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Claims  |
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What is claimed is:
1. A method for detecting a particular polynucleotide sequence in a test
medium containing single stranded nucleic acids, comprising the steps of:
(a) combining the test medium with (i) a nucleic acid probe comprising at
least one single stranded base sequence which is substantially
complementary to hybridization between the sequence to be detected and the
complementary sequence in the probe, and (ii) a nucleic acid intercalator
capable of binding to double stranded nucleic acid in the form of
intercalation complexes, and
(b) detecting hydridized probe by adding an antibody, or a fragment
thereof, capable of binding with intercalation complexes in the
hybridization product resulting from step (a), and determining the
antibody or fragment thereof which becomes bound to such complexes.
2. The method of claim 1 wherein the intercalator is combined with the test
medium as a separate, free compound and noncovalently binds with double
stranded nucleic acid to form intercalation complexes.
3. The method of claim 1 wherein the intercalator is chemically linked to
the probe in the single stranded complementary region of the probe,
whereby upon hybridization said intercalation complexes are formed in such
region.
4. The method of claim 1 wherein the antibody or fragment thereof is
labeled with a detectable chemical group.
5. The method of claim 4 wherein the detectable chemical group is an
enzymatically active group, a fluorescer, a chromophore, a luminescer, a
specifically bindable ligand, or a radioisotope.
6. The method of claim 1 according to a solid phase hybridization technique
wherein one of the probe and the single stranded nucleic acids from the
test medium is immobilized on a solid support and wherein the antibody
associated with the solid support is determined.
7. The method of claim 1 according to a solid phase sandwich hybridization
technique wherein the test medium is combined with first and second
nucleic acid probes each comprising at least one single stranded base
sequence which is substantially complementary to a mutually exclusive
portion of the sequence to be detected and wherein one of the probes is
immobilized on a solid support.
8. The method of claim 1 according to a solution phase hybridization
technique wherein the probe comprises a binding site for a binding
substance and wherein after the hybridization step there is added an
immobilized form of such binding substance.
9. The method of claim 8 wherein the probe comprises a biotin or hapten
moiety and the binding substance is avidin or an anti-hapten antibody,
respectively.
10. The method of claim 1 wherein the probe additionally comprises a double
stranded portion which, upon addition of the intercalator in step(a) as a
separate, free compound, forms said intercalation complexes.
11. The method of claim 1 wherein the intercalator is selected from
acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines
and quinolines.
12. A solid-phase hybridization method for detecting a particular
polynucleotide sequence in a liquid test medium containing single stranded
nucleic acids, comprising the steps of:
(a) forming a reaction mixture by contacting the liquid test medium with a
nucleic acid probe comprising at least one single stranded base sequence
which is substantially complementary to the sequence to be detected, one
of the probe and the single stranded nucleic acids from the test medium
being immobilized on a solid support, such contact being performed under
conditions favorable to hybridization between the sequence to be detected
and the complementary probe sequence,
(b) contacting the solid support carrying resulting immobilized duplexes
with a nucleic acid intercalator and an antibody, or fragment thereof,
capable of binding with intercalation complexes comprising double stranded
nucleic acid complexed with the intercalator,
(c) separating the solid support carrying resulting immobilized antibody or
fragment thereof from the remainder of the reaction mixture, and
(d) determining the separated antibody or fragment thereof on the solid
support as an indication of the presence of the sequence to be detected in
the liquid test medium.
13. The method of claim 12 wherein prior to step (b) the solid support
carrying immobilized duplexes resulting from step(a) is separated from the
remainder of the reaction mixture.
14. The method of claim 12 wherein the antibody or fragment thereof is
labeled with a detectable chemical group and wherein in step(d) such
detectable group is measured on the solid support as an indication of the
presence of the sequence to be detected.
15. The method of claim 12 wherein the probe also comprises at least one
double stranded region which, upon addition of the intercalator in
step(b), forms said intercalation complexes capable of being bound by the
antibody or fragment thereof.
16. The method of claim 12 wherein the liquid test medium comprises a
biological sample which has been subjected to conditions to release and
denature nucleic acids present therein.
17. A solid-phase hybridization method for detecting a particular
polynucleotide sequence in a liquid test medium containing single stranded
nucleic acids, comprising the steps of:
(a) forming a reaction mixture by contacting the liquid test medium with a
nucleic acid probe, the probe comprising at least one single stranded base
sequence substantially complementary to the sequence to be detected and
the probe being chemically linked to a nucleic acid intercalator in the
single stranded complementary region of the probe such that duplex
formation in such region bearing the linked intercalator results in the
formation of intercalation complexes, one of the probe and the single
stranded nucleic acids from the test medium being immobilized on a solid
support, such contact being performed under conditions flavorable to
hybridization between the sequence to be detected and the complementary
probe sequence,
(b) adding to the reaction mixture an antibody, or a fragment thereof,
capable of binding with intercalation complexes comprising double stranded
nucleic acid complexed with the intercalator,
(c) separating from the remainder of the reaction mixture, the solid
support carrying resulting immobilized antibody or fragment thereof bound
to intercalation complexes formed between the intercalator-linked probe
and the sequence to be detected, and
(d) determining the separated antibody or fragment thereof on the solid
support as an indication of the presence of the sequence to be detected in
the liquid test medium.
18. The method of claim 17 wherein the antibody or fragment thereof is
labeled with a detectable chemical group and wherein in step(d) such
detectable group is measured on the solid support as an indication of the
presence of the sequence to be detected.
19. The method of claim 17 wherein the liquid test medium comprises a
biological sample which has been subjected to conditions to release and
denature nucleic acids present therein.
20. A solution-phase hybridization method for detecting a particular
polynucleotide sequence in a liquid test medium containing single stranded
nucleic acids, comprising the steps of:
(a) forming a reaction mixture by contacting the liquid test medium with a
nucleic acid probe comprising at least one single stranded base sequence
which is substantially complementary to the sequence to be detected, the
probe comprising a binding site for a binding substance, such contact
being performed under conditions favorable to hybridization between the
sequence to be detected and the complementary probe sequence,
(b) adding to the reaction mixture simultaneously or in separate steps (i)
a nucleic acid intercalator, (ii) an antibody, or fragment thereof,
capable of binding with intercalation complexes comprising double stranded
nucleic acid complexed with the intercalator, and (iii) an immobilized
form of a binding substance for the probe,
(c) separating the resulting immobilized phase comprising antibody, or
fragment thereof, bound to immobilized intercalation complexes from the
remainder of the reaction mixture, and
(d) determining the separated immobilized antibody, or fragment thereof, as
an indication of the presence of the sequence to be detected in the liquid
test medium.
21. The method of claim 20 wherein the antibody or fragment thereof is
labeled with a detectable chemical group and wherein in step(d) such
detectable group is measured in the immobilized phase as an indication of
the presence of the sequence to be detected.
22. The method of claim 20 wherein the probe comprises a biotin or hapten
moiety and the immobilized binding substance is avidin or an anti-hapten
antibody, respectively.
23. The method of claim 20 wherein the liquid test medium comprises a
biological sample which has been subjected to conditions to release and
denature nucleic acids present therein.
24. A test kit for detecting a particular polynucleotide sequence in a test
medium, comprising in a packaged combination:
(1) a nucleic acid probe comprising at least one single stranded base
sequence which is substantially complementary to the sequence to be
detected,
(2) a nucleic acid intercalator, and
(3) an antibody, or a fragment thereof, capable of binding with
intercalation complexes comprising hybridized double stranded nucleic acid
complexed with the intercalator.
25. The test kit of claim 24 wherein the antibody or fragment thereof is
labeled with a detectable chemical group.
26. The test kit of claim 25 wherein the detectable chemical group is an
enzymatically active group, a fluorescer, a chromophore, a luminescer, a
specifically bindable ligand, or a radioisotope.
27. The test kit of claim 25 wherein the detectable chemical group is an
enzyme.
28. The test kit of claim 24 which additionally comprises a solid support
for immobilizing single stranded nucleic acids from the test medium.
29. The test kit of claim 24 wherein the probe is immobilized on a solid
support.
30. The test kit of claim 24 wherein the probe comprises a binding site for
a binding substance and the reagent system additionally comprises an
immobilized form of such binding substance.
31. The test kit of claim 30 wherein the probe comprises a biotin or hapten
moiety and the immobilized binding substance is avidin or an anti-hapten
antibody, respectively.
32. The test kit of claim 24 wherein the intercalator is a separate, free
compound, substantially uncomplexed with nucleic acids.
33. The test kit of claim 32 wherein the probe additionally comprises at
least one double stranded region.
34. The test kit of claim 24 wherein the intercalator is chemically linked
to a single stranded region of the probe such that duplex formation in
such region results in the formation of intercalation complexes.
35. The test kit of claim 24 for use in a sandwich hybridization format
which comprises a second nucleic acid probe, the first and second probes
respectively comprising at least one single stranded base sequence which
is substantially complementary to a mutually exclusive portion of the
sequence to be detected.
36. The test kit of claim 35 wherein one of the probes is immobilized.
37. The test kit of claim 36 wherein a single stranded region of the probe
that is not immobilized is chemically linked to the intercalator such that
duplex formation in such region results in the formation of intercalation
complexes.
38. The test kit of claim 24 wherein the intercalator is selected from
acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines
and quinolines.
39. The test kit of claim 24 which additionally comprises a denaturation
agent capable of converting double stranded nucleic acids in a test sample
into single stranded form.
40. A method for detecting double stranded nucleic acid in a liquid medium,
comprising the steps of:
(a) adding to said medium (i) a nucleic acid intercalator and (ii) an
antibody, or a fragment thereof, capable of binding with intercalation
complexes comprising double stranded nucleic acid complexed with the
intercalator, and
(b) detecting the binding of said antibody or fragment thereof to said
complex.
41. The method of claim 40 wherein the antibody or fragment thereof is
labeled with a detectable chemical group.
42. The method of claim 41 wherein the detectable chemical group is an
enzymatically active group, a fluorescer, a chromophore, a luminescer, a
specifically bindable ligand, or a radioisotope.
43. The method of claim 40 wherein the intercalator is selected from
acridine dyes, phenanthridines, phenazines, furocoumarins, phenothiazines
and quinolines. |
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Claims |
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Description |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to nucleic acid hybridization assay methods and
reagent systems for detecting specific polynucleotide sequences. The
principle of nucleic acid hybridization assays was developed by workers in
the recombinant DNA field as a means for determining and isolating
particular polynucleotide base sequences of interest. It was found that
single stranded nucleic acids, e.g., DNA and RNA, such as obtained by
denaturing their double stranded forms, will hybridize or recombine under
appropriate conditions with complementary single stranded nucleic acids.
By labeling such complementary probe nucleic acids with some readily
detectable chemical group, it was then made possible to detect the
presence of any polynucleotide sequence of interest in a test medium
containing sample nucleic acids in single stranded form.
In addition to the recombinant DNA field, the analytical hybridization
technique can be applied to the detection of polynucleotides of importance
in the fields of human and veterinary medicine, agriculture, and food
science, among others. In particular, the technique can be used to detect
and identify etiological agents such as bacteria and viruses, to screen
bacteria for antibiotic resistance, to aid in the diagnosis of genetic
disorders such as sickle cell anemia and thalassemia, and to detect
cancerous cells. A general review of the technique and its present and
future significance is provided in Biotechnology (August 1983), pp.
471-478.
2. Description of the Prior Art
The state-of-the-art nucleic acid hybridization assay technqiues involve
chemical modification of either the probe nucleic acid or sample nucleic
acids for the purpose of labeling and detection. The necessity of
chemically modifying nucleic acids severely limits the practical use of
the technique since it requires the large-scale preparation of labeled
probes involving complicated and expensive synthetic and purification
procedures or the in situ synthesis of labeled sample nucleic acids by the
analytical user. In particular, the resulting labeled polynucleotide must
retain the ability to hybridize efficiently with its complementary sample
or probe sequence. Such a requirement severely limits the availability of
useful synthetic approaches to label modification of polynucleotides
intended for use in hybridization assays.
The early hybridization techniques involved the use of radioactive labels
such as .sup.3 H, .sup.32 P, and .sup.125 I. Labeled probes are
synthesized enzymatically from radiolabeled nucleotides and a
polynucleotide by such techniques as nick translation, end labeling,
second strand synthesis, reverse transcription, and transcription. Thus,
an additional requirement of such enzymatic methods is that the modified
or labeled nucleotides must serve as effective substrates for the
polymerase enzymes involved in the assembly of the labeled polynucleotide.
Direct chemical modification of the polynucleotide is also possible,
however, such a method is quite inefficient in incorporating labels into
the polynucleotide and can affect the ability of the polynucleotide to
undergo hybridization.
Because of the handling and storage disadvantages of radiolabeled
materials, there has been considerable continuing efforts to develop
useful nonradioisotopic labeling approaches. Such labels have included
light emitting molecules such as fluorescers and chemiluminescers, and
ligand molecules which are capable of being specifically bound by
counterpart binders which are in turn labeled with detectable chemical
groups such as fluorescers and enzymes. Examples of ligand labels are
haptens, which are specifically bound by antibodies, and other small
molecules for which specific binding proteins exist, e.g., biotin which is
bound by avidin.
British Pat. No. 2,019,408 describes polynucleotide probes which are
labeled with biotin through cytochrome C linking groups and which are then
detectable by enzyme-labeled avidin. An alternative approach to labeling
probes with low molecular weight ligands such as biotin is described in
European Pat. Appln. No. 63,879. In this technique,
5-allylamine-deoxyuridine triphosphate (dUTP) derivatives are condensed
with the desired ligand label and the thus modified nucleotide is
incorporated by standard enzymatic methods into the desired probe. The use
of light emitting labels is suggested by European Pat. Appln. Nos. 70,685
and 70,687. Other representatives of the patent literature pertaining to
hybridization assays are U.S. Pat. Nos. 4,302,204 concerning the use of
certain water soluble polysaccharides to accelerate hybridization on a
solid-phase; 4,358,535 concerning the detection of pathogens in clinical
samples; and 4,395,486 concerning the detection of sickle cell anemia
trait using a synthetic oligonucleotide probe.
Techniques for detecting directly the polynucleotide duplex formed as the
product of hybridization between the sample and probe polynucleotides, and
thereby dispensing with the chemical labeling of one or the other
polynucleotide, have been generally unsuccessful. Attempts to generate
antibodies which will selectively bind double stranded DNA/DNA hybrids
over single stranded DNA have failed [Parker and Halloran, "Nucleic Acids
in Immunology", ed. Plescia and Braun, Springer-Verlag, NY(1969) pp. 18 et
seq]. Some success has been achieved in generating antibodies that will
bind RNA/DNA mixed hybrids and have low affinity for the single stranded
polynucleotides [Rudkin and Stollar, Nature 265:472(1977); Stuart et al,
PNAS(USA)78:3751(1981); Reddy and Sofer, Biochem. Biophys. Res. Commun.
103:959(1981); and Nakazato, Biochem. 19:2835(1980)], however, the
sensitivity of these methods has not reached the levels required for
clinical hybridization tests and one would have to use RNA probes which
are well known to be quite unstable.
Accordingly, there is an established need for a technique for detecting
hybridization without requiring chemical modification of polynucleotides
or involving a labeling method of relative simplicity. Further, such
technique should enable the use of a variety of labels, particularly of
the nonradioisotopic type. A nucleic acid hybridization assay method and
reagent system having these and other advantages are principal objectives
of the present invention.
U.S. Pat. No. 4,257,774 describes a method for detecting various compounds
that interact with nucleic acids, particularly compounds suspected as
possible mutagens or carcinogens, by measuring the ability of such
compounds to inhibit the binding of intercalators such as acridine orange
to nucleic acids. Poirier, M.C. et al (1982) PNAS 79:6443-6447 describe
the preparation of a monoclonal antibody selective for certain
cis-platinum/double stranded DNA complexes over the free cis-platinum
compound and double stranded DNA.
SUMMARY OF THE INVENTION
It has now been found that hybridization which occurs between sample
nucleic acid and the probe in nucleic acid hybridization assays can be
detected advantageously by means of an antibody, or an appropriate binding
fragment thereof, capable of binding with intercalation complexes formed
in association with hybridized probe. In essence, a particular
polynucleotide sequence is detected in a test medium containing single
stranded nucleic acids by forming a hybridization aggregate or product
comprising hybridized probe and a nucleic acid intercalator compound bound
to double stranded nucleic acid in the form of intercalation complexes.
The antibody or fragment thereof is then used to detect intercalation
complexes in the hybridization aggregate.
The use of nucleic acid hybridization as an analytical tool is based
fundamentally on the double stranded, duplex structure of DNA. The
hydrogen bonds between the purine and pyrimidine bases of the respective
strands in double stranded DNA can be reversibly broken. The two
complementary single strands of DNA resulting from this melting or
denaturation of DNA will associate (also referred to as reannealing or
hybridization) to reform the duplexed structure. As is now well known in
the art, contact of a first single stranded nucleic acid, either DNA or
RNA, which comprises a base sequence sufficiently complementary to (i.e.,
"homologous with") a second single stranded nucleic acid under appropriate
solution conditions, will result in the formation of DNA/DNA, RNA/DNA, or
RNA/RNA hybrids, as the case may be.
The present invention enables the detection of formed hybrids by inducing
an immunogenic modification of double stranded nucleic acid in the region
of hybridization or in flanking regions. The resulting product can then be
detected by conventional assay schemes based on the binding of specific
antibody to the epitopes or antigenic determinants formed on the
hybridization product. The requisite immunogenic modification of double
stranded nucleic acid is accomplished principally by binding of a
molecule, usually a low molecular weight compound, to the duplex. Such
binding results in the creation of an antigenic determinant which
distinguishes double stranded nucleic acid from both single stranded
nucleic acid and the free, unbound modifier molecule. Preferably, this is
accomplished by employing a modifier compound which is essentially
incapable of binding with single stranded nucleic acid and which forms a
binding complex with double stranded nucleic acid which alters the normal
helical relationship of the complementary strands of the duplex.
Such modifier molecule as described herein is a nucleic acid intercalator
which preferentially will interact with the normal nucleic acid helix by a
non-covalent insertion between base pairs. Such insertion causes, in this
preferred interaction, the tertiary structure of the helix to change by
unwinding and elongation along the helical axis. A schematic
representation of this preferred intercalation interaction is shown in
FIG. 1 of the drawings. The resulting intercalation complex is
characterized by newly formed antigenic determinants which are understood
to comprise the intercalated modifier compound and the reoriented
phosphodiesterase backbones of the respective strands of the duplex.
Preferably, the intercalator compound is one of the generally planar,
aromatic organic molecules known to form intercalation complexes with
double stranded nucleic acid. Such compounds are exemplified by the
acridine dyes, e.g., acridine orange, the phenanthridines, e.g., ethidium,
the phenazines, furocoumarins, phenothiazines, quinolines, and the like as
are more fully described below. It should be clearly understood that while
the present invention will be hereinafter described with particular
reference to such intercalator compounds, the present invention
contemplates the use of equivalent modifier molecules which, as described
above, will bind to double stranded nucleic acid to induce an immunogenic
change in the duplex.
In accordance with the present invention, the intercalator can be combined
with the test medium, and thereby become exposed to the double stranded
nucleic acids present and/or forming in the hybridization reaction
mixture, as a separate, free compound and bind noncovalently to such
double stranded nucleic acids to form intercalation complexes.
Alternatively, the intercalator can be appropriately linked by chemical
bonds, preferably covalent bonds, to the probe. In the former case, the
present invention provides a method for performing a hybridization assay
without the need to chemically modify either the sample or probe
polynucleotide in order to detect hybridization. In the latter case, a
simple, synthetically straightforward means for labeling polynucleotides
or the hybridization aggregate is provided by the use of photoreactable
forms of the intercalator.
In all embodiments, the present invention provides a highly versatile,
sensitive, and specific method for detecting hybridization based on
antibody binding to the intercalation complexes in the aggregate formed.
Of course, appropriate fragments and polyfunctional forms of the antibody
can be used as described more fully below, and it will be understood that
when used in this disclosure the term antibody will mean its fragmented
and polyfunctional forms as well, unless otherwise noted. Determining the
binding of antibody to intercalation complexes can be accomplished in a
variety of conventional manners and preferably involves the use of
antibody labeled with a detectable chemical group such as an enzymatically
active group, a fluorescer, a luminescer, a specifically bindable ligand,
or a radioisotope.
The invention is applicable to all conventional hybridization assay
formats, and in general to any format that is possible based on formation
of a hybridization product or aggregate comprising double stranded nucleic
acid. In particular, the unique detection scheme of the present invention
can be used in solution and solid-phase hybridization formats, including,
in the latter case, formats involving immobilization of either sample or
probe nucleic acids and sandwich formats.
The hybridization product or aggregate formed according to the present
invention comprises hybridized probe and intercalator bound to double
stranded nucleic acid in the form of intercalation complexes. The
intercalation complexes can involve double stranded regions formed by
hybridization between sample and probe nucleic acids. Alternatively, such
double stranded regions can be comprised in the probe itself and in such
case can additionally be intercalated prior to use of the probe in the
assay. Thus, the detectable intercalation complexes can be formed in situ
during the assay or can be existent in the probe reagent as present to the
test medium. Further, the intercalation complexes can be chemically linked
to one or both of the strands of the intercalated duplex. In general, any
variation can be followed provided that the hybridization product
ultimately comprise intercalation complexes detectable by the antibody
binding phenomenon which is the underlying basis of the present invention.
Thus, the present invention provides an advantageous nucleic acid
hybridization method and reagent system. Additionally, there is provided a
novel antibody reagent capable of binding with intercalation complexes.
Furthermore, besides the detection of particular polynucleotide sequences,
the present invention provides a general method for detecting double
stranded nucleic acid by adding intercalator and the anti-(intercalation
complex) antibody and determining antibody binding.
The advantages of the present invention are significant and many. The
invention is amenable to a wide variety of nonradioactive detection
methods. Further, labeling of nucleic acids is straightforward and uses
easily synthesized reagents. Labeling with the intercalator does not
require expensive polymerases, and the labeling density of the
intercalator can be easily controlled. Certain preferred embodiments have
other advantages. In those embodiments in which the intercalator-nucleic
acid complex is formed in situ, no prior synthesis of the complex is
required and this approach can be used in a format in which a probe is
immobilized on a solid support and immersed in a solution containing the
specimen nucleic acid. In the embodiment where the intercalator is
covalently coupled to the nucleic acid, the intercalator is attached to
the probe during the manufacturing process, resulting in a controlled
level of saturation. This approach also minimizes user exposure to an
intercalating agent, many of which may be potentially hazardous.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1, as described above, is a schematic representation of the preferred
interaction between intercalator and double stranded nucleic acid which
results in an intercalation complex that is detectable by antibody.
FIGS. 2-5 are schematic diagrams of four preferred hybridization formats
for use in the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Intercalator
As described above, the intercalator compound preferably is a low molecular
weight, planar, usually aromatic but sometimes polycyclic, molecule
capable of binding with double stranded nucleic acids, e.g., DNA/DNA,
DNA/RNA, or RNA/RNA duplexes, usually by insertion between base pairs. The
primary binding mechanism will usually be noncovalent, with covalent
binding occuring as a second step where the intercalator has reactive or
activatable chemical groups which will form covalent bonds with
neighboring chemical groups on one or both of the intercalated duplex
strands. The result of intercalation is the spreading of adjacent base
pairs to about twice their normal separation distance, leading to an
increase in molecular length of the duplex. Further, unwinding of the
double helix of about 12 to 36 degrees must occur in order to accomodate
the intercalator. General reviews and further information can be obtained
from Lerman, J. Mol. Biol. 3:18(1961); Bloomfield et al, "Physical
Chemistry of Nucleic Acids", Chapter 7, pp. 429-476, Harper and Rowe,
NY(1974); Waring, Nature 219:1320 (1968); Hartmann et al, Angew. Chem.,
Engl. Ed. 7:693(1968); Lippard, Accts. Chem. Res. 11:211(1978); Wilson,
Intercalation Chemistry(1982), 445; and Berman et al, Ann. Rev. Biophys.
Bioeng. 10:87(1981).
A wide variety of intercalating agents can be used in the present
invention. Some classes of these agents and examples of specific compounds
are given in the following table:
______________________________________
Intercalator Classes
and Representative Compounds
Literature References
______________________________________
A. Acridine dyes Lerman, supra; Bloom-
field et al, supra;
proflavin, acridine orange,
Miller et al, Bio-
quinacrine, acriflavine
polymers 19:2091(1980)
B. Phenanthridines Bloomfield et al, supra;
Miller et al, supra
ethidium
coralyne Wilson et al, J. Med.
Chem. 19:1261(1976)
ellipticine, ellipticine
Festy et al, FEBS
cation and derivatives
Letters 17:321(1971);
Kohn et al, Cancer Res.
35:71(1976); LePecq
et al, PNAS (USA)71:
5078(1974); Pelaprat
et al, J. Med. Chem.
23:1330(1980)
C. Phenazines Bloomfield et al, supra
5-methylphenazine cation
D. Phenothiazines "
chlopromazine
E. Quinolines "
chloroquine
quinine
F. Aflatoxin "
G. Polycyclic hydrocarbons
"
and their oxirane
derivatives
3,4-benzpyrene Yang et al, Biochem.
benzopyrene diol Biophys. Res. Comm.
oxirane 82:929(1978)
benzanthracene-5,6-oxide
Amea et al, Science
176:47(1972)
H. Actinomycens Bloomfield et al, supra
actinomycin D
I. Anthracyclinones "
rhodomycin A
daunamycin
J. Thiaxanthenones "
miracil D
K. Anthramycin "
L. Mitomycin Ogawa et al, Nucl.
Acids Res., Spec.
Publ. 3:79(1977);
Akhtar et al, Can. J.
Chem. 53:2891(1975)
M. Platinium Complexes
Lippard, supra
N. Polyintercalators Waring et al, Nature
echinomycin 252:653(1974);
Wakelin, Biochem. J.
157:721(1976)
quinomycin Lee et al, Biochem. J.
triostin 173:115(1971); Huang
BBM928A et al, Biochem. 19:
tandem 5537(1980); Viswamitra
et al, Nature 289:
817(1981)
diacridines LePecq et al, PNAS
(USA)72:2915(1975);
Carrellakis et al,
Biochem. Biophys.
Acta 418:277(1976);
Wakelin et al, Bio-
chem 17:5057(1978);
Wakelin et al, FEBS
Lett. 104:261(1979);
Capelle et al, Bio-
chem. 18:3354(1979);
Wright et al, Biochem.
19:5825(1980); Bernier
et al Biochem. J.
199:479(1981); King
et al, Biochem. 21:
4982(1982)
ethidium dimer Gaugain et al, Bio-
chem. 17:5078(1978);
Kuhlman et al, Nucl.
Acids Res. 5:2629
(1978); Marlcovits
et al, Anal. Biochem.
94:259(1979); Dervan
et al, JACS 100:1968
(1978); ibid 101:
3664(1979).
ellipticene dimers
Debarre et al, Compt.
and analogs Rend. Ser. D 284:
81(1977); Pelaprat
et al, J. Med. Chem.
23:1336(1980)
heterodimers Cain et al, J. Med.
Chem. 21:658(1978);
Gaugain et al, Bio-
chem. 17:5078(1978)
trimers Hansen et al, JCS
Chem. Comm. 162(1983);
Atnell et al, JACS 105:
2913(1983)
O. Norphillin A Loun et al, JACS 104:
3213(1982)
P. Fluorenes and fluorenones
Bloomfield et al, supra
fluorenodiamines Witkowski et al,
Wiss. Beitr.-Martin-
Luther-Univ. Halle
Wittenberg, 11(1981)
Q. Furocoumarins Venema et al, MGG,
angelicin Mol. Gen. Genet.
179;1 (1980)
4,5'-dimethylangelicin
Vedaldi et al, Chem.-
Biol. Interact. 36:
275(1981)
psoralen Marciani et al, Z.
Naturforsch B 27(2):
196(1972)
8-methoxypsoralen Belognzov et al, Mutat.
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5-aminomethyl-8- Hansen et al, Tet. Lett
methoxypsoralen 22:1847(1981)
4,5,8-trimethylpsoralen
Ben-Hur et al,
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Issacs et al, Biochem.
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26:305(1982)
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Biochem. Biophys.
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R. Benzodipyrones Murx et al, J. Het.
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______________________________________
Several embodiments of the present invention involve the chemical, e.g.,
covalent, linkage of the intercalator to one or both of the complementary
strands of a duplex. Essentially any convenient method can be used to
accomplish such linkage. Conveniently, the linkage is formed by effecting
intercalation with a reactive, preferably photoreactive intercalator,
followed by the linking reaction. A particularly useful method involves
the use of azidointercalators. The reactive nitrenes are readily generated
at long wavelength ultraviolet or visible light and the nitrenes of
arylazides prefer insertion reactions over their rearrangement products
[see White et al, Methods in Enzymol. 46:644(1977)]. Representative
azidointercalators are 3-azidoacridine, 9-azidoacridine, ethidium
monoazide, ethidium diazide, ethidium dimer azide [Mitchell et al, JACS
104:4265(1982)], 4-azido-7-chloroquinoline, and 2-azidofluorene. Other
useful photoreactable intercalators are the furocoumarins which form [2+2]
cycloadducts with pyrimidine residues. Alkylating agents can also be used
such as bis-chloroethylamines and epoxides or aziridines, e.g.,
aflatoxins, polycyclic hydrocarbon epoxides, mitomycin, and norphillin A.
Depending on the hybridization format involved, as will be described in
detail below, chemically linked intercalation complexes can be used in a
variety of manners in the present invention. They can be formed in situ in
the hybridization reaction mixture or in a process step thereafter, or can
be a step in the synthesis of a labeled probe or sample nucleic acid. In
the latter case, where intercalation occurs in the region of
complementarity between the probe and sample nucleic acids, mono-linkages
will be accomplished followed by denaturing of such region to yield single
stranded nucleic acid with chemically linked intercalator oriented such
that upon hybridization, the linked intercalator will assume an
intercalation position.
HYBRIDIZATION FORMATS AND PROBES
The probe will comprise at least one single stranded base sequence
substantially complementary to or homologous with the sequence to be
detected. However, such base sequence need not be a single continuous
polynucleotide segment, but can be comprised of two or more individual
segments interrupted by nonhomologous sequences. These nonhomologous
sequences can be linear, or they can be self-complementary and form
hairpin loops. In addition, the homologous region of the probe can be
flanked at the 3'- and 5'-terminii by nonhomologous sequences, such as
those comprising the DNA or RNA of a vector into which the homologous
sequence had been inserted for propagation. In either instance, the probe
as presented as an analytical reagent will exhibit detectable
hybridization at one or more points with sample nucleic acids of interest.
Linear or circular single stranded polynucleotides can be used as the
probe element, with major or minor portions being duplexed with a
complementary polynucleotide strand or strands, provided that the critical
homologous segment or segments are in single stranded form and available
for hybridization with sample DNA or RNA. Particularly preferred will be
linear or circular probes wherein the homologous probe sequence is in
essentially only single stranded form [see particularly, Hu and Messing,
Gene 17:271-277(1982)].
Where the probe is used in a hybridization format calling for use of an
intercalator-labeled probe, as will be seen below, such probe can be in a
variety of forms such as a completely single stranded polynucleotide
having intercalator chemically linked thereto whereby hybridization
results in formation of intercalation complexes. Alternatively, the probe
can comprise a double stranded portion or portions which have been
intercalated, optionally with covalent linkage of the intercalator to one
or both strands in the duplex.
In terms of hybridization formats, the present invention is focused on
formation of a hybridization aggregate comprising the hybridized probe and
the intercalator bound to duplexes in the form of the antibody-detectable
intercalation complexes. Thus, the event of hybridization is associated
with the formation of the detectable intercalation complexes.
Fundamentally, the resulting intercalation complexes in the aggregate can
be in the region of hybridization between the sample and probe nucleic
acids or can be in a double stranded region remote from the hybridization
region. In such latter case, the intercalated region can be formed during
the assay or can be in the intercalated state when brought to the assay,
e.g., covalently linked or noncovalently intercalated double stranded
regions serving as labels for the probe.
Practice of the present analytical method is not limited to any particular
hybridization format. Any conventionl hybridization technique can be used.
As improvements are made and as conceptually new formats are developed,
such can be readily applied to carrying out the present method.
Conventional hybridization formats which are particularly useful include
those wherein the sample nucleic acids or the polynucleotide probe is
immobilized on a solid support (solid-phase hybridization) and those
wherein the polynucleotide species are all in solution (solution
hybridization).
In solid-phase hybridization formats, one | | |
