing
conditions, wherein one of the nucleic acid molecules is
derivatized with a functionalized linker, (b) depositing this
duplex onto an electrode or an addressable multielectrode array,
(c) contacting the adsorbed duplex which potentially contains a
base-pair mismatch with an intercalative, redox-active moiety under
conditions suitable to allow complex formation, (d) measuring the
amount of electrical current or charge generated as an indication
of the presence of a base-pair mismatch within the adsorbed duplex,
(e) treating the complex under denaturing conditions in order to
separate the complex, yielding a monolayer of single-stranded
oligonucleotides, and (f) rehybridizing the single-stranded
oligonucleotides with another target sequence. Steps (c) through
(f) can then be repeated for a sequential analysis of various
oligonucleotide probes. Attenuated signals, as compared to the
observed signals for fully base-paired, i.e. wild-type, sequences,
will correspond to mutated sequences.
[0023] In some instances, it may be desirable to crosslink the
intercalative, redox-active species to the duplex and perform the
assay comprised of steps (a) through (d) only.
[0024] Another preferred method relates to the detection of point
mutations utilizing electrocatalytic principles. More specifically,
this method utilizes an electrode-bound double-stranded DNA
monolayer which is immersed in a solution comprising an
intercalative, redox-active species, which binds to the monolayer
surface, and a non-intercalative redox-active species which remains
in solution. This method comprises the steps of: (a) contacting at
least one strand of a first nucleic acid molecule with a strand of
a second nucleic acid molecule under hybridizing conditions,
wherein one of the nucleic acid molecules is derivatized with a
functionalized linker, (b) depositing this duplex which potentially
contains a base-pair mismatch onto an electrode or an addressable
multielectrode array, (c) immersing this complex in an aqueous
solution comprising an intercalative, redox-active moiety and a
non-intercalative, redox-active moiety under conditions suitable to
allow complex formation, (d) measuring the amount of electrical
current or charge generated as an indication of the presence of a
base-pair mismatch within the adsorbed duplex, (e) treating the
complex under denaturing conditions in order to separate the
complex, yielding a monolayer of single-stranded oligonucleotides,
and (f) rehybridizing the single-stranded oligonucleotides with
another target sequence. Steps (c) through (f) can then be repeated
for a sequential analysis of various oligonucleotide probes.
Utilizing this method, pronounced currents and thus increased
signals will be observed due to the electrocatalytic reduction of
the non-intercalative, redox-active moiety by the surface-bound,
redox-active moiety.
[0025] Yet another aspect of the invention relates to a method of
detecting the presence or absence of a protein and comprises the
steps of: (a) contacting at least one strand of a first nucleic
acid molecule with a strand of a second nucleic acid molecule under
hybridizing conditions, wherein one of the nucleic acid molecules
is derivatized with a functionalized linker and wherein the formed
duplex is designed such to contain the recognition site of a
nucleic acid-binding protein of choice, (b) depositing this duplex
onto an electrode or an addressable multielectrode array, (c)
contacting the adsorbed duplex with an intercalative, redox-active
moiety under conditions suitable to allow complex formation, (d)
potentially crosslinking the intercalative, redox-active moiety to
the duplex, (e) immersing the complex in a first sample solution to
be analyzed for the presence of the nucleic acid-binding protein,
(f) measuring the amount of electrical current or charge generated
as an indication of the presence or absence of the nucleic
acid-binding protein in the sample solution, (g) treating the
complex under appropriate conditions to remove the nucleic
acid-binding protein, and (h) immersing it in a second sample
solution to be analyzed for the presence of the nucleic
acid-binding protein in order to separate the complex. Steps (e)
through (h) can then be repeated for a sequential analysis of
various sample solutions. Attenuated signals, as compared to
signals measured for a reference solution without the nucleic
acid-binding protein, indicate the presence of the nucleic
acid-binding protein which is binding to its recognition site, thus
causing a perturbation in base-stacking.
[0026] The invention also relates to the nature of the redox-active
moieties. The requirements of a suitable intercalative,
redox-active moiety include the position of its redox potential
with respect to the window within which the oligonucleotide-surface
linkage is stable, as well as the synthetic feasibility of covalent
attachment to the oligonucleotide. In addition, chemical and
physical characteristics of the redox-active intercalator may
promote its intercalation in a site-specific or a non-specific
manner. In a preferred embodiment, the redox-active species is in
itself an intercalator or a larger entity, such as a nucleic
acid-binding protein, that contains an intercalative moiety.
[0027] The nature of the non-intercalative, redox-active species
for the electrocatalysis based assays depends primarily on the
redox potential of the intercalative, redox-active species utilized
in that assay.
[0028] Yet another aspect of the invention relates to the
composition and length of the oligonucleotide probe and methods of
generating them. In a preferred embodiment, the probe is comprised
of two nucleic acid strands of equal length. In another preferred
embodiment the two nucleic acid strands are of uneven length,
generating a single-stranded overhang of desired sequence
composition (i.e. a "sticky end"). The length of the
oligonucleotide probes range preferably from 12 to 25 nucleotides,
while the single-stranded overhangs are approximately 5 to 10
nucleotides in length. These single-stranded overhangs can be used
to promote site-specific adsorption of other oligonucleotides with
the complementary overhang or of enzymes with the matching
recognition site.
[0029] The invention further relates to methods of creating a
spatially addressable array of adsorbed duplexes. A preferred
method comprises the steps of (a) generating duplexes of variable
sequence composition that are derivatized with a functionalized
linker, (b) depositing these duplexes on different sites on the
multielectrode array, (c) treating the complex under denaturing
conditions to yield a monolayer of single-stranded
oligonucleotides, and (d) hybridizing these single-stranded
oligonucleotides with a complementary target sequence. Another
preferred method comprises the steps of (a) depositing 5 to 10
base-pair long oligonucleotide duplexes that are derivatized on one
end with a functionalized linker and contain single-stranded
overhangs (approximately 5 to 10 nucleotides long) of known
sequence composition at the opposite end onto a multielectrode
array, and (b) contacting these electrode-bound duplexes under
hybridizing conditions with single-stranded or double-stranded
oligonucleotides that contain the complementary overhang.
[0030] Another aspect of the invention is directed towards the
nature of the electrode, methods of depositing an oligonucleotide
duplex (with or without a redox-active moiety adsorbed to it) onto
an electrode, and the nature of the linkage connecting the
oligonucleotide duplex to the electrode. In a preferred embodiment,
the electrode is gold and the oligonucleotide is attached to the
electrode by a sulfur linkage. In another preferred embodiment the
electrode is carbon and the linkage is a more stable amide bond. In
either case, the linker connecting the oligonucleotide to the
electrode is preferably comprised of 5 to 20 .sigma. bonds.
[0031] Yet another aspect of the invention relates to various
methods of detection of the electrical current or charge generated
by the electrode-bound duplexes combined with an intercalative,
redox-active species. In a preferred embodiment, the electrical
current or charge is detected using electronic methods, for example
voltammetry or amperommetry, or optical methods, for example
fluorescence or phosphoresence. In another preferred embodiment,
the potential at which the electrical current is generated is
detected by potentiommetry.
6. BRIEF DESCRIPTION OF DRAWINGS
[0032] Table 1 describes the electrochemical detection of
single-base mismatches based on cyclic voltammograms measured for
1.0 .mu.M daunomycin noncovalently bound to duplex-modified
electrodes.
[0033] FIG. 1 is a schematic diagram depicting DNA duplexes used
for study of distance-dependent reduction of daunomycin. The right
insert illustrates the daunomycin-guanine crosslink. The left
insert shows the thiol-terminated tether which connects the duplex
to the electrode surface and provides 16 .sigma.-bonds between the
electrode and the base stack.
[0034] FIG. 2 illustrates cyclic voltammograms of gold electrodes
modified with daunomycin-crosslinked thiol-terminated duplexes (A)
SH-.sup.5'ATGGATCTCATCTAC+complement and (B)
SH-.sup.5'ATCCTACTCATGGAC+co- mplement, where the bold Gs represent
the daunomycin crosslinking site.
[0035] FIG. 3 illustrates cyclic voltammograms of gold electrodes
modified with daunomycin-crosslinked thiol-terminated duplexes
containing TA and CA basepairs. The oligonucleotide
SH-.sup.5'ATTATATAATTGCT was hybridized with the corresponding
complements containing either a T or a C opposite from the
underlined A.
[0036] FIG. 4 describes the charges (Q.sub.c) measured for
daunomycin at DNA-modified electrodes containing different
single-base mismatches. To obtain the seven different mismatched
duplexes the thiol-modified sequence, SH-.sup.5'AGTACAGTCATCGCG,
was hybridized with the following seven different complements (the
mismatch is indicated in bold, and the specific basepair and the
melting temperature of the duplex is given in parentheses):
.sup.5'CGCGATGACTGTACT (TA, T.sub.m=68.degree. C.),
.sup.5'CGCGACGACTGTACT (CA, T.sub.m=56.degree. C.),
.sup.5'CGCGATGTCTGTACT (TT, T.sub.m=57.degree. C.),
.sup.5'CGCGATCACTGTACT (CC, T.sub.m=56.degree. C.),
.sup.5'CGCGATGGCTGTACT (GT, T.sub.m=62.degree. C.),
.sup.5'CGCGATGAATGTACT (GA, T.sub.m=60.degree. C.),
.sup.5'CGCGATGCCTGTACT (CT, T.sub.m=58.degree. C.).
[0037] FIG. 5 describes the charge obtained for DNA-modified
electrodes in the presence of 1.0 .mu.M daunomycin. the identified
duplexes of varying percentages of GC content were either fully
base-paired or contained a single CA mismatch. Mismatch detection
measuring the electrical current or charge generated was
independent of the sequence composition.
[0038] FIG. 6 describes the charges (Q.sub.c) measured during the
in situ detection of a CA mismatch. Electrodes were derivatized
with the sequence SH-.sup.5 AGTACAGTCATCGCG, where either a C or a
T was incorporated into the complement across from the underlined
A. Using cyclic voltammetry, the electrochemical response of
daunomycin non-covalently bound to duplex-modified electrodes was
measured first for the intact TA or CA duplexes (TA vs. CA),
secondly (after denaturation of the duplex) for the single stranded
oligonucleotide (ss), thirdly (after rehybridization with the
opposite complement) again for the duplex (CA vs. TA), and lastly
(after repeating the denaturation step) again for the
single-stranded oligonucleotide (ss).
[0039] FIG. 7 represents a schematic illustration of
electrocatalytic reduction of ferricyanide. Methylene blue
(MB.sup.+) is reduced electrochemically through the DNA base stack
to form leucomethylene blue (LB.sup.+). Ferricyanide is then
reduced by LB.sup.+, causing the regeneration of MB.sup.+ and the
observation of catalytic currents.
[0040] FIG. 8 illustrates cyclic voltammograms of gold electrodes
modified with thiol-terminated duplexes containing TA and CA
basepairs immersed in a solution containing 1.0 .mu.M methylene
blue and 1.0 mM ferricyanide. The oligonucleotide
SH-.sup.5'AGTACAGTCATCGCG was hybridized with the corresponding
complements containing either a T or a C opposite from the
underlined A.
7. DETAILED DESCRIPTION OF THE INVENTION
[0041] The expression "amplification of polynucleotides" includes
methods such as polymerase chain reaction (PCR), ligation
amplification (or ligase chain reaction, LCR) and amplification
methods based on the use of Q-beta replicase. These methods are
well known and widely practiced in the art. See, e.g., U.S. Pat.
Nos. 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and
Wu et al., 1989a (for LCR). Reagents and hardware for conducting
PCR are commercially available. Primers useful to amplify sequences
from a particular gene region are preferably complementary to, and
hybridize specifically to sequences in the target region or in its
flanking regions. Nucleic acid sequences generated by amplification
may be sequenced directly. Alternatively the amplified sequence(s)
may be cloned prior to sequence analysis. A method for the direct
cloning and sequence analysis of enzymatically amplified genomic
segments has been described by Scharf (1986).
[0042] The term "base-stacking perturbations" refers to any event
that causes a perturbation in base-stacking such as, for example, a
base-pair mismatch, a protein binding to its recognition site, or
any other entities that form oligonucleotide adducts.
[0043] The term "denaturing" refers to the process by which strands
of oligonucleotide duplexes are no longer base-paired by hydrogen
bonding and are separated into single-stranded molecules. Methods
of denaturation are well known to those skilled in the art and
include thermal denaturation and alkaline denaturation.
[0044] The term "hybridized" refers to two nucleic acid strands
associated with each other which may or may not be fully
base-paired.
[0045] The term "intercalative moieties" refers to planar aromatic
or heteroaromatic moieties that are capable of partial insertion
and stacking between adjacent base pairs of double-stranded
oligonucleotides. These moieties may be small molecules or part of
a larger entity, such as a protein. Within the context of this
invention the intercalative moiety is able to generate a response
or mediate a catalytic event.
[0046] The term "mismatches" refers to nucleic acid bases within
hybridized duplexes which are not 100% complementary. A mismatch
includes any incorrect pairing between the bases of two nucleotides
located on complementary strands of DNA that are not the
Watson-Crick base-pairs A:T or G:C. The lack of total homology may
be due to deletions, insertions, inversions, substitutions or
frameshift mutations.
[0047] The term "mutation" refers to a sequence rearrangement
within DNA. The most common single base mutations involve
substitution of one purine or pyrimidine for the other (e.g., A for
G or C for T or vice versa), a type of mutation referred to as a
"transition". Other less frequent mutations include "transversions"
in which a purine is substituted for a pyrimidine, or vice versa,
and "insertions" or "deletions", respectively, where the addition
or loss of a small number (1, 2 or 3) of nucleotides arises in one
strand of a DNA duplex at some stage of the replication process.
Such mutations are also known as "frameshift" mutations in the case
of insertion/deletion of one of two nucleotides, due to their
effects on translation of the genetic code into proteins. Mutations
involving larger sequence rearrangement also may occur and can be
important in medical genetics, but their occurrences are relatively
rare compared to the classes summarized above.
[0048] The term "nucleoside" refers to a nitrogenous heterocyclic
base linked to a pentose sugar, either a ribose, deoxyribose, or
derivatives or analogs thereof. The term "nucleotide" relates to a
phosphoric acid ester of a nucleoside comprising a nitrogenous
heterocyclic base, a pentose sugar, and one or more phosphate or
other backbone forming groups; it is the monomeric unit of an
oligonucleotide. Nucleotide units may include the common bases such
as guanine (G), adenine (A), cytosine (C), thymine (T), or
derivatives thereof. The pentose sugar may be deoxyribose, ribose,
or groups that substitute therefore.
[0049] The terms "nucleotide analog", "modified base", "base
analog", or "modified nucleoside" refer to moieties that function
similarly to their naturally occurring counterparts but have been
structurally modified.
[0050] The terms "oligonucleotide" or "nucleotide sequence" refers
to a plurality of joined nucleotide units formed in a specific
sequence from naturally occurring heterocyclic bases and
pentofuranosyl equivalent groups joined through phosphorodiester or
other backbone forming groups.
[0051] The terms "oligonucleotide analogs" or "modified
oligonucleotides" refer to compositions that function similarly to
natural oligonucleotides but have non-naturally occurring portions.
Oligonucleotide analogs or modified oligonucleotides may have
altered sugar moieties, altered bases, both altered sugars and
bases or altered inter-sugar linkages, which are known for use in
the art.
[0052] The terms "redox-active moiety" or "redox-active species"
refers to a compound that can be oxidized and reduced, i.e. which
contains one or more chemical functions that accept and transfer
electrons.
[0053] The term "redox protein" refers to proteins that bind
electrons reversibly. The simplest redox proteins, in which no
prosthetic group is present, are those that use reversible
formation of a disulfide bond between to cysteine residues, as in
thioredoxin. Most redox proteins however use prosthetic groups,
such as flavins or NAD. Many use the ability of iron or copper ions
to exist in two different redox states.
[0054] The present invention provides a highly sensitive and
accurate method based on an electrochemical assay using
intercalative, redox-active species to determine the presence and
location of a single or multiple base-pair mismatches. Briefly, the
system is comprised of (i) a reagent mixture comprising an
electrode-bound oligonucleotide duplex to which an intercalative,
redox-active moiety is associated and (ii) means for detecting and
quantitating the generated electrical current or charge as an
indication for the presence of a fully base-paired versus a
mismatch containing duplex. The present invention is particularly
useful in the diagnosis of genetic diseases that arise from point
mutations. For example, many cancers can be traced to point
mutations in kinases, growth factors, receptors binding proteins
and/or nuclear proteins. Other diseases that arise from genetic
disorders include cystic fibrosis, Bloom's syndrome, thalassemia
and sickle cell disease. In addition, several specific genes
associated with cancer, such as DCC, NF-1, RB, p53, erbA and the
Wilm's tumor gene, as well as various oncogenes, such as abi, erbB,
src, sis, ras, fos, myb and myc have already been identified and
examined for specific mutations.
[0055] The present invention provides methods for detecting single
or multiple point mutations, wherein the oligonucleotide duplex
carrying the redox-active species is adsorbed and therefore
continuously exposed to an electrode whose potential oscillates
between a potential sufficient to effect the reduction of said
chemical moiety and a potential sufficient to effect the oxidation
of the chemical moiety. This method is preferred over other methods
for many reasons. Most importantly, this method allows the
detection of one or more mismatches present within an
oligonucleotide duplex based on a difference in electrical current
measured for the mismatch-containing versus the fully base-paired
duplex. Thus the method is based on the differences in
base-stacking of the mismatches and is independent of the sequence
composition of the hybridized duplex, as opposed to existing
methods that depend on thermodynamic differences in hybridization.
Furthermore, this method is nonhazardous, inexpensive, and can be
used in a wide variety of applications, alone or in combination
with other hybridization-dependent methods.
[0056] One particular aspect of the invention relates to the method
for sequential detection of mismatches within a number of nucleic
acid samples which comprises the following steps. At least one
strand of a nucleic acid molecule is hybridized under suitable
conditions with a first nucleic acid target sequence forming a
duplex which potentially contains a mismatch, and wherein one of
the nucleic acids is derivatized with a functionalized linker. This
duplex is then deposited onto an electrode or an addressable
multielectrode array forming a monolayer. An intercalative,
redox-active species (e.g., daunomycin) is noncovalently adsorbed
(or crosslinked, if desired) onto this molecular lawn, and the
electrical current or charge generated is measured as an indication
of the presence of a base pair mismatch within the adsorbed
oligonucleotide complex. Subsequent treatment of the duplexes
containing the intercalative, redox-active species under denaturing
conditions allows separation of the complex, yielding a
single-stranded monolayer of oligonucleotides which can be
rehybridized to a second oligonucleotide target sequence. The steps
of duplex formation, adsorption of the intercalative, redox-active
species, measurement of the electrical current or charge, and
denaturation of the complex to regenerate the single-stranded
oligonucleotides may be repeated as often as desired to detect in a
sequential manner genetic point mutations in a variety of
oligonucleotide probes.
[0057] The charges passed at each of the electrodes is measured and
compared to the wild-type, i.e. fully base-paired, sequences.
Electrodes with attenuated signals correspond to mutated sequences,
while those which exhibit no change in electrical current or charge
are unmutated. Furthermore, the intensity of the signal compared to
the wild-type sequence not only reports the presence of the
mismatch but also describes the location of the disruption within
the analyzed duplex.
[0058] Another aspect of the invention relates to the method of
detecting mutations utilizing electrocatalysis. Briefly, the
modification of electrode surfaces with oligonucleotide duplexes
provides a medium that is impenetrable by negatively charged
species due to the repulsion by the high negative charge of
oligonucleotides. However, electrons can be shuttled through the
immobilized duplexes to redox-active intercalators localized on the
solvent-exposed periphery of the monolayer, which in turn can
catalytically reduce these negatively charged species. More
specifically, this electrocatalytic method comprises the following
steps. At least one strand of a nucleic acid molecule is hybridized
under suitable conditions with a first nucleic acid target sequence
forming a duplex which potentially contains a mismatch, and wherein
one of the nucleic acids is derivatized with a functionalized
linker. This duplex is then deposited onto an electrode or a
multielectrode array forming a monolayer. The assembly is immersed
into an aqueous solution containing both an intercalative,
redox-active species (e.g., methylene blue) and a
non-intercalative, redox-active species (e.g., ferricyanide). The
electrical currents or charges corresponding to the catalytic
reduction of ferricyanide mediated by methylene blue are measured
for each nucleic acid-modified electrode and compared to those
obtained with wild-type, i.e. fully base-paired sequences.
Subsequent treatment of the duplexes under denaturing conditions
allows separation of the complex, yielding a single-stranded
monolayer of oligonucleotides which can be rehybridized to a second
oligonucleotide target sequence. The steps of duplex formation,
measurement of the catalytically enhanced electrical current or
charge, and denaturation of the complex to regenerate the
single-stranded oligonucleotides may be repeated as often as
desired to detect in a sequential manner genetic point mutations in
a variety of oligonucleotide probes. This particular method based
on electrocatalysis at oligonucleotide-modified surfaces is
extremely useful for systems where attenuated signals resulting
from the presence of mismatches are small. The addition of a
non-intercalative electron acceptor amplifies the signal intensity,
and allows more accurate measurements. This approach may be
particularly useful to monitor assays based on redox-active
proteins which bind to the oligonucleotide-modified surface, but
are not easily oxidized or reduced because the redox-active center
is not intercalating.
[0059] The present invention further relates to the nature of the
redox-active species. These species have a reduced state in which
they can accept electron(s) and an oxidized state in which they can
donate electron(s). The intercalative, redox-active species that
are adsorbed or covalently linked to the oligonucleotide duplex
include, but are not limited to, intercalators and nucleic
acid-binding proteins which contain a redox-active moiety.
[0060] An intercalator useful for the specified electrochemical
assays is an agent or moiety capable of partial insertion between
stacked base pairs in the nucleic acid double helix. Examples of
well-known intercalators include, but are not limited to,
phenanthridines (e.g., ethidium), phenothiazines (e.g., methylene
blue), phenazines (e.g., phenazine methosulfate), acridines (e.g.,
quinacrine), anthraquinones (e.g., daunomycin), and metal complexes
containing intercalating ligands (e.g., phi, chrysene, dppz). Some
of these intercalators may interact site-selectively with the
oligonucleotide duplex. For example, the chrysene ligand is known
to intercalate at the mispaired site of a duplex itself (Jackson,
1997), which can be exploited for selective localization of an
intercalator. This can be in particular useful to construct a
duplex monolayer which contains the intercalative, redox-active
species exclusively at its periphery.
[0061] In the case of redox-active nucleic acid-binding proteins,
differences in DNA-mediated electron transfer between the
duplex-bound protein and the electrode allow for the detection of
base-pair mismatches or other base-stacking perturbations. Examples
of redox-active proteins include, but are not limited to, mut Y,
endonuclease III, as well as any redox-active cofactor-containing
DNA-binding protein. Such proteins convert to one of an oxidized
and reduced form upon reacting with a selected substrate,
whereafter the operation of the electrode regenerates the other of
the oxidized and reduced forms.
[0062] The choice of a protein depends partially on its adsorption
and binding properties to biological macromolecules, i.e. nucleic
acids, and with non-biological macromolecules, whether in a
homogeneous solution, or when immobilized on a surface. By changing
the absorption or binding characteristics, selectivity, signal to
noise ratio and signal stability in these assays can be improved.
The charge of a protein affects its adsorption on surfaces,
absorption in films, electrophoretic deposition on electrode
surfaces, and interaction with macromolecules. It is, therefore, of
importance in diagnostic and analytical systems utilizing proteins
to tailor the charge of the protein so as to enhance its adsorption
or its binding to the macromolecule of choice, i.e the nucleic
acid. In other cases, i.e when the detection assay is used during
several cycles, it is of equal importance to be able to facilitate
desorption, removal, or stripping of the protein from the
macromolecule. These assays require oligonucleotide duplexes that
are designed such as to allow for site-specific binding of the
protein of choice, which may require a single-stranded overhang.
Once the protein is adsorbed onto the nucleic acid, electrons are
relayed via the oligonucleotide duplex to the electrode.
[0063] The nature of the non-intercalative, redox-active species
used in a particular ectrocatalytic assay depends primarily on the
redox potential of the intercalating, redox-active species utilized
in that same assay. Examples include, but are not limited to, any
neutral or negatively charged probes, for example
ferricyanide/ferrocyanide, ferrocene and derivatives thereof (e.g.,
dimethylaminomethyl-, monocarboxylic acid-, dicarboxylic acid-),
hexacyanoruthenate, and hexacyanoosmate.
[0064] Yet another aspect of the invention relates to a method of
detecting the presence or absence of a protein inducing
base-stacking perturbations in DNA duplexes, this method comprising
the following steps. At least one strand of a nucleic acid molecule
is hybridized under suitable conditions with a second strand of
nucleic acid molecule forming a duplex, wherein one of the nucleic
acids is derivatized with a functionalized linker. This duplex is
designed such to contain the recognition site of a protein of
choice at a distinct site along that duplex. This duplex is then
deposited onto an electrode or an addressable multielectrode array
forming a monolayer and an intercalative, redox-active species is
adsorbed onto this molecular lawn. In a preferred embodiment, the
intercalative, redox-active species is site-specifically localized.
In another preferred embodiment, the intercalative, redox-active
species is crosslinked to the oligonucleotide duplex. This formed
complex is then exposed to a sample solution that potentially
contains the specific protein and the electrical current or charge
generated is measured as an indication of the presence or absence
of the protein. Subsequently, the protein is removed under
appropriate conditions to regenerate the duplex containing the
intercalative, redox-active moiety. The steps of duplex formation,
adsorption or crosslinking of the intercalative, redox-active
species, measurement of the electrical current or charge, and
regeneration of the duplex containing the intercalative,
redox-active moiety may be repeated as often as desired to detect
in a sequential manner the presence of a specific protein in
multiple sample solutions.
[0065] The charges passed at each of the electrodes are measured
and compared to the charges measured in a reference solution
without the protein. Electrodes with attenuated signals indicate
the presence of the protein in question which is binding to its
recognition site, thus causing a perturbation in base-stacking.
Examples of proteins that can be used for this assay include, but
are not limited to, restriction enzymes, TATA-binding proteins, and
base-flipping enzymes (e.g., DNA methylase).
[0066] The present invention also relates to the choice of nucleic
acid probes. Any nucleic acid, DNA or RNA, can be subjected to this
mismatch detection method, provided that the mismatch(es) to be
detected lie within the region between the attachment site of the
intercalative, redox-active moiety and the electrode in order to be
able to measure a difference in electrical current. The nucleic
acid probes to be compared may comprise natural or synthetic
sequences encoding up to the entire genome of an organism. These
probes can be obtained from any source, for example, from plasmids,
cloned DNA or RNA, or from natural DNA or RNA from any source,
including bacteria, yeast, viruses, organelles and higher organisms
such as plants and animals. The samples may be extracted from
tissue material or cells, including blood cells, amniocytes, bone
marrow cells, cells obtained from a biopsy specimen and the like,
by a variety of techniques as described for example by Maniatis et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Cold Spring Harbor, New York (1982), incorporated herein
by reference.
[0067] Alternatively, the sequences of choice can also be prepared
by well known synthetic procedures. For standard DNA and RNA
synthesis methods, see for example "Synthesis and Applications of
DNA and RNA" ed. S. A. Narang, Academic Press, 1987, M. J. Gait,
"Oligonucleotide Synthesis", IRL Press, Wash. D.C. U.S.A., 1984,
and "Oligonucleotides and Analogues"ed. F. Eckstein, IRL Press,
Wash. D.C. U.S.A., 1991, as incorporated herein by reference.
Briefly, oligonucleotides and oligonucleotide analogs may be
synthesized, conveniently through solid state synthesis of known
methodology. In a preferred embodiment, the monomeric units are
added to a growing oligonucleotide chain which is covalently
immobilized to a solid support. Typically, the first nucleotide is
attached to the support through a cleavable linkage prior to the
initiation of synthesis. Step-wise extension of the oligonucleotide
chain is normally carried out in the 3' to 5' direction. When the
synthesis is complete, the polymer is cleaved from the support by
hydrolyzing the linkage mentioned above and the nucleotide
originally attached to the support becomes the 3' terminus of the
resulting oligomer. Nucleic acid synthesizers such as the Applied
Biosystems, Incorporated 380B are commercially available and their
use is generally understood by persons of ordinary skill in the art
as being effective in generating nearly any oligonucleotide or
oligonucleotide analog of reasonable length which may be desired.
Triester, phosphoramidite, or hydrogen phosphonate coupling
chemistries are used with these synthesizers to provide the desired
oligonucleotides or oligonucleotide analogs.
[0068] In addition, the invention also relates to nucleic acid
probes that are constructed with a defined sequence comprised of
nucleotide and non-natural nucleotide monomers to restrict the
number of binding sites of the intercalative, redox-active agent to
one single site. For example, in the case of the redox-active
intercalator daunomycin mixed nucleotide/non-natural nucleotide
oligomers were prepared containing A-T and/or I-C basepairs and one
discrete guanine binding site to which daunomycin is crosslinked.
The non-natural nucleotides are constructed in a step-wise fashion
to produce a mixed nucleotide/non-natural nucleotide polymer
employing one of the current DNA synthesis methods well known in
the art, see for example "Synthesis and Applications of DNA and
RNA" ed. S. A. Narang, Academic Press, 1987, M. J. Gait,
"Oligonucleotide Synthesis", IRL Press, Wash. D.C. U.S.A., 1984,
and "Oligonucleotides and Analogues" ed. F. Eckstein, IRL Press,
Wash. D.C. U.S.A., 1991.
[0069] Methods and conditions used for contacting the
oligonucleotide strands of two DNAs, two RNAs or one DNA and one
RNA molecule under hybridizing conditions are widely known in the
art. Suitable hybridization conditions may be routinely determined
by optimization procedures well known to those skilled in the art
to establish protocols for use in a laboratory. See e.g., Ausubel
et al., Current Protocols in Molecular Biology, Vol. 1-2, John
Wiley & Sons (1989); Sambrook et al., Molecular Cloning A
Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Springs Harbor Press
(1989); and Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Cold Spring Harbor, New York
(1982), all of which are corporated by reference herein. For
example, conditions such as temperature, concentration of
components, hybridization and washing times, buffer components, and
their pH and ionic strength may be varied.
[0070] Another aspect of the invention relates to a
surface-modified electrode and its use in a bioelectrochemical
process, in which electrons are transferred directly between an
electrode and an electroactive biological material which is capable
of accepting or donating one or more electrons. Such a
bioelectrochemical process can be in either direction. In
particular, the invention provides electrodes having their surface
modified with oligonucleotide duplexes carrying an intercalative,
redox-active moiety. The electrode can be of any material
compatible with the surface modifier being adsorbed or bound
thereon, including, but not limited to noble metals such as gold,
silver, platinum, palladium, as well as carbon. The preferred
material for the electrodes are gold and carbon.
[0071] The oligonucleotide duplex can be adsorbed onto the
electrode in any convenient way. Preferably, the process of
preparing such a modified electrode comprises adsorbing the
oligonucleotide duplex which is derivatized at the 5'-end with a
functionalized linker chains onto the electrode surface in one
monolayer to obtain a uniform lawn. These linkers include, but are
not limited to, thiol- or amine-terminated chains. This process is
generally understood by persons of ordinary skill in the art as and
is relatively simple, reproducible and can easily be automated.
[0072] Furthermore, the density and composition of the monolayer is
subject to variation depending on the selected assay. Methods of
detecting single base-pair mismatches using intercalative,
redox-active moieties require a densely packed monolayer to prevent
the adsorbed intercalative, redox-active moieties from diffusing
into the lawn. The method for detecting the presence or absence of
a protein requires preferably an uneven monolayer comprised of
duplexes of variable length to allow the protein to bind
effectively to its recognition site along the duplex.
[0073] In addition, the present invention further relates to
methods of creating a spatially addressable array of adsorbed
duplexes. In a preferred embodiment, oligonucleotide duplexes of
variable sequence composition that are derivatized at the 5'-end
with a functionalized linker are deposited onto a multielectrode
array. Subsequent treatment of these electrode-bound duplexes under
denaturing conditions yields a monolayer of single-stranded
oligonucleotides, which can then be hybridized with a complementary
oligonucleotide probe that potentially contains a mismatch. In
another preferred embodiment, short oligonucleotide duplexes (5 to
10 base-pairs in length) that are derivatized on one end with a
functionalized linker and contain single-stranded overhangs (5 to
10 nucleotides in length) of designed sequence composition at the
opposite end are deposited onto a multielectrode array to generate
a spatially addressable matrix. These electrode-bound duplexes can
then be hybridized with single-stranded or double-stranded
oligonucleotides that contain the complementary overhang.
[0074] Solid supports containing immobilized molecules have been
extensively used in research, in clinical analyses and in the
commercial production of foods and chemicals (see e.g., U.S. Pat.
No. 5,153,166; Akashi, 1992). Immobilized nucleic acids are used in
hybridization assays (Lamture, 1994) and immobilized proteins in
radioimmuno or ELISA assays (see, U.S. Pat. No. 5,314,830). In
addition, enzymes have been immobilized to facilitate their
separation from product and to allow for their efficient and
repetitive use. A number of important factors have to be considered
in the development of an effective immobilization procedure. First,
the procedure must minimize non-specific adsorption of molecules.
Second, the procedure must maintain the functional integrity of the
immobilized molecules. Third, the stability of the bond between the
support and the immobilized molecule must be such to avoid leaching
which would lead to reduced accuracy and sensitivity. Finally, the
coupling procedure must be efficient enough to result in a support
with a high capacity for the target molecules as well as be cost
effective.
[0075] Another aspect of the invention relates to measuring the
electrical current as a function of degree of hybridization of the
oligonucleotide duplex adsorbed onto the electrode. When the
intercalative, redox-active species is exposed to electrochemical
or chemical energy, the electrical current may be continuously
detected using techniques well known in the art. These include, but
are not limited electronic methods, for example voltammetry or
amperommetry, or optical methods, for example fluorescence or
phosphoresence.
[0076] Generally, photoluminescence excitation and emission occur
with electromagnetic radiation of between about 200 nanometers and
about 900 nanometers in wavelength. Likewise, chemiluminescent and
electrochemiluminescent emission generally occur with the emitted
electromagnetic radiation being between about 200 nanometers and
about 900 nanometers in wavelength. The potential at which the
reduction or oxidation of the chemical moiety occurs depends upon
its exact chemical structure as well as factors such as the pH of
the solution and the nature of the electrode used. It is well known
how to determine the optimal emission and excitation wavelengths in
a photoluminescent system and the optimal potential and emission
wavelength of an electrochemiluminescent and chemiluminescent
system.
[0077] There are many methods for quantifying the amount of
luminescent species present. The rate of energy input into the
system can provide a measure of the luminescent species. Suitable
measurements include, for example, measurements of electric current
when the luminescent species is generated electrochemically, the
rate of reductant or oxidant utilization when the luminescent
species is generated chemically or the absorption of
electromagnetic energy in photoluminescent techniques. In addition,
the luminescent species can be detected by measuring the emitted
electromagnetic radiation. All of these measurements can be made
either as continuous, event-based measurements, or as cumulative
methods which add the signal over a long period of time.
Event-based measurements may be carried out with photomultiplier
tubes, photodiodes or phototransistors to produce electric currents
proportional in magnitude to the incident light intensity, or by
using charge couple devices. Examples of cumulative methods are the
integration of event-based data, and the use of photographic film
to provide cumulative data directly.
[0078] The publications and other reference materials referred to
herein describe the background of the invention and provide
additional detail regarding its practice and are hereby
incorporated by reference. For convenience, the reference materials
are referenced and grouped in the appended bibliography.
[0079] The present invention is further described in the following
examples. These examples are for illustrative purposes only, and
are not to be construed as limiting the scope of the invention as
set forth in the appended claims.
EXAMPLES
[0080] Materials. Phosphoramidite reagents (including the
C.sub.6S-S thiol modifier) were obtained from Glen Research.
[.gamma.-.sup.32P]dATP was obtained from NEN-DuPont. Potassium
ferrocyanide (Fisher) was recrystallized from aqueous solution
prior to use. Daunomycin was obtained from Fluka.
[0081] Synthesis of Derivatized Duplexes. Oligonucleotides
immobilized on a controlled pore glass resin were treated in
succession with carbonyldiimidazole and 1,6-diaminohexane (1 g/10
ml dioxane, 30 min/ea.) at the 5'-hydroxy terminus before cleavage
from the resin (Wachter, 1986). After deprotection, the free amine
was treated with 2-pyridylthiopropionic acid N-succinimide ester to
produce a disulfide (Harrison, 1997). The sequences were purified
by reverse-phase HPLC, converted to free thiols using
dithiothreitol, and repurified before hybridization to their
complements. Derivatized oligonucleotides were characterized by
mass-assisted laser desorption ionization time-of-flight mass
spectrometry and HPLC retention times. Duplexes were hybridized in
deoxygenated 5 mM phosphate/50 mM NaCl (pH 7) by heating to
90.degree. C. followed by slow cooling to room temperature.
Unprotected duplexes were stored frozen under argon to prevention
oxidation of the thiol.
[0082] Atomic Force Microscopy (AFM). All AFM images were collected
using a MultiMode AFM running on the NanoScope IIIa controller
(Digital Instruments, Santa Barbara, Calif.). A glass AFM chamber
(Digital Instruments, Santa Barbara, Calif.) and a fluid volume of
approximately 50 microliters were used for the experiments.
Si.sub.3N.sub.4 cantilevers (spring constant, 0.06 N/m) with
integrated, end-mounted oxide-sharpened Si.sub.3N.sub.4 probe tips
were used. The applied vertical force of the AFM probe during
imaging was minimized to beneath 100 pN. Continually adjusting the
cantilever deflection feedback setpoint compensated for thermal
drifting of the cantilever and a consistent, minimum force was
maintained AFM height calibrations were carried out on a
NIST-traceable 180-nm height standard and then confirmed by
measuring a single-atom step in the Au gold surface. The AFM images
were recorded in "Height" (or constant force) mode. Holes in the
monolayer used to determine monolayer thicknesses were prepared by
decreasing the scan size to approximately 100-150 nm, increasing
the scan rate to 24-30 Hz, and increasing the vertical force by
advancing the setpoint several units. After about one minute, the
scan size, scan rate, and setpoint were returned to their previous
values, and images featuring a bare gold square were captured. All
images captured for height-contrast analysis were recorded at
minimum vertical tip forces. This was accomplished by decreasing
the set-point until the tip disengaged from the surface, then
reintroducing it with the minimum force required to achieve a
stable image. In several cases, the film height was also measured
in tapping mode, and gave the same result as the contact-mode
experiments.
[0083] Electrochemistry. Cyclic voltammetry (CV) was carried out on
0.02 cm.sup.2 polycrystalline gold electrodes using a Bioanalytical
Systems (BAS) Model CV-50W electrochemical analyzer at
20.+-.2.degree. C. in 100 mM phosphate buffer (pH 7). A normal
three-electrode configuration consisting of a modified gold-disk
working electrode, a saturated calomel reference electrode (SCE,
Fisher Scientific), and a platinum wire auxiliary electrode was
used. The working compartment of the electrochemical cell was
separated from the reference compartment by a modified Luggin
capillary. Potentials are reported versus SCE. Heterogeneous
electron-transfer rates were determined and analyzed by CV (Nahir,
1994; Weber, 1994; Tender, 1994).
[0084] Ellipsometry. Optical ellipsometry (.lambda.=632.8 nm) was
carried out on dried samples at 25.degree. C. using a Gaertner
Model L116C ellipsometer.
Example 1
Site-specific Incorporation of a Redox-Active Intercalator into a
DNA Duplex
[0085] The redox-active intercalator daunomycin (DM) (Arcamone,
1981) was incorporated into the DNA duplex to investigate charge
transduction through these duplexes (FIG. 1). DM undergoes a
reversible reduction (Molinier-Jumel, 1978; Berg, 1981) within the
potential window of the monolayers (Kelley, 1997a), and covalent
adducts of intercalated DM crosslinked to the 2-amino group of
guanine (Leng, 1996) have been crystallographically characterized
within duplex DNA (Wang, 1991). Thus, a series of oligonucleotides
primarily containing A-T or inosine (I)-C pairs were constructed
with discrete guanine binding sites to which DM was crosslinked.
Preferably, thiol-terminated duplexes (0.1 mM) containing an
adjacent pair of guanines were hybridized, incubated with 0.2%
formaldehyde and 0.2 mM DM in 5 mM phosphate, 50 mM NaCl, pH 7 for
1 h, and phenol extracted to remove excess DM.
[0086] Moving the guanine site along the duplex resulted in a
systematic variation of the through-helix DM/gold separation, and
allowed an investigation of the effect of distance on the dynamics
of charge transport through the monolayers (FIG. 1).
Example 2
Characterization of DNA Duplexes Modified with a Redox-Active
Intercalator
[0087] Modified duplexes were characterized by mass spectrometry,
ultraviolet/visible absorption spectroscopy, and thermal
denaturation experiments, all of which were consistent with a 1:1
DM-duplex stoichiometry. For example, the duplex
SH--(CH.sub.2)CONH(CH.sub.2).sub.6-
NHCO.sub.2-.sup.5'ATCCTACTCATGGAC with its inosine complement
modified with DM was analyzed by MALDI-TOF spectrometry.
Mass-to-charge ratios (found/calc) of 5284/(5282) (DM+SH strand),
4541/(4540) (complement), and 4742/(4742) (SH strand) were
detected. These values correspond to the calculated masses for
fragments expected from this duplex. UV-visible absorption
spectroscopy also revealed a 1:1 duplex/DM stoichiometry based upon
comparison of the duplex absorbance at 260 nm
(M=14.9.times.10.sup.3 M.sup.-1cm.sup.-1) and the absorbance of
intercalated DM at 480 n (M=5.1.times.10.sup.3 M.sup.-1 cm.sup.-1.
In the presence of 100 mM phosphate, 100 mM MgCl.sub.2, and at pH
7, thermal denaturation studies of 5 DM duplexes (monitored by
absorbance at 260 nm) revealed melting temperatures of 48 and
50.degree. C. for the native and daunomycin-crosslinked duplexes,
respectively. A similar melting profile was obtained by monitoring
hypochromicity at 482 nm for the DM duplex.
Example 3
Preparation of Gold Electrodes Derivatized with DNA Duplexes
[0088] Electrodes were conveniently prepared by modifying gold
surfaces with 15 base-pair DNA duplexes derivatized at the 5' end
with a thiol-terminated alkane chain. Bulk gold electrodes were
polished successively with 0.3- and 0.5-.mu.M alumina (Buhler),
sonicated for 30 min, and etched in 1.0 M sulfuric acid. Au(111)
surfaces were prepared by vapor deposition onto mica or glass
(Widrig, 1991; Zei, 1983). Electrodes were then modified by
incubation in 0.1 mM solutions of derivatized DNA duplexes in 5 mM
phosphate/50 mM NaCl (pH 7) for 12-48 h at ambient temperature.
Modified electrodes were rinsed in buffer prior to use.
[0089] Before deposition of the duplexes onto the gold surfaces,
the presence of the free thiols was confirmed using a spectroscopic
assay based on dithionitrobenzene (Riddles, 1979). Subsequently,
the samples were deposited onto the gold surfaces for 12-24 h.
[0090] Electrochemical assays, radioactive tagging experiments, and
atomic force microscopy (AFM) (Kelley, 1997a, 1997b) all indicate
that the oligonucleotides form densely packed monolayers oriented
in an upright position with respect to the gold surface.
Example 4
Characterization of Modified DNA Duplexes Monolayers on Gold
Surfaces
[0091] The DM-modified duplexes readily formed self-assembled
monolayers on gold. AFM studies of modified films reveal densely
packed monolayers with heights greater than 45 .ANG. at open
circuit. More specific, AFM studies were carried out under
electrochemical control, and revealed that the DNA films undergo a
potential-dependent change in structure. At open circuit, the
monolayer film height is 45(3) .ANG.. Based on the anisotropic
dimensions of the 15-base pair duplexes (20 .ANG. in diameter vs.
60 .ANG. in length), this thickness indicates that the helical axis
is oriented .about.45" from the gold surface. At applied voltages
negative of the potential of zero charge, film thickness of
.about.60 .ANG. are observed; more positive potential cause a drop
in the film height to a limiting value of 20 .ANG. at low surface
coverages.
[0092] Based on the crossectional area of DNA (.about.3 nm.sup.2)
and the geometrical area of the gold electrodes (0.02 cm.sup.2),
the maximum surface coverage of DNA was calculated as
.about.6.times.10.sup.-11 mol/cm. Coulometry at electrodes modified
with duplexes containing crosslinked DM revealed a DM surface
coverage of 7.5(7).times.10.sup.-11 mol/cm .sup.2, indicating that
the surface is densely packed with the modified duplexes. The DM
value appeared to exceed slightly the theoretical .UPSILON. for
DNA, and likely resulted from additional electrode surface
roughness.
[0093] To assess routinely the surface coverage of DM-derivatized
DNA on gold, the electrochemical response of Fe(CN).sub.6.sup.4- (2
mM) was monitored. This negatively charged ion is repelled from the
modified-electrode surface by the polyanionic DNA, and exhibits
essentially no response when the surface is well covered. While not
a direct measure of surface coverage, this technique allowed the
convenient assay of individual electrodes for adequate
modification.
[0094] Cyclic voltammograms of these surfaces showed the reversible
reduction of DM at -0.65 V versus SCE (Molinier-Jumel, 1978; Berg,
1981). These films were extremely stable and exhibited responses
characteristic of surface-bound species (e.g., linear plots of peak
current versus scan rate) (Bard, 1980).
Example 5
Measurement of Electrochemical Response of a Redox-Active
Intercalator Crosslinked to a Fully Base-Paired DNA Duplex on a
Gold Surface
[0095] Integration of the electrochemical response yielded a
surface coverage (.UPSILON.) of electroactive daunomycin of
7.5(7).times.10.sup.-11 mol/cm.sup.2, a value in good agreement
with the coverages of 15-base pair duplexes previously measured via
.sup.32P labeling (Kelley, 1997a). However, significant
fluctuations in the surface coverages of DM-modified duplexes were
observed. Therefore, only electrodes which exhibited both large
integrated currents for the reduction of crosslinked DM and an
attenuated responses for the oxidation of ferrocyanide in solution
were studied.
[0096] Given the 1:1 stoichiometry of crosslinked DM to DNA, the
observed data indicated that all of the bound DM was
electrochemically reduced. Doping these films with increasing
percentages of DM-free duplexes resulted in a linear decrease in
the observed electrochemical signals (as determined from
coulometric assays), consistent with each of the bound
intercalators being electrochemically active.
[0097] Remarkably, efficient reduction of DM was observed
regardless of its position along the 15-base-pair sequence as
illustrated in FIG. 2. Based on molecular modeling, the DM/gold
separations span .about.25 .ANG.. The through-helix DM-electrode
separation is >10 .ANG. for DM bound at the end of the duplex
closest to the electrode (FIG. 2A), and the DM-electrode separation
is >35 .ANG. (FIG. 2B) for DM crosslinked to the end of the
duplex farthest from the electrode. The surface coverage of
electroactive daunomycin for these 15 base-pair duplexes as
measured by integrating the currents within the illustrated
voltammograms were 0.65.times.10.sup.-10 mol/cm.sup.2 and
0.80.times.10.sup.-10 mol/cm.sup.2, respectively. The DM:DNA
stoichiometry for these same samples, measured by absorption
spectroscopy were 0.9:1 and 1.1:1, respectively. Thus, the charge
did not depend on distance, but did reflect the yield of
crosslinking.
Example 6
Measurement of Electrochemical Response of a Redox-Active
Intercalator Crosslinked to a Mismatch-Containing DNA Duplex on a
Gold Surface
[0098] Electrochemical responses of a redox-active intercalator
crosslinked to a mismatch-containing DNA duplex on a gold surface
were measured to determine whether these observed rates were a
result of direct contact between the redox-active cofactor and the
electrode surface (which has previously been shown to yield
apparently distance-independent heterogeneous electron transfer
(Feng, 1995, 1997)). A single site within the 15-base-pair duplex
was mutated to produce a CA mismatch (known to cause local
disruptions in the DNA base stack (Patel, 1984; Aboul-ela, 1985)
between the intercalated DM and the electrode surface. FIG. 3
illustrates that such a simple change virtually eliminated the
electrochemical response.
[0099] The coulometry of DM at electrodes modified with
CA-containing duplexes varied to some degree as a function of the
surface coverage. At high surface coverages (as determined by the
ferrocyanide assay), essentially no signal was observed with the
mismatched duplexes. However, at more moderate surface coverages,
small signals corresponding to the reduction of DM were found.
These typically did not exceed 30% of the signals found for the TA
duplexes. The morphology of partial DNA monolayers is unknown.
[0100] Significantly, sequences in which the positions of the DM
and CA mismatch were reversed (such that the mismatch was located
above the DM relative to the gold) showed no diminution in the
electrochemical response. AFM images of the CA-mutated sequences
were identical to those of the TA analogs (monolayer thicknesses of
.about.40 .ANG. at open circuit), revealing that the bulk structure
of the DNA films was not significantly altered by the presence of a
mismatch. Moreover, the oxidation of ferrocyanide was similarly
attenuated at both surfaces. Expected masses for DM-crosslinked DNA
duplexes (accounting for the single base change) were measured by
mass spectrometry, and spectrophotometric assays revealed that the
extent of crosslinking was identical in both fully paired and
mismatched sequences.
[0101] The exquisite sensitivity of the electrochemistry of DM to
intervening lesions in the base stack provides therefore the basis
for an exceptionally versatile DNA-mismatch sensor.
Example 7
Analysis of the Electrochemical Behavior of Fully Base-Paired or
Mismatch-Containing DNA Duplexes Containing Non-Crosslinked
Intercalators
[0102] A practical method to detect mismatches utilizes a system
based on non-crosslinked, intercalative, redox-active species. The
electrochemistry of DM non-covalently intercalated into
DNA-modified films was studied in order to develop a general
approach to test heterogeneous sequences that may possess more than
one guanine-binding site. Coulometric titrations confirmed that DM
strongly binds to surfaces modified with fully base-paired
duplexes, and yielded affinity constants very similar to those
determined for homogeneous solutions (Arcamone, 1981;
Molinier-Jumal, 1978; Berg, 1981). At bulk DM
concentrations.gtoreq.1 .mu.M, the modified electrodes were
saturated with intercalator, and hold approximately one
intercalator per surface-confined duplex. Furthermore,
intercalators non-covalently bound to these films exhibited
electrochemical properties quite similar to those described for
crosslinked DM, with the exception that the binding was reversible,
i.e. in pure buffer solutions, decreasing voltammetric signals were
observed until total dissociation was evident.
[0103] In accord with the studies of covalently bound DM,
incorporation of a single CA mismatch into these duplexes
dramatically decreased the electrochemical response (Table 1). The
magnitude of this mismatch effect depended strongly on the location
of the CA base step along the sequence: when the mutation was
buried deep within the monolayer, the measured charge drops by a
factor-of 3.5(5) (relative to the Watson-Crick duplex), but by only
2.3(4) when it was located near the solvent-exposed terminus. These
observations were consistent with DM occupying sites near the top
of the densely packed monolayer, as suggested in earlier studies of
methylene blue bound to these same surfaces (Kelley, 1997b). The
intensity of the electrochemical signals therefore not only reports
the presence of the mismatch but also may describe the location of
the disruption.
[0104] In addition, lateral charge diffusion within these
monolayers was analyzed. For example, a series of fully base-paired
films (sequence: SH-.sup.5'AGTACAGTCATCGCG) doped with increasing
fractions of CA-mismatched helices were prepared (the mismatch was
localized at the base step denoted by the bold C in the above
sequence.) The coulometric response of DM non-covalently bound to
these surfaces was strongly dependent on the film composition such
that the electrochemical signals decreased linearly with increasing
percentages of mutated duplexes. As there is no measurable
difference in the affinities of DM toward TA- versus CA-containing
films, this linear response indicated that the electroinactive
intercalators (presumably those molecules bound to mutated helices)
are not reduced by lateral charge transfer from the electroactive
species. This result further supports a through-helix pathway for
charge transduction, as intermolecular interactions between
intercalators bound to different duplexes in the film evidently do
not mediate efficient electron transfer.
Example 8
Analysis of Mutation Dependence of Electrochemical Response
[0105] To explore the scope of this mismatch detection strategy,
the charge (Q.sub.c) for DM at DNA-modified electrodes containing
different single-base mismatches was analyzed (FIG. 4). The seven
different mismatched duplexes were obtained by hybridization of the
thiol-modified sequence, SH-.sup.5 AGTACAGTCATCGCG, with the
following seven different complements (the mismatch is indicated in
bold, and the specific basepair and the melting temperature of the
duplex is given in parentheses): .sup.5'CGCGATGACTGTACT (TA,
T.sub.m=68.degree. C.), .sup.5'CGCGACGACTGTACT (CA,
T.sub.m=56.degree. C.), .sup.5'CGCGATGTCTGTACT (TT,
T.sub.m=57.degree. C.), .sup.5'CGCGATCACTGTACT (CC,
T.sub.m=56.degree. C.), .sup.5'CGCGATGGCTGTACT (GT,
T.sub.m=62.degree. C.), .sup.5'CGCGATGAATGTACT (GA,
T.sub.m=60.degree. C.), .sup.5'CGCGATGCCTGTACT (CT,
T.sub.m=58.degree. C.). The charges were then calculated by
integrating background-subtracted cyclic voltammograms. The
obtained values were based on >5 trials, and the results were
comparable for experiments run side-by-side or utilizing different
sample preparations. The melting temperatures of the oligomers in
solution were measured by monitoring duplex hypochromicity at 260
nm using samples that contained 10 .mu.M duplex, 100 mM MgCl.sub.2,
and 100 mM phosphate at pH 7.
[0106] Coulometric analysis confirmed that the attenuation of the
characteristic DM response was strongly dependent upon the identity
of the mutation. In general, pyrimidine-pyrimidine and
purine-pyrimidine mismatches caused marked decreases in the
electrochemical signals; the one purine-purine pair studied (a GA
mismatch, which is notoriously well-stacked within duplex DNA
(Patel, 1984; Aboul-ela, 1985)) did not show a measurable effect.
Surprisingly, a significant decrease was caused by a GT pair, which
is also not highly disruptive to the helix. This wobble base pair,
although thermodynamically stable, appears to mediate electron
transfer poorly.
[0107] FIG. 4 illustrates that across a very narrow range of duplex
thermal stabilities, large differences in the electrochemical
response were observed. Overall, the electrochemical properties of
films containing the different mismatches correlated with the
degree of disruption to base stacking with the individual duplexes.
These results underscore the sensitivity of this electrochemical
assay to base stacking within DNA, and demonstrate the viability of
detecting mismatches based upon charge transduction through thin
films.
Example 9
Analysis of Sequence Dependence of Mismatch Detection Assay
[0108] A single CA mismatch was incorporated into three different
DNA duplexes to test for the sequence dependence of the assay. The
duplexes featured varying percentages of GC content, representing a
wide range of duplex stabilities. The melting temperatures for
these duplexes, as determined by thermal denaturation measurements
obtained by monitoring hypochromicity at 260 nm in duplex solutions
containing 10 .mu.M duplex, 100 mM phosphate, and 100 mM MgCl.sub.2
were: (SH-.sup.5'-ATATAATATATGGAT- ): TA=47.degree. C.,
CA=32.degree. C.; (SH-.sup.5'-AGTACAGTCATCGCG): TA=68.degree. C.,
CA=56.degree. C.; (SH-.sup.5'-GGCGCCCGGCGCCGG): GC=82.degree. C.,
CA=69.degree. C. The charge was quantitated from integrating
background-subtracted cyclic voltammograms obtained at
.upsilon.=100 mV/s and was corrected for electrode area. As
illustrated in FIG. 5, the characteristic drop in coulometric
signals for DNA duplexes containing a single CA mismatch compared
to fully base-paired DNA films was essentially invariant across
AT-rich to GC-rich sequences. This sequence-independent response is
not achievable using traditional mismatch detection assays based
upon differential hybridization.
Example 10
Analysis of Electrochemical Response During Repeated Cycles
[0109] To extend this methodology to single-stranded targets,
techniques for in situ hybridization were developed. Thiol-modified
duplexes were deposited on the gold surface, heat denatured,
thoroughly rinsed, then rehybridized with the desired target by
incubation in .gtoreq.50 pmol of single-stranded oligonucleotide.
The electrochemical properties of the resulting surfaces were
identical to those described above, suggesting the suitability of
this system for genomic testing.
[0110] For example, a 15-base-pair oligonucleotide,
.sup.5'AGTACAGTCATCGCG, which was derivatized with a
thiol-terminated linker, was hybridized both to its native
complement and to a mutated complement (at the site underlined in
the sequence), generating a fully base-paired duplex and a CA
mismatch-containing duplex, respectively (FIG. 6). These duplexes
were deposited on separate electrodes and the electrochemical
responses of DM non-covalently bound to these duplexes were
measured using cyclic voltammetry (.upsilon.=100 mV/s, 1.0 .mu.M
DM). FIG. 6 illustrates that DM exhibited electrochemical responses
characteristic of fully base-paired and CA-mutated films,
respectively. The surfaces were then denatured by immersing the
electrodes in 90.degree. C. pure buffer for 2 min to yield
single-stranded monolayers of identical sequence. Cyclic
voltammetry of DM at these electrodes now revealed nearly identical
responses, with the reduction appearing highly irreversible,
broadened, and becoming smaller as a function of increasing scans.
Importantly, the electrode that initially possessed the CA mismatch
displayed a large signal (for the first scan) after denaturation,
while the reverse was true for the corresponding TA analog. New
duplexes were formed by incubating the electrodes with 100 pmol of
the opposite complement in the presence of buffered 100 mM
MgCl.sub.2 such that the complements were traded (TA.fwdarw.CA,
CA.fwdarw.TA), and the electrochemistry at the duplex-modified
films again showed the characteristic behavior expected for fully
base-paired and CA-mutated films. Finally, the electrodes were
again heated to denature the duplexes and quantitation of the
response showed again the characteristics for single-stranded
oligonucleotides. Thus, electrodes can be cycled through this
sequence of events repeatedly, indicating a practical means to
detect point mutations within natural DNAs.
Example 11
Detection of Genetic Mutations Within a Specific Region of the p53
Gene Using Direct Current Measurement of Thiol-Modified Duplexes on
Gold Surfaces
[0111] A specific embodiment utilizes a gold-microelectrode array
with approximately thirty addressable sites. A different 20-base
pair duplex derivatized with a hexylthiol linker is attached to
each of these sites by deposition form a concentrated duplex
solution overnight. The sequences are chosen to correspond to the
600-base pair region within exons 5 through 8 of the p53 gene where
most of the cancer-related mutations are found. The array is
immersed in aqueous solution at 90.degree. C. for 60 seconds to
denature the immobilized duplexes and remove the complementary
strands. The human sample containing the p53 gene is fragmented
either before or after amplification. A solution containing the
fragmented genomic single-stranded DNA is deposited on the array
for one hour to allow hybridization to occur. Then, in the presence
of a 1.0 .mu.M DM solution, the charge passed at each of the
electrodes is measured, and the response for each sequence is
compared to that obtained from the wild-type (i.e. fully
base-paired) sequences. Electrodes with attenuated signals
correspond to mutated subsequences, while those which exhibited the
expected charge are classified as unmutated.
Example 12
Detection of Mutations Using Electrocatalytic Currents Generated at
DNA-Modified Surfaces
[0112] The signals corresponding to mismatched and fully-paired
sequences can be more highly differentiated by monitoring catalytic
currents at DNA-modified surfaces. Electrons can be shuttled
through the immobilized duplexes to redox-active intercalators
localized on the solvent-exposed periphery of the monolayer, and
then negatively-charged solution-borne species (which are
electrostatically prohibited from the interior of the monolayer)
are catalytically reduced by the intercalating mediators. Since the
catalytic reaction essentially amplifies the signal corresponding
to the intercalator, the attenuation of this response in the
presence of the mismatch is significantly more pronounced. In a
specific embodiment, the sequence SH-.sup.5'AGTACAGTCATCGCG was
deposited on an electrode both hybridized with a fully base-paired
complement, and with a complement containing a CA mismatch (the
position of the mismatch is denoted in bold). These duplexes were
immersed in a solution containing 1.0 .mu.M methylene blue and 1 mM
ferricyanide. In the presence of either of these reagents alone,
only small direct currents were measured. However, in the presence
of a mixture of the intercalator and the negatively charged probe,
pronounced currents were measured corresponding to the
electrocatalytic reduction of ferricyanide by methylene blue. The
amount of current observed for the TA and CA containing films
differ dramatically; using electrocatalysis, the mismatched duplex
can be differentiated from the fully base-paired duplexes by a
factor of approximately 100. Moreover, as illustrated in FIG. 8,
the peak potentials for the TA and CA duplexes are significantly
separated, allowing the presence of the mismatch to be detected
potentiometrically. This approach therefore represents an extremely
sensitive means to detect genetic mutations electrochemically.
Example 13
Detection of Genetic Mutations Within a Specific Region of the p53
Gene Using Electrocatalytic Current Measurement of Thiol-Modified
Duplexes on Gold Surfaces
[0113] Another specific embodiment involves detecting the mutations
within the p53 gene using electrocatalysis. A different 20-base
pair duplex derivatized with a hexylthiol linker is attached to
each of approximately thirty addressable sites of a
gold-microelectrode array by deposition form a concentrated duplex
solution overnight. The sequences are chosen to correspond to the
600-base pair region within exons 5 through 8 of the p53 gene where
most of the cancer-related mutations are found. The array is
immersed in aqueous solution at 90.degree. C. for 60 seconds to
denature the immobilized duplexes and remove the complementary
strands. The human sample containing the p53 gene is fragmented
either before or after amplification. A solution containing the
fragmented genomic single-stranded DNA is deposited on the array
for one hour to allow hybridization to occur. The array is rinsed
and submerged in a solution containing 1.0 .mu.M methylene blue and
1.0 mM ferricyanide. The pronounced currents that are observed
result from the electrocatalytic reduction of the solution-borne
ferricyanide by methylene blue adsorbed at the solvent-exposed
duplex sites. These catalytic currents are measured for each
addressable electrode and compared with those obtained with the
wild-type sequence to detect potential sites of mutations.
Example 14
Detection of Genetic Mutations Within a Gene of Interest Using
Direct or Electrocatalytic Current Measurement of Amine-Modified
Duplexes on Carbon Surfaces
[0114] Another embodiment utilizes a carbon electrode. The
electrode is oxidized at +1.5 V (vs. Ag/AgCl) in the presence of
K.sub.2Cr.sub.2O.sub.7 and HNO.sub.3, and treated with
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC)
and N-hydroxysulfosuccinimide (NHS). Duplexes corresponding to
mutated sequences of a specific gene of interest are derivatized
with a hexylamine linker and applied to the electrode surface. The
device is immersed in aqueous solution at 90.degree. C. for 60
seconds to, generate a single-stranded monolayer, and the
fragmented genomic DNA sample is hybridized to the immobilized
probes at room temperature for 1 hour. The detection of mutations
is accomplished by (i) measuring direct currents in the presence of
1.0 .mu.M daunomycin solution, or (ii) by measuring catalytic
currents in the presence of 1.0 .mu.M methylene blue and 1.0 mM
ferricyanide. Charges passed at each electrode are measured, and
the response for each sequence is compared to that obtained with
wild-type, i.e. fully base-paired, sequences. Attenuated signals
correspond to mutated subsequences, while those which exhibit no
change in current are classified unmutated.
[0115] Although the invention has been described with reference to
particular applications, the principles involved may be used in
other applications which will be apparent to those skilled in the
art. The invention is accordingly to be limited only by the scope
of the claims which follow.
1TABLE I Electrochemical Detection of Single-Base Mismatches.sup.a
Q.sub.c(int) (nC).sup.b T.sub.m (.degree. C.).sup.c
SH-.sup.5'AGTACAGTCATCGCG 165(37) 68 TCATGTCAGTAGCGC
SH-.sup.5'AGTACAGTCATCGCG 56(15) 56 TCATGTCAGCAGCGC
SH-.sup.5'AGTACAGTCATCGCG 95(18) 57 TCATGTCTGTAGCGC
SH-.sup.5'AGTACAGTCATCGCG 51(23) 56 TCATGTCACTAGCGC
SH-.sup.5'AGTACAGTCATCGCG 49(30) 62 TCATGTCGGTAGCGC
SH-.sup.5'AGTACAGTCATCGCG 153(38) 60 TCATGTAAGTAGCGC
SH-.sup.5'AGTACAGTCATCGCG 93(17) 58 TCATGTCCGTAGCGC .sup.aBased on
cyclic voltammograms measured for 1.0 .mu.M daunomycin
noncovalently bound to duplex-modified electrodes (0.1 M phosphate
buffer, pH 7). Values are based on > 5 trials each, and results
were comparable for experiments run side-by side, or from different
sample preparation as long as electrodes exhibited high surface
coverages. Electrodes with lower surface coverages yielded higher
charges (>1 DM/duplex), and decreased attenuations in the
presence of mismatches. .sup.bIntegrated background-subtracted
cathodic charge. .sup.cMeasured by monitoring duplex hypochromicity
at 260 nm. Samples contained 10 .mu.M duplex, 100 mM MgCl.sub.2,
100 mM phosphate, pH 7.
* * * * *