U.S. patent application number 09/128732 was filed with the patent office on 2001-05-31 for triple helix recognition of dna.
This patent application is currently assigned to FLEHR HOHBACH TEST ALBRITTON & HERBERT LLP. Invention is credited to DERVAN, PETER B., MOSER, HEINZ E..
Application Number | 20010002314 09/128732 |
Document ID | / |
Family ID | 26813714 |
Filed Date | 2001-05-31 |
United States Patent
Application |
20010002314 |
Kind Code |
A1 |
DERVAN, PETER B. ; et
al. |
May 31, 2001 |
TRIPLE HELIX RECOGNITION OF DNA
Abstract
Probes and processes for their use for specific recognition
and/or cleavage of double-stranded DNA or RNA at sequence specific
desired loci through the intermediacy of a triple helix are
disclosed. These probes may also be used as diagnostic
chemotherapeutic agents through incorporation of a radiolabeled,
fluorescing, or otherwise detectable molecule. Preferred assay
conditions are also provided for recognition of
homopurine-homopyrimidine double-helical tracts within large DNA by
triple helix formation under physiological conditions.
Hybridization probes for double-stranded recognition with binding
site sizes that range >8 base pairs are also provided.
Inventors: |
DERVAN, PETER B.; (PASADENA,
CA) ; MOSER, HEINZ E.; (SOUTH PASADENA, CA) |
Correspondence
Address: |
FLEHR HOHBACH TEST
ALBRITTON & HERBERT
SUTIE 3400
FOUR EMBARCADERO CENTER
SAN FRANCISCO
CA
941114187
|
Assignee: |
FLEHR HOHBACH TEST ALBRITTON &
HERBERT LLP
|
Family ID: |
26813714 |
Appl. No.: |
09/128732 |
Filed: |
August 4, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09128732 |
Aug 4, 1998 |
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08152250 |
Nov 12, 1993 |
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5789155 |
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08152250 |
Nov 12, 1993 |
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07614205 |
Nov 16, 1990 |
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07614205 |
Nov 16, 1990 |
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07115922 |
Oct 30, 1987 |
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Current U.S.
Class: |
435/375 ; 435/29;
435/6.1; 435/6.18; 514/44R; 536/24.3; 536/24.31; 536/24.5 |
Current CPC
Class: |
C12Q 1/6839
20130101 |
Class at
Publication: |
435/375 ; 435/6;
435/29; 514/44; 536/24.3; 536/24.31; 536/24.5 |
International
Class: |
C12N 005/00; A61K
031/7088; C07H 021/00; C12Q 001/02; C12Q 001/68 |
Claims
What is claimed is:
1. A polynucleotide hybridization probe for a specific nucleic acid
sequence within a large double-helical nucleic acid, comprising a
nucleotide sequence complementary to a specific nucleic acid
sequence within a large double-stranded nucleic acid, said
nucleotide sequence containing at least one nucleoside to which is
attached at least one molecule adapted to cleave said
double-stranded nucleic acid.
2. The probe of claim 1, wherein said at least one molecule adapted
to cleave said double-stranded nucleic acid contains at least one
metal chelator.
3. The probe of claim 2 wherein said metal chelator is attached to
a nucleoside within said nucleotide sequence by a linker group.
4. The probe of claim 1 wherein said large double-stranded nucleic
acid sequence comprises deoxyribonucleic acid.
5. The probe of claim 1 wherein said large double-stranded nucleic
acid sequence comprises ribonucleic acid.
6. The probe of claim 1 wherein said double-stranded nucleic acid
sequence comprises a hybrid duplex with one strand of
deoxyribonucleic acid and one strand of ribonucleic acid.
7. A homopyrimidine polydeoxyribonucleotide hybridization probe for
a specific sequence within a large double-helical nucleic acid
comprising a homopyrimidine nucleotide sequence complementary to a
specific corresponding homopyrimidine-homopurine tract within a
large double-helical nucleic acid, said nucleotide sequence
containing at least one nucleoside to which is attached at least
one molecule adapted to cleave said double-stranded nucleic
acid.
8. The probe of claim 7, wherein said at least one molecule adapted
to cleave said double-stranded nucleic acid contains at least one
metal chelator.
9. The probe of claim 7 wherein said metal chelator is attached to
a nucleoside within said nucleotide sequence by a linker group.
10. The probe of claim 7 wherein said large double-stranded nucleic
acid sequence comprises deoxyribonucleic acid.
11. The probe of claim 7 wherein said large double-stranded nucleic
acid sequence comprises ribonucleic acid.
12. The probe of claim 7 wherein said double-stranded nucleic acid
sequence comprises a hybrid duplex with one strand of
deoxyribonucleic acid and one strand of ribonucleic acid.
13. A process for identifying the presence of a specific nucleic
acid sequence within a large double-helical nucleic acid,
comprising the steps of: a. hybridizing a specific
homopyrimidine-homopurine tract within a large, double-helical
nucleic acid with a corresponding polynucleotide hybridization
probe, said polynucleotide probe containing at least one nucleoside
to which is attached at least one detectable label molecule capable
of being detected upon the binding of said nucleotide to said
tract; b. permitting said hybridization to proceed to formation of
a triple-helix; and c. detecting said label molecule.
14. The process of claim 13, wherein said nucleic acid sequence
codes for a virus.
15. The process of claim 13, wherein said nucleic acid sequence
codes for a oncogene.
16. The process of claim 13 wherein said label molecule contains a
radioactive isotope.
17. The process of claim 13 wherein said label molecule contains a
fluorescing molecule.
18. The process of claim 13 wherein said label molecule contains at
least one enzyme that reacts with a substrate to produce an
observable color change.
19. The process of claim 13 wherein said large double-stranded
nucleic acid sequence comprises deoxyribonucleic acid.
20. The process of claim 13 wherein said large double-stranded
nucleic acid sequence comprises ribonucleic acid.
21. The process of claim 13 wherein said double-stranded nucleic
acid sequence comprises a hybrid duplex with one strand of
deoxyribonucleic acid and one strand of ribonucleic acid.
22. A process for cleaving large double-helical nucleic acids,
comprising the steps of: a. hybridizing a homopyrimidine-homopurine
tract within a large double-helical nucleic acid with a
corresponding homopyrimidine polynucleotide containing at least one
nucleoside to which is attached at least one molecule adapted to
cleave said homopyrimidine-homopurine tract in the major groove; b.
permitting said hybridization to proceed to formation of a
triple-helix; and c. cleaving said tract.
23. The process of claim 22, wherein said at least one molecule
adapted to cleave said double-stranded nucleic acid contains at
least one metal chelator.
24. The process of claim 23 wherein said metal chelator is attached
to a nucleoside within said nucleotide sequence by a linker
group.
25. The process of claim 22 wherein said large double-stranded
nucleic acid sequence comprises deoxyribonucleic acid.
26. The process of claim 22 wherein said large double-stranded
nucleic acid sequence comprises ribonucleic acid.
27. The process of claim 22 wherein said double-stranded nucleic
acid sequence comprises a hybrid duplex with one strand of
deoxyribonucleic acid and one strand of ribonucleic acid.
28. A process for treatment of gene-based disease states such as
those caused by viruses, genetic defects, and oncogenes, comprising
the steps of: a. identifying and sequencing a specific
homopyrimidine-homopurine tract within a double-stranded nucleic
acid sequence involved in a gene-based disease; b. delivering to
said tract an effective amount of a corresponding homopyrimidine
polynucleotide containing at least one nucleoside to which is
attached at least one molecule adapted to alter the natural
expression of any products from said tract; c. hybridizing said
tract with said corresponding homopyrimidine polynucleotide; and d.
permitting said hybridization to proceed to formation of a
triple-helix.
29. The process of claim 28 wherein said large double-stranded
nucleic acid sequence comprises deoxyribonucleic acid.
30. The process of claim 28 wherein said large double-stranded
nucleic acid sequence comprise ribonucleic acid.
31. The process of claim 28 wherein said double-stranded nucleic
acid sequence comprises a hybrid duplex with one strand of
deoxyribonucleic acid and one strand of ribonucleic acid.
Description
FIELD OF THE INVENTION
[0001] This invention relates to nucleic acid probes for
sequence-specific recognition and cleavage of double-helical
nucleic acids through the intermediacy of a triple-helix.
BACKGROUND OF THE INVENTION
[0002] The sequence-specific cleavage of double-helical
deoxyribonucleic acid (hereafter "DNA") by naturally occurring
restriction endonucleases is essential for many techniques in
molecular biology including gene isolation, DNA sequence
determinations chromosome analysis, gene isolation and recombinant
DNA manipulations. Other applications include diagnostic reagents
to detect pathogens and aberrant DNA molecules as well as
chemotherapeutics. The
[0003] The usefulness of DNA cleavage by these naturally recurring
restriction enzymes is limited. The binding site sizes of naturally
occurring restriction enzymes are typically in the range of four to
eight base pairs, and hence their sequence specificities may be
inadequate for mapping genomes (10.sup.5-10.sup.7 base pairs) over
very large distances. For unique recognition of DNA in the
10.sup.5-10.sup.7 base pair range. Sequence specificities at the
8-15 base pair level must be obtained. In addition, there are a
limited number of known restriction endonucleases. Thus, they
cannot be used to specifically recognize a particular piece of DNA
(or RNA) unless that piece of DNA contains the specific nucleic
acid sequences recognized by the endonucleases.
[0004] With the advent of pulsed field gel electrophoresis,
separation of large (up to at least one million base pair) pieces
of DNA is now possible. The design and synthesis of
sequence-specific DNA recognition and cleaving molecules that go
beyond the specificities of the natural restriction enzymes is
obviously desirable, as they would provide valuable tools for
further research, diagnostics, and chemotherapeutics.
[0005] Synthetic sequence-specific binding moieties for
double-helical DNA that have been studied are typically coupled
analogs of natural products (P. D. Dervan, Science 232, 464
(1986)), transition metal complexes (J. K. Barton, Science 233, 727
(1986)), and peptide fragments derived from DNA binding proteins
(J. Sluka, et al., Science, in press). Additionally,
methidiumpropyl-EDTA (hereafter "MPE"), which contains the metal
chelator ethylenediaminetetraacetic acid ("EDTA") attached to the
DNA intercalator methidium, has been shown to cleave double-helical
DNA efficiently in a reaction dependent on ferrous iron (Fe(II))
and dioxygen (O.sub.2). This mechanism is thought to occur by
binding in the minor groove of the right-handed DNA helix. Addition
of reducing agents such as dithiothreitol (hereafter "DTT")
increases the efficiency of DNA cleavage, as reported by Hertzberg
and Dervan, J. Am. Chem. Soc. 104, 313-315 (1982); and Hertzberg
and Dervan, Biochemistry, supra). MPE-Fe(II) cleaves DNA in a
relatively non-sequence specific manner, and with significantly
lower sequence specificity than the enzyme DNAseI, and therefore is
useful in experiments to identify binding locations of small
molecules such as antibiotics, other drugs, and proteins on DNA,
Hertzberg and Dervan, Biochemistry, supra.
[0006] The most sequence-specific molecules characterized so far,
with regard to the natural product analog approach is
bis(EDTA-distamycin) fumaramide which binds in the minor grove and
cleaves at sites containing nine base-pair (hereafter "bp") of
contiguous A,T DNA (Youngquist and Dervan, J. Am. Chem. Soc. 107,
5528 (1985)). A synthetic peptide containing 52 residues from the
DNA binding domain of Hin protein with EDTA at the amino-terminus
binds and cleaves at the 13 bp Hin site (Bruist, et al., Science
235, 777 (1987); Sluka, et al., supra). Another known DNA cleaving
function involves the attachment of a DNA-cleaving moiety such as a
ethylenediaminetetraacetic acid-iron complex (hereafter
"EDTA-Fe(II)"), to a DNA binding molecule which cleaves the DNA
backbone by oxidation of the deoxyribose with a short-lived
diffusible hydroxyl radical (Hertzberg and Dervan, Biochemistry 23,
3934 (1984)). The fact that the hydroxyl radical is a relatively
non-specific cleaving species is useful when studying recognition,
because the cleavage specificity is due to the binding moiety
alone, not some combination of cleavage specificity superimposed on
binding specificity.
[0007] Despite this progress, the current understanding of
molecular recognition of DNA is still sufficiently primitive that
the elucidation of chemical principles involved in creating
specificity in sequence recognition at the .gtoreq.15 base pair
level has been slow in development in comparison to the interest in
the field for mapping large genomes.
[0008] Recognition of single-stranded nucleic acids by nucleic
acid-hybridization probes consisting of sequences of DNA or RNA are
well known in the art. Typically, to construct a DNA hybridization
probe, selected target DNA is obtained as a single-strand and
copies of a portion of the strand are synthesized in the laboratory
and labeled using radioactive isotopes, fluorescing molecules,
photolytic dyes or enzymes that react with a substrate to produce a
color change. When exposed to complementary strands of target DNA,
the labeled DNA probe binds to (hybridizes) its complementary
single-stranded DNA sequence. The label on the probe is then
detected and the DNA of interest is thus located. Probes may
similarly be used to target RNA sequences. DNA probes are currently
well known in the art for locating and selecting genes of known
sequence, and in the diagnosis and chemotherapy of genetic
disorders and diseases.
[0009] Oligonucleotides (polynuclectides containing between 10 and
50 bases) equipped with a DNA cleaving moiety have been described
which produce sequence-specific cleavage of single-stranded DNA.
Examples of such moieties include oligonucleotide-EDTA-Fe
hybridization probes ("DNA-EDTA") which cleaves the complementary
single strand sequence (Dreyer and Dervan, Proc. Natl. Acad. Sci.
USA. 82, 968 (1985); Chu and Orgel, Proc. Natl. Acad. Sci. USA. 82,
963 (1985)). Such probes are disclosed in co-owned [the co-pending]
U.S. Pat. No. 4,795,700.
[0010] In addition to double and single-stranded configurations, it
is also well known in the art that triplexes of nucleic acids
naturally exist (Howard, et al., Biochem. BioPhys. Res. Commun. 17,
93 (1964)). Poly(U) and poly(A) were found to form a stable 2:1
complex in the presence of MgCl.sub.2. After this, several
triple-stranded structures were discovered (Michelson, et al.,
Prog. Nucl. Acid Res. Mol. Biol. 6, 83 (1967): Felsenfeld and
Miles, Annu. Rev. Biochem. 36, 407 (1967)). Poly(C) forms a
triple-stranded complex at pH 6.2 with guanineoligoribonucleotides.
One of the pyrimidine strands is apparently in the protonated form
(Howard, et al., supra). In principle, isomorphous base triplets
(T-A-T and C-G-C.sup.+) can be formed between any
homopyrimidine-homopurine duplex and a corresponding homopyrimidine
strand (Miller and Sobell, Proc. Natl. Acad. Sci. U.S.A. 55, 1201
(1966); Morgan and Wells, J. Mol. Biol. 37, 63 (1968); Lee et al.,
Nucleic Acids Res. 6, 3073 (1979)). The DNA-duplex
poly(dT-dC)-poly(dG-dA) associates with poly(U-C) or poly(dT-dC)
below pH 6 in the presence of MgCl.sub.2 to afford a
triple-stranded complex. Several investigators have proposed an
anti-parallel orientation of the two polypyrimidine strands based
on an anti conformation of the bases, ibid. X-ray defraction
patterns of triple-stranded fibers (poly(A)-2poly(U) and
poly(dA)-2poly(dT)) supports this hypothesis (Arnott and Bond,
Nature New Biology 244, 99 1973); Arnott and Selsing, J. Mol. Biol.
85, 509 (1974); and Arnott et al., Nucleic Acids Res. 3, 2459
(1976)), and suggested an A'-RNA-like conformation of the two
Watson-Crick base paired strands with the third strand in the same
conformation, bound parallel to the homopurine strand of the duplex
by Hoogsteen-hydrogen bonds. (Hoogsteen, Acta Cryst. 12, 822
(1959)). The twelve-fold helix with dislocation of the axis by
almost three angstroms, the C3'-endo sugar puckering and small
base-tilts result in a large and deep major groove that is capable
of accommodating the third strand (Saenger, Principles Of Nucleic
Acid Structure, edited by C. R. Cantor, Springer-Verlag, New York,
Inc. (1984). A high resolution X-ray structure of a triple-helical
DNA or RNA is not known in the art. Importantly, there are no
techniques described in the literature for determining whether a
specific homopyrimidine-homopurine tract (e.g. 15 bp) within a
large duplex DNA (e.g. >10.sup.3 bp can form a triple helix as a
method of recognition at that site.
[0011] No analytical or recombinant DNA applications of
triple-helical DNA or RNA have been reported. Although
triple-stranded structures of polynucleotides were discovered
decades ago, the biological significance has remained obscure. Such
triplexes were proposed to be involved in processes such as
regulation of gene expression, maintenance of folded chromosome
conformations, chromosome condensation during mitosis, and
induction of local conformational changes in B-DNA (Morgan, Trends
Biochem. Sci. 4, N244 (1979); Hopkins, Comments Mol. Cell Biophys.
2, 153 (1984); Minton, J. Exp. Path. 2, 135 (1985)).
[0012] The above-described methods for sequence-specific DNA
recognition and cleavage have been limited to single-stranded DNA
hybridization probes, to natural or synthetic restriction
endonucleases, and to those molecules which recognize sequences of
DNA directly such as antibiotics, and DNA lntercalators such as
methidium.
[0013] Surprisingly, the present inventors have discovered
compositions and methods of specifically tailored recognition of a
significantly larger number of double-stranded DNA and RNA sites
than was previously possible, utilizing triple helix formation
(.gtoreq.15 bp recognition) at discrete highly specific sites
within large DNA. The compositions of the present invention,
utilized by the methods set forth herein, will provide useful tools
for chromosome analysis, gene mapping and isolation. Moreover, as
molecular biology defines specific disease states at the DNA level,
the present invention finds usefulness in diagnostic strategy, as
well as chemotherapeutics.
DISCLOSURE OF THE INVENTION
[0014] The present invention relates to recognition of
homopyrimidine-homopurine double-helical tracts within large DNA,
RNA, and in DNA-RNA double-helical hybrid duplexes, by triple-helix
formation under physiologic conditions. The present invention also
relates to cleavage of said discrete, double-helical tracts.
[0015] One object of the present invention is to provide
homopyrimidine oligonucleotides and their neutral or cationic
analogs equipped with markers, lables, chemotherapeutic agents
and/or efficient DNA cleaving moieties at the 5' end, which are
capable of forming triple helices, which can be produced in
sufficient quantities to provide pharmaceutical, laboratory, or
industrial compositions useful for chromosome analysis, gene
mapping and isolation, diagnostics and chemotherapeutics. An
additional object of the present invention is to utilize precisely
tailored polynucleotide hybridization probes adapted for automated
synthesis and which afford control over the precise location in a
large double-helical nucleic acid of a label or DNA cleaving moiety
at any base position in the polynucleotide probe strand.
[0016] Another object of the present invention is to provide
polynucleotide hybridization probes and methods for their use in
the recognition of any specific sequence within a large
double-helical nucleic acid. Such probes are designed and adapted
as described above, with the substitution of a radioactive label,
photolytic dye, enzyme, or a fluorescing, or otherwise detectable
molecule for the DNA cleaving moiety.
[0017] One object of this invention is to provide a method for
delivering chemotherapeutic agents in vivo that eliminates the need
to denature the DNA before the agent can act. Yet another object of
this invention is to provide a method for precisely locating a
chemotherapeutic agent or replacement gene sequence at a specific
homopyrimidine-homopurine tract anywhere in a large double-stranded
nucleic acid. This invention also finds application in diagnostics
for gene-based diseases, and eliminates the need for many steps in
the commonly used diagnostic processes.
[0018] It is also an object of the present invention to provide a
new assay for triple helices.
[0019] Additional objects and advantages of the invention will be
set forth in part in the description which follows, and in part
will be obvious from the description or may be learned from
practice of the invention. The objects and advantages may be
realized and obtained by means of the instrumentalities and
combinations particularly pointed out in the appended claims.
[0020] To achieve the objects and in accordance with the purposes
of the present invention, homopyrimidinepoly-deoxyribonucleotide
probes with at least one detectable marker, chemotherapeutic agent
or a DNA-cleaving moiety attached to at least one predetermined
position are set forth which are capable of binding the
corresponding homopyrimidine-homopurine tracts within large
double-stranded nucleic acids by triple-helix formation at a
predetermined site.
[0021] The compositions and the methods of the present invention
allow for cleavage of one or both strands of the Watson-Crick DNA.
It is this cleavage event by bifunctional DNA-EDTA probes (i.e.,
recognition and cleavage) that allowed the triple helix formation
at discrete locations to be mapped on large DNA using gel
electrophoresis.
[0022] The polynucleotide sequences of the invention may be either
synthetic sequences or restriction fragments ("natural") DNA
sequences. The compositions and methods of this invention are
understood to apply equally for double-helical RNA, as well as to
hybrid duplexes with one strand of DNA and one strand of RNA.
[0023] Also to achieve the objects of this invention, an assay for
triple helices of up to at least 15 bp is disclosed.
[0024] Additionally, to achieve the objects and in accordance with
the purposes of the present invention, a method is disclosed which
results in recognition, chemotherapeutic alteration, and if
desired, cleavage of homopyrimidine-homopurine tracts within a
large double-stranded nucleic acid by triple helix formation at a
particular, predetermined site. This method comprises:
[0025] (a) hybridizing a specific homopyrimidine-homopurine tract
within a large double-stranded nucleic acid with a corresponding
polynucleotide hybridization probe, said nucleotide containing at
least one nucleoside to which is attached at least one of the
following:
[0026] (i) at least one detectable label molecule capable of being
detected upon the binding of said nucleotide to said tract,
[0027] (ii) at least one molecule adapted to cleave at least one
strand in said homopyrimidine-homopurine tract,
[0028] (iii) at least one molecule of a chemotherapeutic agent;
[0029] (b) permitting said hybridization to proceed to formation of
a triple-helix; and
[0030] (c) at least one of the following:
[0031] (i) detecting said label;
[0032] (ii) cleaving one or both strands of the nucleic acid;
or
[0033] (iii) permitting said chemotherapeutic agent to act.
[0034] It is understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention as
claimed.
[0035] The accompanying drawings, which are incorporated in and
constitute a part of the specification, illustrate some embodiments
of the invention and, together with the description, serve to
explain the principles of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 depicts the bonding of two Watson-Crick base pairs,
and also the bonding of isomorphous base triplets of TAT and
C.sup.+GC.
[0037] FIG. 2 is a schematic representation of the cleavage of
double-helical DNA by a triple-helix-forming DNA-EDTA-Fe probe, and
the generation of a localized hydroxyl radical.
[0038] FIG. 3A is an autoradiogram showing the cleavage products of
a double-stranded DNA containing (dA-dT).sub.15 after exposure to
DNA-EDTA probes 1-3 as analyzed by Maxam-Gilbert sequencing
methods.
[0039] FIG. 3B shows the nucleotide sequence of DNA-EDTA probes
1-3. This Figure also presents histograms of the DNA cleavage
patterns derived by densitometry of the autoradiogram of FIG. 3A
(lanes 3-5 and 8-10).
[0040] FIG. 4A is an autoradiogram showing the cleavage products of
a 628 bp EcoRI/BgII restriction fragment of plasmid pDMAG10 after
exposure to DNA-EDTA probes 4-9, as analyzed by Maxam-Gilbert
sequencing methods. FIG. 4B shows the nucleotide sequence of
DNA-EDTA probes 4-9. This figure also represents histograms of the
DNA cleavage patterns derived by densitometry of the autoradiogram
of FIG. 4A from the cleavage of the restriction fragment with
DNA-EDTA probes 4 and 9.
[0041] FIG. 5A is a bar graph presenting the absolute cleavage
efficiencies obtained with DNA-EDTA probe 4 under various
conditions.
[0042] FIG. 5B is a bar graph presenting relative cleavage
efficiencies obtained with DNA-EDTA probes 4-8 at three
temperatures.
[0043] FIG. 6A is an autoradiogram showing the cleavage products of
plasmid pDMAG10 after exposure to DNA-EDTA probe 9 under various
conditions, as analyzed on a nondenaturing agarose gel.
[0044] FIG. 6B (left) is a schematic representation of the course
resolution cleavage pattern from FIG. 6A. FIG. 6B (middle) is a
simplified schematic model depicting a triple helix complex with
the Hoogsteen bound DNA-EDTA probe 9 at one unique site within
plasmid pDMAG10 DNA.
BEST MODES FOR CARRYING OUT THE INVENTION
[0045] Reference will now be made in detail to the presently
preferred embodiments of the invention, which, together with the
drawings and the following examples, serve to explain the
principles of the invention.
[0046] FIG. 1 shows typical binding of base pairs and triplets as
referred to herein. Structure 1 of FIG. 1 shows a standard
representation of Watson-Crick base pairing of nucleotide bases A
(adenine) and T (thymine). Major groove 2 and minor groove 3 are
shown where they would appear in an A-helical structure; B-Helical
structures (not shown) are also encompassed within the scope of
this invention.
[0047] Structure 4 of FIG. 1 shows Watson-Crick base pairing of G
(guanine) and C (cytosine). Major groove 5 and minor groove 6 are
indicated.
[0048] Structure 7 of FIG. 1 shows isomorphous base triplets of
TAT; the additional pyrimidine strand is bound by
Hoogsteen-hydrogen bonds in the major groove to the complementary
purine strand shown in Watson-Crick duplex 1.
[0049] Structure 8 of FIG. 1 shows isomorphous base triplets of
C.sup.+GC. The additional pyrimidine is bound as described
above.
[0050] As described generally above, the polynucleotide
hybridization probes of the present invention are designed to be
used to detect specific nucleic acid sequences within large double
helical nucleic acids through the formation of triple-helices.
[0051] Construction of hybridization probes for recognition of
single-stranded nucleic acids, consisting of sequences of
deoxyribonuceotides (DNA) or ribonucleotides (RNA) are well-known
in the art. Typically, to construct a hybridization probe, selected
target double-stranded DNA or RNA is obtained as a single strand
and copies of a portion of the strand are synthesized in the
laboratory and labeled using radioactive isotopes, fluorescing
molecules, or enzymes that react with a substrate to produce color
change. When exposed to complementary strands of target DNA, for
example in a sample of tissue fluid taken from a patient, the
labeled DNA or RNA probe binds to (hybridizes) its complementary
single-stranded sequence. The label on the probe is then detected
and the sequence of interest is thus located. Probes constructed of
RNA sequences may be used to hybridize with a single complementary
strand of double-helical DNA forming heteroduplexes without
necessitating denaturating of the double-helical DNA (Thomas, et
al., Proc. Natl. Acad. Soc. USA, 73, 2294-2298 (1976)).
[0052] The hybridization probes as used in the practice of this
invention are unique in that they hybridize with double-stranded
helices. They form a true triple helix, not a D-loop nor other
triple-stranded structure.
[0053] The probes of the present invention may be constructed
complementary to a fragment of a natural sequence, i.e., a fragment
of a polynucleotide which occurred in nature, or which has been
cloned and expressed in the laboratory. Additionally, the
polynucleotide probes of the present invention may be synthetically
created, by hand or with automated apparatus. It is believed that
the means for synthetic creation of polynucleotide sequences are
generally known to one of ordinary skill in the art, particularly
in light of the teachings herein; see, e.g., Horvath, et al.,
Methods In Enzymology 154, 313-326 (1987). Because the probe can
contain any predetermined complementary nucleotide sequence, it is
specifically targetable and can deliver a marker or label,
chemotherapeutic agent or cleavage moiety to the exact site desired
in a specific homopyrimidine-homopurine tract within even a very
large double-stranded nucleic acid.
[0054] In one embodiment of this invention, a polynucleotide
hybridization probe is tethered to a metal chelator for cleaving a
specific nucleic acid sequence. For example, FIG. 2 depicts
oligonucleotide-directed cleavage of double-helical DNA by a
triple-helix forming DNA-EDTA-Fe probe. One thymidine has been
replaced by thymidine with the iron chelator EDTA covalently
attached at C-5. Reduction of dioxygen generates a localized
hydroxyl radical at this point.
[0055] DNA and RNA hybridization probes are selected, or
constructed, to be complementary to any target DNA or RNA. Thus,
the label, chemotherapeutic agent, or cleavage moiety of the
present invention may be incorporated along the length of any such
probe so as to provide precisely the major groove cleavage desired
by the practitioner. Also, more than one label, agent or moiety may
be included in the probe. Previously known and
familiar-hybridization sequences can be assembled, even by
currently available automated technology, incorporating the label,
agent, or cleavage moiety of the present invention. Therefore, the
choice of probes would not require undue experimentation. The
probes of this invention can be of any operable length, with a
preferred range being between fifteen and three billion
nucleosides. The present invention offers the flexibility.
precision, and efficiency long needed in the art.
[0056] In a preferred embodiment, a nucleic acid-cleaving moiety,
such as a metal chelator, is attached to a nucleoside base during
synthesis of a novel nucleoside and the so-modified nucleoside is
then incorporated into a selected polynucleotide using standard
procedures. This polynucleotide containing the chelator-modified
nucleoside recognizes the corresponding complementary sequence of
double-helical homopyrimidine-homopurine DNA for which a probe is
desired. Alternatively, the metal chelator may be attached to a
selected nucleotide located within a given polynucleotide sequence.
In the presence of dioxygen (O.sub.2), an appropriate metal ion,
and a reducing agent, the DNA-chelator probe yields a strand break
at the target complementary DNA sequence, cleaving one or both
strands at that site.
[0057] Oligonucleotides equipped with a DNA cleaving moiety have
been described which produce sequence-specific cleavage of
single-stranded DNA, supra. Examples of such moieties include
oligonucleotide-EDTA-Fe hybridization probes (DNA-EDTA) which
cleave a complementary single strand sequence (Dreyer and Dervan,
Proc. Natl. Acad. Sci. USA 82, 968 (1965); and Chu and Orgel, Proc.
Natl. Acad. Sci. USA 82, 963 (1965)). One example of a DNA-EDTA
probe is synthesized using a novel nucleoside,
5'-DMT-T*-triethylester derived from deoxyuridine to which is
attached the metal chelator EDTA as described in detail in the
co-pending application mentioned above. Such probes are also
described in Dreyer and Dervan, Proc. Natl. Acad. Sci. USA, supra.
These references disclose an EDTA-nucleoside composition
incorporated into a 19-nucleotide base pair sequence of DNA
complementary to a 19 bp sequence in a 167 bp restriction fragment
of DNA from the plasmid pBR322. This DNA-EDTA probe was then used
in the presence of the metal ion Fe(II), atmospheric dioxygen, and
the reducing agent dithiothreitol (DTT) to afford specific cleavage
at its complementary 9 bp complement in single-stranded plasmid
DNA.
[0058] Chelators or other cleavage moieties, as well as marker
labels and chemotherapeutic agents may be incorporated into the
polynucleotide sequences of the probes of the present invention at
various positions for which the chemistry for attachment at such
positions is known, provided that such attachment is accomplished
so as not to disrupt the hydrogen-base pair bonding between the DNA
or RNA sequences during hybridization of the probes.
[0059] A labeled, cleavage- or otherwise-adapted nucleoside may be
incorporated into the polynucleotide sequence of a probe chemically
using known oligonucleotide synthesis methodology, or enzymatic
procedures well known in the art.
[0060] The probes may be labeled in various well known ways for
detection and diagnostic applications, for example with radioactive
metal such as .sup.99Tc following the procedures of D. R. Elmalch,
et al., Proc. Natl. Acad. Sci. 81, 918 (1984) and EDTA or with
fluorescent elements such as the lanthanides Tb.sup.+3 or
Eu.sup.+3. Leung, et al., Biochem. Biophys. Res. Comm. 75, 15
(1977). If a chelator is desired to be used in a cleavage moiety,
other metal chelators may be used in place of EDTA such as
polyamines or other chelators capable of binding Fe(II-III) or
Cu(I-II). Other polyamino carboxylic metal chelators may be
utilized in place of EDTA such as 1,2-diamino-cyclohexane
tetraacetic acid, diethylenetriamine pentaacetic acid,
ethylenediamine di-(-O-hydroxyphenol-acetic acid), and
hydroxyethylene diamine trlacetic acid. A metal chelator may be
attached to the nucleotide probe during synthesis via a
hydrocarbon-amide linkage which may consist of several carbon
atoms. The specificity of the probe for the reaction site is
prescribed by the nucleotide sequence within which the metal
chelator or other cleavage moiety is attached. The moiety can be
incorporated into polydeoxyribonucleotides or polyribonucleotides
of any desired length and sequenced using routine phosphoramidite
or phosphotriester procedures.
[0061] One convenient synthesis of DNA-EDTA probes involves the
incorporation of a modified thymidine into an oligonucleotide by
chemical methods. This approach allows for automated synthesis and
affords-control-over the precise location of the EDTA moiety at any
thymidine position in the oligonucleotide strand, Felsenfeld, et
al., supra. Homopyrmidine oligonucleotides and their neutral or
cationic analogs may also be used in this invention. In the
examples which follow, oligonucleotides-EDTA probes of different
length, composition, and EDTA-thymidine position were synthesized
in this manner. Each of the DNA-EDTA probes described in the
Examples was purified by gel electrophoresis.
[0062] In a preferred embodiment of this invention, bifunctional
DNA-EDTA probes are used for recognition and cleaving of the target
double-stranded nucleic acid. These probes allow triple helix
formation at a discrete location to be mapped on large DNA using
gel electrophoresis. An important part of the present invention
involves the development of preferred assay conditions for
measuring formation and cleavage of the triple helix. This will be
discussed in more detail in Example 2 below. However, the preferred
general conditions for the cleavage reactions are as follows:
approximately 100 nM in bp radio labeled restriction fragment
(approximately 10,000 cpm), 25 nM tris/acetate, pH 7.0, 1 nM
spermine, (MY), 100 nM NaCl, 100 .mu.molar in vp sonicated,
deproteinized cath-thimus DNA, 20 volume-percent ethyleneglycol, 1
.mu.molar DNA-EDTA probe, 25 .mu.molar Fe(II) and 2 nM DTT. The
cleavage reactions were run for approximately 16 hours at
0-25.degree. C. These conditions may be varied without departing
from the scope of this invention.
[0063] As described in the examples below, the affinity cleaving
method utilizing DNA, EDTA and known in the art allows the effect
of reaction conditions, probe length, and single base mismatches on
triple-helix formation to be analyzed on high resolution sequencing
gels. Precise methods for quantitation and measurement and
determination of the presence and orientation of triple helices is
set out in more detail in the Examples below.
[0064] As will be seen in the Examples, the directional orientation
of the third strand as well as the identity of the grooves in
right-handed DNA-helix occupied by the bound DNA-EDTA probe can be
analyzed by high resolution gel electrophoresis (FIG. 2).
Additionally, the location of triple helices within large pieces of
DNA can be mapped by double strand breaks analyzed by nondenaturing
agarose gel electrophoresis.
[0065] Practice of this invention includes several processes: a
process for identifying and recognizing formation of triple helices
at a discrete highly specific site, a process for cleaving
double-stranded DNA through the formation of a triple helix, and
processes for diagnostic and chemotherapeutic use of the triple
helices of this invention.
[0066] Additionally, to achieve the objects and in accordance with
the purposes of the present invention, a process is disclosed which
results in recognition, chemotherapeutic use, and if desired,
cleavage of homopyrimidine-homopurine tracts within a large
double-stranded nucleic acid by triple helix formation at a
particular, predetermined site. This method comprises:
[0067] (a) hybridizing a specific homopyrimidine-homopurine tract
within a large double-stranded nucleic acid with a corresponding
polynucleotide hybridization probe, said nucleotide containing at
least one nucleoside to which is attached at least one of the
following:
[0068] (i) at least one detectable label molecule capable of being
detected upon the binding of said nucleotide to said tract,
[0069] (ii) at least one molecule adapted to cleave at least one
strand in said homopyrimidine-homopurine tract,
[0070] (iii) at least one molecule of a chemotherapeutic agent;
[0071] (b) permitting said hybridization to proceed to formation of
a triple-helix; and
[0072] (c) at least one of the following:
[0073] (i) detecting said label;
[0074] (ii) cleaving one or both strands of the nucleic acid;
or
[0075] (iii) permitting said chemotherapeutic agent to act.
[0076] More details on the methods for recognition and cleavage are
set forth in the Examples.
[0077] Synthesis and the preparation of necessary and desired
component parts of the probes of the present invention, and their
assembly is believed to be within the duties and tasks performed by
those with ordinary skill in the art and, as such, are capable of
being performed without undue experimentation.
[0078] The probes described in this invention generally range in
length from 11 to 15 bases. This size increase relative to
restriction endonucleases permits sequence recognition orders of
magnitude greater than that possible with restriction
endonucleases. From the disclosure and Examples herein, it is clear
that larger, as well as smaller, probes would work in similar
fashion as disclosed herein.
[0079] The probes of the present invention are not limited to the
production of sequence-specific cleavage of double-stranded DNA by
triple-helix formation, but may also be utilized as diagnostic
agents when a radio-isotope labeled, fluorescing, or otherwise
detectable metal ion is attached to the probe. The probes of the
present invention may also be used as target-specific
chemotherapeutics with the attachment of an "artificial" or natural
gene repressor or other effective agent to the polynucleotide.
[0080] It is to be understood that various other modifications will
be apparent to and can readily be made by those skilled in the art,
given the disclosure herein, without departing from the scope and
spirit of this invention. Accordingly, it is not intended that the
scope of the claims appended hereto be limited to the description
as set forth herein, but rather that the claims be construed as
encompassing all the features of patentable novelty which reside in
the present invention, including all features which would be
treated as equivalents thereof by those skilled in the art to which
this invention pertains.
[0081] Examples of the application of the probes of the present
invention, and representative protocols and processes for their use
appear below.
INDUSTRIAL APPLICABILITY
[0082] This invention finds usefulness in a range of medical and
laboratory applications. It greatly enhances current capabilities
for recognition of a small homopyrimidine-homopurine tract in a
large double-helical nucleic acid without requiring denaturation of
the double-helix or its digestion into smaller pieces before the
identification. The probes of the current invention and the method
for their use could become useful tools in chromosome analysis,
gene mapping and isolation, as well as other laboratory procedures.
Moreover, as molecular biology defines specific disease states at
the DNA level, a chemotherapeutic strategy of "artificial
repressors" based on triple-helix-forming nucleic acid analogs
becomes a possibility.
EXAMPLES
[0083] For the following examples, nine homopyrimidine DNA probes,
11-15 nucleotides in length, described in more detail below, each
containing a single thymidine with EDTA covalently attached at C-5
(labeled T*), were synthesized for binding and cleavage studies
with two different duplexed target DNA's.
[0084] Generally, unless specifically controverted below, the
following definitions apply: DMT refers to 4,4-dimethoxytrityl; DTT
refers to dithiothreitol; DNA-EDTA 1-9, the probes examined below,
refers to oligodeoxy-ribonucleotides with an EDTA-modified
thymidine at positions 1, 5, or 8; Spermine indicates
spermine-4-HCl (Aldrich, 98% pure) which was dissolved in water and
then pH adjusted with NaOH to 7.4; TBE-buffer includes 0.89 nM TRIS
(meaning TRIS(hydroxy-methyl)amino-methane), 0.89 mM Boric acid,
and 1 mM EDTA-disodium salt; Fe(II) refers to
Fe(NH.sub.4).sub.2(SO.sub.4).sub.2-6H.sub.2O. Aqueous solutions of
DTT and Fe(II) were freshly prepared before use.
EXAMPLE 1
Determination Of Orientation And Groove Location Of Hoogsteen
Strand Binding Watson-Crick DNA
[0085] Although it is widely believed that the two homopyrimidine
strands in triple-helical DNA or RNA are antiparallel a definite
proof is lacking in the published literature. A double-stranded DNA
was examined that contains (dA-dT).sub.15 as a target sequence
which could, in principle, bind the d(T).sub.15 probe-strand in
parallel or antiparallel orientation. A 30-base pair duplex of DNA
containing the tract (dA-dT).sub.15 was labeled separately at the
5' end of each strand. This was allowed to incubate with
(T).sub.15-EDTA probes 1 to 3 (shown in FIG. 3B) with the
thymidine-EDTA located at oligonucleotide positions 8, 5, and 1,
from the 5'-end, respectively. The .sup.32P-labeled DNA was
dissolved in buffer containing calf-thymus DNA, NaCl, TRIS,
spermine and ethylene glycol and was mixed with the DNA-EDTA-Fe(II)
probes, previously equilibrated with Fe(II) for 1 minute. After
incubation at 0.degree. C. for 10 minutes, the reactions were
initiated by addition of an aqueous solution of DTT, such that the
final concentrations were 10 mM TRIS/HCl (pH 7.4), 1 mM spermine,
100 mM NaCl, 40 vol-% ethylene glycol, 100 .mu.M (bp) of
calf-thymus DNA, 0.67 .mu.M DNA-EDTA probe, 25 .mu.M Fe(II) and 1
mM DTT(33). The cleavage reactions were allowed to proceed for 15
hours at 0.degree. C. and then stopped by freezing and
lyophilization. The resulting cleavage products were separated by
electrophoresis on a denaturing 20 percent polyacrylamide gel and
visualized by autoradiography (FIG. 3A).
[0086] FIG. 3A shows an autoradiogram of the 20 percent
Maxam-Gilbert sequencing gel. Lanes 1 to 5 contain 5'-End-labeled
d(A.sub.5T.sub.15G.sub.10); lanes 6 to 10 contain 5'-End-labeled
d(C.sub.10A.sub.15T.sub.5). The Maxam-Gilbert G+A sequencing
reactions used for lanes 1 and 6 are disclosed in Maniatis et al.,
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory (1982), and Maxam and Gilbert, Methods Enzymol. 65, 499
(1987). Controls in lanes 2 and 7 showing the two 5'-labeled 30-bp
DNA standards were obtained by treatment according to the cleavage
reactions in the absence of DN-EDTA-Fe probes. Lanes 3 to 5 and 8
to 10 are the DNA cleavage products in the presence of DNA-EDTA-Fe
probes 1 to 3, approximately 0.5 .mu.M (bp) 5' .sup.32P-labeled
DNA, (approximately 10,000 cpm), 10 mM TRIS/HCl, pH 7.4, 1 mM
spermine, 100 mM NaCl, 100 .mu.M (bp) sonicated, deproteinized
calf-thymus DNA, 40 percent by volume ethylene glycol, 0.67 .mu.M
probe, 25 .mu.M Fe(II) and 1 mM DTT; incubated for 15 hours at
0.degree. C. Lanes 3 and 8 contain DNA-EDTA-Fe 1, lanes 4 and 9
contain DNA-ET-Fe 2, lanes 5 and 10 are DNA-EDTA-Fe 3.
[0087] On the d(T).sub.15 strand of the Watson-Crick duplex, one
major cleavage site is observed for each DNA-EDTA probe 1-3 with
the maximum cleavage site shifted to the 5'-side of T*. The
location of the cleavage patterns on Watson-Crick DNA produced by
the probes 1-3 with respect to the position of T* reveal the
orientation of the DNA-EDTA probe on the duplex DNA (FIG. 3B).
[0088] FIG. 3B shows the (T).sub.15-EDTA probes 1-3 where T* is the
position of the thymidine-EDTA. Also shown are histograms of the
DNA cleavage patterns for these probes, derived by densitometry of
the autoradiogram shown in FIG. 3A (lanes 3-5 and 6-10). The
heights of the arrows represent the relative cleavage intensities
at the indicated bases. Arrows are shown if the cleavage intensity
at a particular nucleotide was greater than 5% when compared to
that of the nucleotide cleaved the most efficiently. The box in
each histogram indicates the double-stranded sequence which is
bound by the DNA-EDTA-Fe(II) probes 1-3. The Watson-Crick base-pair
to which T* is Hoogsteen hydrogen bonded in the triple-strand helix
is shaded.
[0089] As seen from the orientations, the homopyrimidine-EDTA
strand binds parallel to the homopurine and anti-parallel to the
homopyrimidine strands of Watson-Crick DNA. These observations rule
out strand displacement (D-loop) as the mode of binding. The
asymmetry of the cleavage patterns on opposite strands of DNA
reveals the identity of the groove in right handed DNA occupied by
EDTA-Fe. An asymmetric cleavage pattern with maximal cleavage
shifted to the 5' or 3' side on opposite strands corresponds to the
diffusible hydroxyl radical being generated in the major or minor
groove, respectively. The cleavage patterns shown in FIG. 3B reveal
that the DNA-EDTA-Fe(II) probe is located in the major groove of
the Watson-Crick DNA.
[0090] Homopyrimidine probes 1 and 2 which bear the EDTA at an
internal base position cleave exclusively the homopyrimidine strand
of the target DNA. A model of the triple-helix between these
homopyrimidine-EDTA-Fe(II) probes and the double-helical DNA (not
shown) reveals that the homopurine Watson-Crick strand in the
triple-helix is protected from the hydroxyl radical by the
sugar-phosphate backbones of the Hoogsteen-paired strand. In effect
there are now three grooves in the triple helix and EDTA-Fe is
exposed to only one groove (FIG. 1). The nucleotides 3 to 4 bases
on the 5'-side of T* in the right-handed triple helix are proximal
to the EDTA-Fe(II) and are therefore expected to be cleaved most
efficiently. DNA-EDTA-Fe(II)3, which carries the cleaving moiety at
the 5'-end, should form a triplex with no flanking nucleotides on
the 5'-side of T*. A homopyrimidine probe with the cleaving
function at the 5'-end should generate cleavage on both strands.
Indeed, the d(T).sub.15-EDTA-Fe(II)3, carrying the ETA at the 5'
end, cleaves both strands of the target duplex DNA (FIG. 3B).
EXAMPLE 2
Specific Cleavage Of A DNA Restriction Fragment And Determination
of Assay Conditions
[0091] This example illustrates two important aspects of the
present invention. In the first part, it is shown that an
unsymmetrical mixed probe can go into and be precisely located
within any large target homopyrimidine-homopurine tract, for
example a target sized a factor of 50 over the probe. In this
example, restriction fragments with mixed bases are used which vary
in length, have single mismatches with the known target, and vary
in the positions of the EDTA (FIG. 4B).
[0092] In the second part preferred assay conditions are
determined, as well as the effect of varying those conditions with
probes of 11-15 bp in length.
[0093] Specific Cleavage Of A DNA Restriction Fragment. Cleavage by
triple helix formation with DNA-EDTA-Fe(II) probes 4-9 was examined
on a restriction fragment 628 base pairs in length that contained
the sequence d(AAAAAGAGAGAGAGA). This sequence was obtained from
plasmid pDMAG10 which was a gift from D. Mendel, who constructed it
by inserting the d(AAAAAGAGAGAGAGA) containing duplex in the large
BamHI-HindIII restriction fragment of pBR322 (Mendel and Dervan,
Proc. Natl. Acad. Sci. USA., 84, 910 (1987)). A single site labeled
628 bp EcoRI-BgII restriction fragment containing the target
sequence was obtained by linearizing pDMAG10 with EcoRI, labeling
with .sup.32P (Van Dyke and Dervan, Science, 225, 1122 (1984)), and
cleaving with BgII. This sequence represents a mixed homopurine
target which is located 47 nucleotides from the 3'-(and 5') P-label
of the DNA fragment. The concentration of the single stranded
oligodeoxynucleotides were determined using the following epsilon
values (260 mm) for each base: 15400(A), 11700(G), 7300(C) and
8800(T).
[0094] FIG. 4A shows an autoradiogram of the 8 percent
Maxam-Gilbert high-resolution polyacrylamide sequencing gel run on
probes 4-9. The EcoRI/BgII restriction fragment of plasmid pDMAG10
is labeled at the 3' end with .sup.32P. The Maxam-Gilbert G+A
sequencing reactions described in the previous example were used
here for lane 20. In general, the cleavage reactions were carried
out as follows: a mixture of DNA-EDTA probe (1 .mu.M) and Fe(II)
(25 .mu.M) was combined with the .sup.32P-labeled restriction
fragment (approximately 100 .mu.M (bp)) in a solution of
calf-thymus DNA (100 .mu.M (bp)), NaCl (100 mM), TRIS/acetate, pH
7.4 (25 mM TRIS), spermine (1 mM) and ethyleneglycol (20 vol-%) and
incubated for 10 minutes at 0.degree. C. Cleavage reactions were
initiated by addition of 2 mM DTT, proceeded 16 hours at 0.degree.
C. to 25.degree. C., and stopped by precipitation with ethanol. The
reaction products were analyzed on a high resolution polyacrylamide
gel. For each lane the parameters differing from these general
conditions are given below. (lane 21): Control, minus DNA-EDTA;
(lanes 22, 27 and 32): 1 .mu.M DNA-EDTA 6; (lanes 23, 28 and 33): 1
.mu.M DNA-EDTA 5; (lanes 24, 29 and 34): 1 .mu.M DNA-EDTA 4; (lanes
25, 30 and 35): 1 .mu.M DNA-EDTA 7 (Hoogsteen-type TG-mismatch);
(lanes 26, 31 and 36): 1 .mu.M DNA-EDTA 8 (Hoogsteen-type
CA-mismatch). The reactions were run for 16 hours at 0.degree. C.
(lanes 22 to 26), 12.5.degree. C. (lanes 27 to 31) and 25.degree.
C. (lanes 21 and 32 to 36) respectively. Electrophoresis on a 5
percent polyacrylamide gel separated the radiolabeled 628 bp
fragment from other digest products.
[0095] FIG. 4B shows the sequence of DNA-EDTA probes 4-9 where T*
is the position of the thymidine-EDTA. Histograms shown in this
figure of the DNA-cleavage patterns were determined by densitometry
of the autoradiogram from the cleavage of the 628 bp restriction
fragment with DNA-EDTA probes 4 and 9.
[0096] On the 3' end-labeled DNA-strand, carrying the
homopyrimidine target sequence, DNA-EDTA-Fe(II) 4 and 9 both
produce sequence specific cleavage patterns shifted to the 5' side
of the T* position (FIG. 4B) consistent with major groove binding.
The efficiency of the sequence specific cleavage of the DNA
restriction fragment by DNA-EDTA-Fe(II) 4 is dependent on spermine
and/or Co(NH.sub.3).sub.6.sup.3+-concentrations, ethylene glycol,
pH and probe concentration (FIG. 5A).
[0097] The cleavage efficiency of probes 4-6 which differ in length
(15, 13 and 11 nucleotides) and probes 7 and 8 which differ in
sequence (each contain one Hoogsteen base mismatch in the triple
helix complex) were examined under identical conditions at
different temperatures. Identical cleavage patterns are observed
for the DNA-EDTA-Fe(II) probes 4-8. At 0.degree. C., probes 4-6
which differ in length but have in common T* at position each
produce a cleavage pattern of the same intensity. At 25.degree. C.
probe 6 which is 11 nucleotides in length cleaves the target DNA 3
times less efficiently than probes 5 or 4 which are 13 and 15
nucleotides in length, respectively. DNA-EDTA 7 and 8 which contain
a single base mismatch at position 10 and 11 generate cleavage
patterns of reduced intensity and is temperature sensitive.
Compared to DNA-EDTA probe 4, the relative cleavage efficiency
decreases for the single base mismatch probes 7 from 0.4 (at
0.degree. C.) to 0.08 (25.degree. C.) and 8 from 0.5 (at 0.degree.
C.) to 0.13 (25.degree. C.) (FIG. 5B).
[0098] Optimization Of Assay Conditions. In this example, the
effect of added cations, organic solvents, pH, temperature, probe
length and sequence homology were studied. The results, detailed
below, are summarized in FIG. 5.
[0099] FIG. 5A shows a bar graph presenting the absolute cleavage
efficiencies obtained with DNA-EDTA-Fe 4 under various conditions.
The values were determined by cutting out the corresponding pieces
of the dried gel and measuring their radioactivity by scintillation
counting. The numbers given are calculated by dividing the
radioactivity of the cleavage site (sum of 5 most efficiently
cleaved nucleotides) with the total radioactivity obtained from the
uncleaved fragment, the cleavage site and the background, which is
corrected for the background that resulted from the untreated 628
bp fragment. The remaining values were assigned by correlation of
absolute with relative cleavage efficiencies determined by
densitometry of the autoradiogram. FIG. 5(B) shows a bar graph
presenting the relative cleavage efficiencies (sum of 6 most
efficiently cleaved nucleotides) obtained with DNA-EDTA-Fe 4-8
(FIG. 4A) at three temperatures as determined by densitometry. The
data is reproducible within .+-.10% of reported values.
[0100] Importance Of Added Cations. The importance of added cations
for formation of triple-stranded DNA or RNA has been known since
the discovery of those structures. To bind double-helical DNA, the
DNA-EDTA-Fe(II) probe must overcome the repulsion between two
anionic chains of the Watson-Crick duplex and its own negatively
charged phosphodiester backbone. One way to achieve this is to use
multivalent cations (Michelson, et al., Prog. Nucl. Acid. Res. Mol.
Biol. 6, 83 (1967) and Felsenfeld and Miles, supra.). The naturally
occuring polyamines and their derivatives are known to stabilize
double-and triple-helical structures of nucleic acids. (Blaser and
Gabbay, Biopolymers 6, 243 (1968)). We find preferred cleaving
efficiencies for DNA-EDTA-Fe(II) 4 in the presence of mM
concentrations of spermine or Co(NH.sub.3).sub.6.sup.3+. No
cleavage occurs in the absence of spermine or
Co(NH.sub.3).sub.6.sup.3+ which demonstrates the importance of
these or similar cations for triple-helix formation (FIG. 5A).
Spermine appears to be ideal for the stabilization of the
triple-stranded complex with DNA-EDA-Fe(II) probes. It efficiently
neutralizes the negative charges of the sugar-phosphate backbones
and does not compete with the Fe(II) for the EDTA-moiety. No
cleavage is observed if MgCl.sub.2 or CaCl.sub.2 (up to 8 mM) are
substituted for spermine which could also be due to competition
with Fe(II) for the metal chelator EDTA (Hertzberg and Dervan,
Biochemistry 23, 3934 (1984).
[0101] Role Of Organic Solvents. According to x-ray fiber
diffraction studies, the three strands of a triple helix occur in a
A'RNA-like conformation (Arnott, et al., Nucleic Acids Res. 3, 2459
(1976)). A conformational transition may be necessary to allow the
binding of the DNA-EDTA-Fe(II) probe. It is established that a B to
A conformational change takes place on lowering the relative
humidity. This transformation is dependent on the ratio of (A+T) to
(G+C) and can be achieved by the addition of a variety of organic
solvents to the DNA aqueous solution. The increase in organic
solvent concentration should favor the B to A conformational
transition and suggest that triple helices should form more readily
(Saenger, Principles of Nucleic Acid Structure, edited by C. R.
Cantor, Springer-Verlag, New York, Inc. (1984)). As a result, the
cleavage due to the DNA-EDTA probe should increase correspondingly.
We find that the efficiency of oligonucleotide duplex cleavage by
(T).sub.15-EDTA-Fe(II) 1-3 is increased by a factor of 10 upon
addition of ethyleneglycol (40 percent by volume). Other organic
solvents such as methanol, ethanol, dioxane or DMF give rise to
similar behavior. In the presence of ethylene-glycol,
DNA-EDTA-Fe(II) probes provide cleavage patterns without detectable
background, a result that may be due to radical scavenging by this
solvent.
[0102] Curiously, the mixed T and C homopyrimidine EDTA-Fe(II) 4
demonstrates different behavior. The addition of 20 vol-%
ethyleneglycol is not necessary and does not increase the cleavage
efficiency as found in the (T).sub.15 case. One explanation for
this difference is that a mixed T, C probe may have a higher
affinity than the oligo T probe to the corresponding Watson-Crick
target sequence due to the protonated cytosines required to form
the Hoogsteen-hydrogen bonds in the triple helix. The alternative
explanation is that the target Watson-Crick sequences differ in
conformation and one may be more A like than the other.
[0103] The pH-Dependence Of Cleavage Efficiency. Mixed
homopyrimidine DNA-EDTA-Fe(II) 4 cleaves the target-DNA over a
relatively narrow range of pH-values producing the maximum cleavage
at pH 7.0 (FIG. 5A)(the pH values are not corrected for temperature
or different ethyleneglycol percentage and are given for the
tenfold concentrated buffer solutions at 25.degree. C.). This
behavior could be caused by two independent properties of the
oligonucleotide-EDTA probes. On one hand, triplex formation
requires protonation of cytosines at N-3 in the third strand to
enable the Hoogsteen hydrogen bonds between DNA-EDTA-Fe(II) probes
and the target Watson-Crick DNA. It was previously demonstrated
that complexes of triple-helical nucleic acids, containing
cytosines in the homopyrimidine strands, are stable in slightly
acidic to neutral solutions and start to dissociate on increasing
pH (Lipsett, J. Biol. Chem. 239, 1256 (1964); Morgan and Wells, J.
Mol. Biol. 37, 63 (1969)). Therefore it seems not unreasonable that
the DNA-EDTA-Fe(II) probes do not bind Watson-Crick DNA in slightly
basic solutions (pH.gtoreq.8) and consequently do not produce
cleavage under these conditions. On the other hand, studies with
methidiumpropyl-EDTA-Fe indicate that the cleavage efficiency of
EDTA-Fe decreases sharply below pH 7 (Hertzberg and Dervan, supra),
presumably due to either partial protonation of the EDTA and the
resulting loss of Fe(II) or some pH-dependence of the cleavage
reaction. Based on known EDTA cleavage chemistry, it is anticipated
that at slightly acidic pH-values, DNA-EDTA-Fe(II) probes do not
produce efficient cleavage. In data not shown, footprinting
experiments confirm that the triple helix is forming at acidic pH
values.
[0104] Influence Of Probe Length, Temperature, and Sequence
Homology. At 1 .mu.M concentration the DNA- EDTA probe approaches
the maximum cleavage efficiency on the 628 bp restriction fragment
(FIG. 5A). We chose DNA-EDTA probes 15 nucleotides in length for
our initial studies to attain reasonable binding affinities at the
double-helical target sequence (Cassani and Bollum, Biochemistry 8,
3928 (1969); Raae and Kleppe, Biochemistry 17, 2939 (1978)). Having
determined the preferred cleavage conditions for DNA-EDTA-Fe(II) 4,
we focused on the size dependence for DNA-EDTA-Fe(II) probes to
form a triple-helix complex wash the Watson-Crick DNA. DNA-
EDTA-Fe) probes 5 and 6, which are 13 and 11 nucleotides in size,
produce cleavage patterns of similar intensities at 0.degree. C.,
indicating that homopurine-homopyrimidine sequences as short as 11
nucleotides can specifically bind the 628 bp restriction fragment.
The influence of oligonuclectide length becomes more apparent if
the cleavage reactions are allowed to proceed at higher
temperatures. DNA-EDTA 4 and 5 cleave the target duplex DNA at
25.degree. C. with approximately the same efficiency, whereas the
relative intensity of the cleavage pattern produced by the shorter
6 becomes significantly weaker (FIG. 4A, 5B).
[0105] In order to test the importance of sequence homology for
triple helix formation and cleavage, two probes, DNA-EDTA-Fe(II) 7
and 8, were synthesized that contained single base mismatches
compared to DNA-EDTA-Fe(II) 4 but had in common the location of T*
at position 5. When bound to the double-helical target sequence,
probes 7 and 8 should give rise to one mismatched base-triplet with
respect to the Hoogsteen hydrogen bonding. The mismatching bases in
the probe-strands were chosen so that the corresponding tautomeric
or protonated structures of the mismatching pyrimidine base could
still allow the formation of isomorphous base triplets. Compared to
DNA-EDTA-Fe(II) 4, both single mismatch probes 7 and 8 generate
weaker cleavage patterns at 0.degree. C. and the difference becomes
more apparent for the cleavage patterns produced at 25.degree. C.
(FIG. 5B). Probes 7 and 8 cleave the target DNA less efficiently
than the corresponding homologous DNA-EDTA-Fe(II) 4. This result
indicates that a single base-mismatch in a DNA-EDTA-Fe(II) probe,
15 nucleotides in length, can lower the cleavage efficiency by at
least a factor of 10. Clearly, the sequence specific recognition of
large double-helical DNA by DNA-EDTA-Fe(II) probes is sensitive to
single base mismatches indicating the importance of the correct
homopyrimidine probe sequence for the formation of a
triple-stranded complex with the target-DNA.
EXAMPLE 3
Site Specific Double-Strand Cleavage Of Plasmid DNA
[0106] The ability of DNA-EDTA-Fe(II) 9 to cause double strand
breaks at a homopurine-homopyrimidine insert in large DNA is
presented in FIG. 6A. This figure shows double-strand cleavage of
plasmid DNA analyzed on a nondenaturing 0.9% agarose gel. The
plasmid pDMAG10 (Mendel and Dervan, Proc. Natl. Acad. Sci. USA 84,
910 (1987)) was digested with StyI restriction endonuclease to
produce a linear DNA fragment 4.06 kb in size which contains the
homopurine site d(A.sub.5(AG).sub.5) located 1.0 kb upstream from
the restriction site. This affords heterogenous overhangs and each
end could be labeled separately using either a-.sup.32P-ATP or
a-.sup.32-P-TTP according to standard procedures. Lanes 1-3 of FIG.
6A shows plasmid pDMAG10 linearized with StyI and labeled at the
downstream end of the restriction site with a-.sup.32P-ATP. Lanes
4-6 show the same plasmid with the other end labeled with
a-.sup.32P-TTP.
[0107] The .sup.32P-end-labeled DNA was allowed to incubate with
DNA-EDTA-Fe(II) 9 (5 .mu.M) for 10 minutes at 0.degree. C. as
previously described and the cleavage reaction was initiated by the
addition of DTT (2 mM) and run at 0.degree. C. for 16 hours.
Cleavage conditions included .sup.32P labeled DNA plasmid, 100 mM
NaCl, 1 mM spermine, 25 mM TRIS/acetate pH 7.0, 100 .mu.M (bp)
sonicated, deproteinized calf thymus DNA, 5 .mu.M DNA-EDTA-Fe(II)
9, 25 .mu.M Fe(II) and 2 mM DTT. Lanes 1 and 4 are controls
containing no DA-EDTA-Fe(II) 9. Lanes 2 and 5 are DNA size markers
obtained by digestion of StyI linearized pDMAG10 with EcoRI, PvuI,
SalI (both ends labeled), and Xmn I labeled with a-.sup.32P-TTP):
4058 (undigested DNA), 3338, 2994, 2368, 1690, 1460, 1064, and 720.
Lanes 3 and 6 are DNA-EDTA-Fe(II) probe 9 at 5 .mu.M added.
[0108] Separation of the cleavage products by agarose gel
electrophoresis followed by autoradiography reveals only one major
cleavage site producing two DNA fragments, 3.04 and 1.02 kb in size
as determined by comparison with comigrating DNA size markers (FIG.
6A, lanes 3 and 6).
[0109] FIG. 6B (left) shows the course resolution cleavage pattern
from gel 6A. FIG. 6B (middle) depicts a simplified model of the
triple helix complex with the Hoogsteen bound DNA-EDTA-Fe(II) 9 at
one unique site within 4.06 kb of plasmid DNA. The high resolution
cleavage pattern at that site is shown in FIG. 4B.
[0110] This example demonstrates that the probes of this invention
can go into a very large piece of double-stranded DNA and precisely
locate a double-strand cleavage. This technique has tremendous
potential for mapping chromosomes. The work reported here
demonstrates that homopyrimidine-homopurine double-helical tracts
can be recognized within large DNA by triple helix formation under
physiological conditions.
* * * * *