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TECHNICAL FIELD
The field of this invention is fluorescent compositions.
BACKGROUND
Detection of fluorescent signals finds wide application in a variety of
situations and under a variety of conditions. Fluorescence has many
advantages as a means of generating a detectable signal. Fluorescence does
not suffer from the many disadvantages of a radioactive label, while in
many cases it provides for a high level of sensitivity. Instrumentation
for detection of fluorescence is readily available and fluorescent labels
have found application in such diverse situations as immunodiagnostics,
detection of nucleic acid bands and protein bands in gel electrophoresis
and in fluorescence activated cell sorters. The sensitivity of the
fluorescent signal depends upon a number of factors: the possibility of
self-quenching; the effect of other molecules associated with the
fluorescent molecule on the quantum efficiency of the fluorescence; the
effect of the medium on the quantum efficiency and fluorescence
characteristics of the fluorescer; the stability of the fluorescer to
light; the ability to remove background fluorescence; and the nature of
the light source.
For many applications one wishes to have a number of distinguishable
fluorescers, so that one can detect different characteristics of a system.
For example, in the FACS, there may be an interest in identifying the
presence of two characteristics of the cell or other composition. In the
hybridization of DNA, one may wish to observe two different DNA sequences,
as observed in a gel, on a plate, or the like. Frequently, it is very
difficult to obtain fluorescers having emission maxima which are
sufficiently different so as to be differentiable while allowing for
excitation at the same wave length. There is, therefore, substantial
interest in identifying fluorescent markers which permit multiplex
determinations by providing for readily differentiable, fluorescent
emission maxima, while allowing for excitation with a narrow band
radiation source.
Relevant Literature
The following references describe DNA intercalating fluorescent dimers and
their physical characteristics: Gaugain et al., Biochemistry 17,
5071-5078, 1978; Gaugain et al., Biochemistry 17, 5078-5088, 1978;
Markovits et al., Anal. Biochemistry 94, 259-269, 1979; Markovits
Biochemistry 22, 3231-3237, 1983; and Markovits et al., Nucl. Acids Res.
13, 3773-3788, 1985. Interaction of various intercalating compounds with
nucleic acids is reviewed by Berman and Young, Ann. Rev. Biophys. Bioeng.
(1986) 10:87-224. Retention of ethidium bromide on electrophoresis of the
dye with DNA or RNA is described by Angemuller and Sayavedra-Soto,
Biotechniques 8, 36, 1990 and Rosen and villa-Komaroff, Focus 12, 23, 1990
(see also Glazer et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:3851-3855;
Rye et al. (1991) Nucl. Acids Res. 19:327-333; Quesada et al. (1991)
BioTechniques 10:616-625; Rye et al. (1992) Nucl. Acids Res. 20:2803-2812.
SUMMARY OF THE INVENTION
Novel fluorescent compositions and their use are provided, where the
fluorescent compositions are characterized by strongly binding to double
stranded DNA having a multiplicity of positive charges, having at least
two fluorophoric moieties, where one moiety is capable of efficiently
quenching the fluorescence (excitation energy) of the moiety emitting at a
lower wave length; the intercalated fluorescent composition provides for
emission efficiencies substantially in excess of the parent fluorophore;
and ratios of observed fluorescence emission between the higher wave
length emitting fluorophore and the lower wave length emitting fluorophore
are at least about 1.2 to 1. These compositions find use as fluorescent
markers for DNA, in combination with DNA as labels for other molecules,
and in the analysis of various systems, where multiplex fluorescent
analysis is desired.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a synthetic scheme for the synthesis of thiazole monomer
intermediates;
FIG. 2 is a synthetic scheme for the synthesis of thiazole dimers (TOTO and
TOTAB);
FIG. 3 is a synthetic scheme for the synthesis of thiazole-ethidium dimer
(TOED); and
FIG. 4 is a synthetic scheme for the synthesis of fluorescein-ethidium
heterodimer (FED).
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Novel methods are provided employing heterodimeric fluorescent compounds,
by themselves or intercalated into double stranded nucleic acids, where
the compositions are characterized by having two different fluorophore
moieties joined by a linking group comprising at least two positive
charges, advantageously used together or in conjunction with a homodimer.
The fluorophore moieties of the heterodimer are related in that a first
fluorophore moiety has an absorption maximum in solution at less than
about 500 nm, usually less than about 450 nm, and generally greater than
about 275 nm, usually greater than about 300 nm. The emission maximum for
the second fluorophore moiety in solution will generally be greater than
350 nm, usually greater than about 400 nm, and less about 600 nm, usually
less than about 550 nm. By contrast, the absorption maximum when
intercalated in DNA for the first fluorophore moiety will usually be at
least about 375 nm, usually at least about 400 nm, while, more usually at
least about 450 nm. The emission maximum for the second fluorophore moiety
when intercalated in DNA will generally be at least about 450 nm, more
usually at least about 500 nm and usually less than about 750 nm, more
usually less than about 700 nm. At least one of the fluorophores will be
capable of strongly binding to, usually intercalating, dsDNA.
The second fluorophore moiety will be further characterized, when
associated with dsDNA, by having an absorption maximum at least 25 nm
greater than the first fluorophore moiety to which it is combined and
usually not greater than about 100 nm, more usually not greater than about
75 nm. The second fluorophore will be capable of quenching at least about
80% of the fluorescence which would otherwise be observed from the first
fluorophore under the conditions of irradiation, when the subject
composition is intercalated into dsDNA. In order to have efficient
quenching, there will be overlap between the emission spectrum of the
donor and the absorption spectrum of the recipient or second fluorophore.
Usually, the ratios of observed fluorescence emission between the higher
wave length emitting fluorophore and the lower wave length emitting
fluorophore will be at least 1.2, more usually at least about 2, and may
be 5 to 1, or more. The Stokes shift between the wavelength of the
irradiation, where a narrow wavelength band is employed, particularly a
laser, and the fluorescent emission maximum will be at least about 75 nm,
more usually at least about 100 nm, preferably greater than about 125 nm.
It is advantageous to have a donor chromophore with an extinction
coefficient at the excitation wavelength to maximize the effectiveness of
light absorption.
While donor fluorophores may have extinction coefficients as low as about
2000, desirably the extinction coefficient will be at least 5000,
preferably at least 10,000, and may be 50,000 or more.
The subject heterodimers have novel fluorescent properties. The emission of
the absorbing fluorophore is at least about 80%, usually at least about
85% quenched. In addition, when bound solely to primary binding sites of
dsDNA, the emission of the emitting fluorophore is at least about 2 times,
usually at least about 7 times, brighter (as great) as the emission
maximum of the homodimer of the emitting fluorophores under the same
conditions, except for the wavelength of the excitation light. Thus,
fluorescence yield can be greatly increased with a weakly absorbing
fluorescer.
The subject compositions may comprise two or more fluorophore moieties,
where a pair of the fluorophores are related in accordance with the ranges
indicated above. The other fluorophores may be the same or different from
the pair of fluorophores. If different, the fluorescent properties will
usually be governed by the ranges indicated for the pair of fluorophores.
Where the fluorophores are different, each fluorophore would have a
differential absorption maximum in relation to the next successive
fluorophore in accordance with the above-indicated ranges. That is, if
there were three different fluorophore moieties, there would be a
difference in absorption maxima of at least 25 nm between the first and
second and at least 25 nm between the second and third, and so on.
Usually, the molecules would not have more than about four different
fluorophores, more usually not more than about three different
fluorophores. It is not essential that there be any particular order in
which the fluorophores are linked, so that they may be linked in a linear
manner, from a central linking group having spokes radiating from a
center, to a cyclic molecule or other convenient synthetic intermediate,
so long as the distance between the fluorophores allows for energy
transfer.
The linking group will be characterized by having at least two groups or
functionalities that can carry a positive charge or are charged, for
example, amino groups or ammonium groups, sulfonium groups, etc., for the
most part comprising nitrogen or sulfur as the positive heteroatom. The
linking chain may be of a length to allow for simultaneous intercalation
to adjacent monomeric units dsDNA, in this case usually providing a length
of at least about 10 .ANG.ngstroms, usually having at least about 9 atoms,
more usually at least about 10 atoms in the chain, and usually not more
than about 26, usually not more than about 20 atoms, between fluorescent
units, counting the shortest chain where a cyclic linking group is
involved.
The linking group will usually be aliphatic or alicyclic, having from 0-8,
more usually from 0-6, preferably from about 2-6 heteroatoms in the chain,
particularly heteroatoms that would provide for a positive charge. There
will normally be at least a total of two positive charges, more usually
not more than eight positive charges, more usually not more than about six
positive charges on the linking group.
The fluorophoric moieties may be cyclic, polycyclic, particularly
polycyclic aromatic having at least two rings, and not more than about six
rings, more usually not more than about five rings, where at least two of
the rings are fused, usually not more than four of the rings being fused.
The aromatic compound may be carbocyclic or heterocyclic, particularly
having from 1-3, more usually 1-2 nitrogen atoms as heteroannular atoms.
Other heteroannular atoms include oxygen and sulfur (chalcogen).
The rings may be substituted by a wide variety of substituents, which
substituents may include alkyl groups of from 1-4 carbon atoms, usually
1-2 carbon atoms, oxy, which includes hydroxy, alkoxy and carboxy,
generally of from 1-4 carbon atoms, amino, including mono- and
disubstituted amino, particularly mono- and dialkyl amino, of from 0-8,
usually 0-6 carbon atoms, thio, particularly alkylthio from 1-4, usually
1-2 carbon atoms, cyano, non-oxo-carbonyl, such as carboxy and derivatives
thereof, particularly carboxamide or carboxyalkyl, from 1-8, usually 1-6
carbon atoms, oxo-carbonyl or acyl, generally from 1-4 carbon atoms, halo,
particularly of atomic number 9-35, etc.
Fluorophore moieties of particular interest will involve two ring systems,
which are joined by a bond or a linking group having one or more ethylenic
groups which are in conjugation with the aromatic moieties. Aromatic
groups of interest include benzimidazole, benzthiazole, benzoxazole,
quinoline and acridine. Illustrative groups include thiazole orange,
thiazole blue, ethidium, fluorescein, acridine, phenanthridine, xanthenes,
and particularly fluorones.
Couples of fluorophore moieties include thiazole orange and thiazole blue,
thiazole orange and ethidium, fluorescein and ethidium, acridine and
ethidium, etc. However, acridine has a very low extinction coefficient
above 320 nm and is not a donor of choice. Acridine could serve as a donor
for thiazole orange or for fluorescein, but would give marginal gains in
fluorescence emission.
Compounds can be prepared from alkylene polyamines, where the alkylene
groups are from 2-10 usually 2-6 carbon atoms, and haloalkyl- or
pseudohaloalkyl substituted fluorescent polycyclic aromatic compounds, to
provide for ternary or quaternary amino groups. The amino groups may be
quaternized with any convenient alkylation agent, either before or after
reaction with the fluorescent compound or may be prepared initially as
ternary amines using alkyl amines, where the alkyl group will be of from
about 1-6, usually 1-3 carbon atoms.
These compounds find use as labeling agents, where the compounds are used
in a process for detection of nucleic acid or as a label which is prepared
for labeling a compound to provide a fluorescent signal. By having
multiple markers capable of simultaneous detection, many analytical
applications are attainable. For example, such applications include
fluorescence immunoassay, fluorescence in situ hybridization, and flow
cytometric analysis of cell populations, to list a few.
By appropriate combinations of the subject compositions, one can employ
dyes which share a common excitation wavelength, exploiting energy
transfer to achieve readily resolvable emission wavelengths. In addition,
with the dsDNA-dye complex, excitation of the donor leads to greatly
enhanced emission from the acceptor, with the donor fluorescence
substantially quenched as compared to the appropriate monomer dye in the
same environment. Thus, by using combinations of the subject dyes,
high-sensitivity multiplex detection of different dsDNA fragments can be
achieved.
The subject compositions can find use in separations employing an
electrical field, e.g. electrophoresis. In employing the subject
compounds, the nucleic acid, usually DNA, and the dye may be brought
together in appropriately buffered medium and incubated for sufficient
time for the dye to non-covalently bind and intercalate in the nucleic
acid. The ratio of dye to double stranded nucleic acid may be varied
widely ranging from about one molecule of dye per base pair to as little
as one molecule of dye per 400 base pairs, or fewer, usually as few as one
molecule of dye per 100 base pairs depending upon the desired degree of
sensitivity. Below about 15 bp/dye molecule, the increase in emission upon
further addition of dye is not as efficient as above 15 bp/dye. Dye
present in excess of one dye for four base pairs or more, may result in
total quenching, so that any increase in the amount of dye above a molar
ratio of one dye molecule for four base pairs may not be desirable.
However, the amount of dye combined with the DNA may be in a ratio of 1
per 2 base pairs or even 1 per 1 base pair or even greater ratios, where
quenching is not observed. Generally, the amount of dye will range from
about one molecule for 4 to 100 base pairs, usually about 10 to 50 base
pairs, for optimum results.
One may combine different samples with different dyes, followed by
combining the different samples to be electrically separated. Thus, in the
same channel, where an electrophoresis is carried out, one can detect the
various bands with light of the same wavelength used for irradiation, by
detecting the differences in fluorescent wavelength from the various
bands.
The amount of nucleic acid will generally be conventional amounts employed
for electrophoresis, generally ranging from about 5 pg/.mu.l to 5
ng/.mu.l. Because of the fluorescent efficiency, capillary electrophoresis
can be performed efficiently. Various conventional buffers may be
employed, such as tris-acetate or tris-borate, generally present in the
range of about 1-50 mM, more usually in the range of about 1-20 mM, to
provide a pH in the range of about 5-10, more usually about 7-9. Also, a
metal ion chelator may be present in a minor amount, generally from about
0.05-0.5 mM. Conveniently, EDTA may be employed.
The dye and nucleic acid may be incubated, usually for at least about 5
minutes and not more than about 2 hours, where complex formation will
normally be complete in less than about 1 hour, usually in about 30 min.,
at room temperature. The incubated solution may be used directly or
further diluted, as appropriate, prior to application to the gel.
The electrophoresis may be performed in any convenient and conventional
manner, where the bands may now be detected by fluorescence of the
non-covalently bound and intercalated dye. The electrophoresis ensures
that unbound dye is removed from the region of the bands and the dye is
found to be retained in the nucleic acid, so that individual bands may
readily be detected by fluorescence scanning.
Instead of incubating the nucleic acid with the dye prior to applying the
nucleic acid to the gel, one may apply the dye after having carried out
the separation. Since the intercalated dye will have a substantially
different absorption-emission range (and much enhanced fluorescence
intensity) from the unintercalated dye, one can readily detect the
intercalated dye, even in the presence of significant amounts of the
non-intercalated dye.
Any conventional detection system may be employed for detecting the
individual bands. Depending on the particular dye employed, the excitation
light will be chosen to be within a major absorption band of the absorbing
dye.
Of particular interest is the use of a confocal laser scanning fluorescence
imaging system. A system which has been found to be convenient employs a
long-pass dichroic beam splitter to reflect the laser beam down through a
microscope objective and onto the sample. The fluorescence emission is
collected by the objective and passed through the beam splitter to a
photodetector. The fluorescence emission is then passed through a spatial
filter to effect confocal detection in a long-pass or band-pass color or
interference filter before reaching a photomultiplier tube. An appropriate
servomotor-driven XY translation stage is employed with a 2.5 .mu.m
resolution to translate the gel past the laser beam at a convenient speed,
nearly about 1-5 cm/sec. A microcomputer may be employed to control the XY
translation stage and to acquire and display images. The fluorescence
images may then be pseudo-colored and coded to represent different
intensity levels and contrast stretched with a histogram equalization
method to enhance the images. To quantitate the image data, the image
columns that enclose the nucleic acid bands may be extracted and
integrated.
The nucleic acid may be readily isolated free of the intercalated
fluorescent dye for further use. One may use the Geneclean.RTM. kit for
recovery of 50% or better of the nucleic acid. By combining the
intercalated dye containing nucleic acid with Glassmilk in an aqueous
solution of alkali metal iodide, e.g. 1-10 ng nucleic acid (1-5 .mu.g/ml
nucleic acid) and about 1-10 .mu.g/ml of Glassmilk, incubating with
agitation for about 5-60 mins. followed by centrifugation, the resulting
pellet is isolated. After resuspending the pellet in an appropriate
ethanolic buffered aqueous solution (e.g. 1:1) followed by centrifugation
and repeating this washing procedure, the nucleic acid is obtained
substantially free of the fluorescent dye.
By virtue of the use of the subject intercalating fluorescent dyes in the
electrophoresis, greatly enhanced sensitivities are achieved due to the
much higher level of fluorescence intensity which is obtained. Sizes and
amounts of DNA fragments in mixtures of unknown composition can be
determined with a total amount of material ranging from 100 pg to 1 ng
depending on the complexity of the mixture and the size range of the
fragments. Thus, the subject method can find application in the detection
of nucleic acid of less than about 5 ng, particularly less than about 1
ng, frequently less than about 100 pg, even less than about 50 pg.
Instead of employing the subject dyes for detection of nucleic acid bands
in electrophoresis, compositions comprising dsDNA and the subject dyes at
substantial saturation may be employed, where the dsDNA is joined to an
entity for binding to another entity, either covalently or non-covalently.
The entities will be either referred to as specific binding pairs, since
the entities will have specific affinity for a complementary entity, as
compared to diverse other types of molecules, or covalently binding
functionalities for reacting with other molecules, such as polypeptides or
saccharides.
The specific binding pairs may involve a wide variety of molecules, which
are arbitrarily called ligands and receptors. For the subject invention,
the ligands and receptors may include a wide variety of proteins, such as
antibodies, specific binding proteins, such as surface membrane protein
receptors, lectins, blood proteins, and the like, carbohydrates, small
organic molecules, both naturally occurring and synthetic to which
proteins specifically bind, either naturally occurring protein receptors
or antibodies, nucleic acids which may hybridize or specifically bind to
an homologous or partially homologous sequence usually having at least
about 30% complementarity, preferably at least about 50% complementarity
over the complementary region, and the like. In effect, any two molecules
which have a specific binding affinity may be employed, so that the label
may be used for detection of the presence of the complementary member. The
desired specificity may be varied widely, depending upon the particular
nature of the molecules to be detected, the information desired about the
nature of the sample, or the like.
The labels may be used for detecting any of a wide variety of molecules in
a wide variety of samples, which includes physiological samples, e.g.
blood, plasma, urine, spinal fluid, saliva, feces, mucus, etc., waste
samples, from processing, garbage, soil, water, etc., contaminants in
products, such as food, drugs, etc.
Depending upon the fluorescence intensity one desires, one can vary the
length of the dsDNA and the level of non-covalent binding and
intercalation to increase the fluorescence intensity per molecule.
Usually, there will be at least about 16 base pairs, more usually at least
20 base pairs, and one may have dsDNA of at least about 1 kbp or even 2
kbp or more. The particular length of the dsDNA is not critical to this
invention and may be varied in accordance with the fluorescence intensity
desired per molecule, purpose of the label, convenience, and the like. It
is found that with some dyes, e.g. ethidium-acridine heterodimer, there is
an increase in fluorescence intensity by having A-T pairs. Thus, one may
provide for a poly A-T.poly A-T dimer to be used as the label. However, if
one wishes to further increase the stability of the dsDNA, beyond that
which the intercalating dimer provides, one can use a combination of AT
and GC pairs or a poly G-C.poly G-C dsDNA. Alternatively, one may use any
source of random DNA, such as calf thymus DNA, E. coli DNA, etc.
The dsDNA should provide for means for binding to another molecule. This
can be achieved in a wide variety of ways, depending upon the manner in
which the label is to be employed. For example, the dsDNA may include
biotin conjugated nucleotides, one or more biotins, where the biotin will
bind to avidin or streptavidin (hereafter both will be referred to as
"avidin"). The biotins may vary from one biotin per nucleotide to 0.1% of
the nucleotides depending on the nature of the procedures, conditions,
etc. Alteratively, any molecule may be employed, particularly a small
organic molecule (less than about 2 kdal) which is unlikely to be
encountered in the sample of interest, where the small organic molecule
has a specific receptor or antibody, particularly monoclonal antibody, to
which it specifically binds. Thus, thyroxine, corticosteroids, estrogens,
retinoic acid, mannose and the like may be used with proteins which bind
specifically to such molecules. Alternatively, synthetic molecules may be
employed for which antibodies have been produced, such as
2,4-dinitrophenyl, barbiturate, phosphatidylcholine, etc. These molecules
may be included during synthesis of the DNA by being linked to an internal
or terminal nucleotide, where the DNA is synthesized in accordance with
conventional automatic procedures, or may be added after synthesis of the
DNA by linking to either available hydroxyl or amino groups.
The binding entity may be an active functionality for covalently bonding to
a molecule having a functionality capable of forming a stable covalent
link, such as amino, hydroxyl, thio, carboxyl, activated olefin or aryl,
or the like where the functionality is other than a naturally occurring
functionality of the nucleotide. The label may be modified with an
activated olefin, such as maleyl, for reaction with a thiol group, a
carboxyl for reaction with an amine, or the like. In this manner, many
different types of molecules may be fluorescently labeled for use in
diagnostics, both competitive assays and non-competitive assays,
histology, cytology, separations e.g. electrophoresis, HPLC, FACS, and the
like.
The strands of DNA may take various structures. In many situations, the
dsDNA may comprise two strands, where the strands may be completely or
only partially overlapping, where the ends may extend in the 5' and/or 3'
directions, so that one strand may be substantially longer than the other
strand, where the other strand may bind either 5' proximal, 3' proximal or
centrally. Alternatively, the two strands may overlap to provide for
staggered ends, where the single stranded portions of the DNA may then be
used to bind to complementary sequences. Alternatively, one may provide a
single strand with an inverted repeat, so that the strand loops back on
itself to provide the double stranded portion. The hairpin structure may
be used solely for labeling, or a single stranded portion of the hairpin
may be employed for hybridizing to a complementary sequence. The
hybridizing single stranded portion may be an extension at either the 5'
or 3' end to provide for a staggered terminus or may be present in the
loop of the hairpin.
The subject labels may be used in a wide variety of environments and
contexts to provide for high levels of fluorescence intensity without
interference from the molecules to which the labels bind, either directly
or indirectly, the media employed, the conditions employed, and the like.
Thus, the subject labels may be employed in specific binding pair assays,
where the label may be readily linked to another molecule through a
specific binding pair combination. For example, in diagnostic assays, one
may combine an avidin conjugated antibody, where the antibody binds to a
molecule of interest, to a biotin labeled DNA dye composition to provide
for fluorescent labeled antibody.
Alternatively, the antibody may be labeled with biotin, so that avidin may
act as a bridge between the biotin labeled antibody and the biotin labeled
DNA dye composition. In this way, the fluorescent label may be added after
combining the sample with a complementary specific binding pair member and
carrying out the assay, followed by addition of label and removal of any
nonspecifically bound label.
Where a single stranded DNA sequence is provided as part of the label, this
can be used for hybridizing to complementary DNA or RNA sequences. The
presence of the non-covalently bound and intercalated dye greatly enhances
the stability of the dsDNA. Thus, one can introduce the subject labels
into a denaturation medium under conditions where the non-covalently bound
and intercalated dsDNA will be stable, while the sample DNA may be
denatured to provide for single strands. Where single stranded DNA or RNA
is present, there will be no need for providing for denaturation
conditions. Therefore, the subject molecules may be used as probes to
identify DNA sequences under a wide variety of conditions, including
electrophoresis, polymerase chain reactions, where the single stranded
sequence may serve as a primer, in Southern blotting, Northern blotting
and the like.
Instead of having non-covalent complexes between the non-nucleic acid
specific binding pair member and the DNA dye aggregate, one can provide
for covalent bonding. Thus, by providing for activated groups such as
carboxy, diazo, azido, activated ethylene, or the like, the fluorescent
moiety may be readily linked to other molecules, such as proteins, sugars,
lipids, or the like by employing conventional linking groups resulting in
amides, amines, diazo, esters, thioethers, or insertion into a
carbon-hydrogen bond or addition to a double bond, and the like. For
example, one may introduce a thiol group at either the 3' or 5' terminus
of a synthetic oligonucleotide, synthesize the complementary strand and
form a non-covalently bound and intercalated dye complex. The thiol group
on the DNA can then be reacted with a maleimide modified protein, e.g. an
antibody. One can add an acylmethylazide and upon phototypes produce a
nitrene which would randomly insert or add to a double bond. Other
techniques may follow conventional procedures found in the literature.
The subject DNA dye composition may also be used in situations where one
wishes to transfer energy or receive energy from another molecule. Thus,
the subject compositions may be used with other fluorescent dye
substituted molecules, e.g. dye intercalated DNA molecules, for receipt or
transfer of excitation energy, or with other fluorescent molecules, so as
to extend the shift between the excitation light and the emission light.
This technique may be used in diagnostic assays, or where one wishes to
determine the spatial relationship between two entities, e.g. epitopes,
surface membrane receptors, etc.
One may also use the subject labels in a fluorescence activated cell sorter
to provide for greatly enhanced sensitivity as a result of the
substantially increased fluorescence intensity. Again, one may use ligands
for surface membrane receptor proteins, sugars for lectins, antibodies for
epitopes present on the surface of the cell, or the like, where the
subject labels may be bound covalently or non-covalently, to the molecule
which binds to the cell component.
With the subject compositions one can also detect proteins to
transcriptional initiation elements, e.g. promoters, operators, enhancers,
etc. By having labeled dsDNA, according to the subject invention, mixed
with labeled proteins, labeled with a fluorescent molecule emitting at a
different wavelength from the non-covalently bound and intercalated
fluorescer, or other appropriate label, one can determine the presence of
transcription factors and cofactors. For example, one can gel
electrophorese the mixture and identify the presence of the protein bound
to DNA by virtue of the double labelling and band shift.
One may also use the subject fluorescent non-covalently bound and
intercalated DNA for in situ hybridization studies, intermolecular
transfer of fluorescent molecules from one doubly stranded nucleic acid
molecule to another, e.g. for transferring fluorescent dye without the
fluorescer being transferred to the medium. This may find use in making
chromosomes with triplex formation, in transferring to nucleic acid in a
gel or on a membrane, etc. The fluorescer intercalated DNA may be bound to
a particle, e.g. magnetic, to be removed after use as a transfer agent.
The subject compounds may be used with advantage with a confocal
fluorescence imaging system, where less than 100 pg of DNA can be detected
with some dyes while with other combinations, less than about 5 pg of DNA
can be detected. In histology and cytology, the subject fluorescent labels
provide for high-sensitivity in detecting target epitopes, particularly at
low levels.
Besides using the dyes individually, the dyes may be used in combination
for a wide variety of applications, where one wishes to obtain at least
two bits of information concerning the sample or composition of interest.
Since the intercalated dyes have different absorption and emission spectra
from the non-intercalated dyes, one can detect the presence of the two
dimeric compounds when intercalated into DNA, while in the absence of DNA,
there would be substantial overlap, so that only poor discrimination would
be obtained. Furthermore, one need not use the heterodimers of the subject
invention solely, since the heterodimers may be used with homodimers,
where the heterodimer and homodimer have the same absorbing fluorophore or
different absorbing fluorophore excited by a common wavelength range, but
obviously differing in the emitting fluorophore. Thus, ethidium dimer,
thiazole orange dimer, thiazole blue dimer, oxazole yellow dimer, and the
like may be used in conjunction with heterodimers, where usually the same
absorbing fluorophore is present, although in many instances, a different
absorbing fluorophore will suffice, where the two absorbing fluorophores
have overlapping absorption peaks.
By using the combinations employing the subject dyes, detection of two
different events may be obtained in a number of different environments. Of
particular interest is flow cytometry, where a single exciting light may
be used and the fluorescence determined as to two or more events. The
events may involve binding events to two different epitopes of the same or
different protein, where a single protein may be involved or an
aggregation of proteins, as is present in viruses and cells or significant
fragments thereof. In this way, one may select for particles such as
cells, by virtue of the presence of two different markers, using a single
exciting laser. Similarly, in histology and cytology, one may determine
the presence of different proteins which may be present on the same or
different cells or present extracellularly. As appropriate, one may inject
the dyes bound to a specific binding molecule into a laboratory animal to
follow the migration of different molecules or cells, where one is
interested in the presence of the two different targets at a particular
site. Numerous other applications, where single excitation light is
desired, while a plurality of different information values is desired
concurrently, have been generally described in literature and will be
further developed as the subject invention becomes available.
For many applications, a plurality of fluorescent molecules will be
desirable. Kits can be provided where the fluorescent molecules in the kit
are characterized by having absorption maxima within about 25 nm, so that
excitation light of a relatively narrow bandwidth may be used, generally
of not more than about 30 nm, usually of not more than about 20 nm. The
absorbing fluorophore may be the same or different.
The kit will have two or more fluorescent multimers, each having from two
to four, usually two to three, fluorophores, wherein at least one of the
fluorophores is able to bind, usually intercalate, into dsDNA. At least
one of the multimers will be a heteromultimer being characterized by
having a Stokes shift of at least about 25 nm, capable of binding dsDNA at
a ratio of at least one dye per 200 bp dsDNA without diminishing the
fluorescence as compared to one fluorophore per dsDNA molecule on a per
dye basis, and the fluorophores absorb and emit at different wavelengths,
when bound or unbound to dsDNA. Besides the at least one heteromultimer,
the other multimers may be homomultimers, of particular interest are homo-
and heterodimers and heterotrimers.
The following examples are offered by way illustration and not by way
limitation.
EXPERIMENTAL
Synthesis of Dyes
Materials and Methods. The starting materials including 3
-methyl-benzothiazole-2-thione, lepidine,
3,8-diamino-9-phenylphenanthridine, fluorescein isothiocyanate isomer I,
diethylenetriamine, tetramethyl-1,3-diaminopropane, 1,3-diaminopropane,
anhydrous HBr/acetic acid were purchased from Aldrich and used without
further purification. Anhydrous methanol, triethylamine, and pyridine were
distilled from sodium and stored under nitrogen. Anhydrous nitrobenzene
was freshly distilled from P.sub.2 O.sub.5. All anhydrous reactions were
run in oven dried glassware under a nitrogen atmosphere. Reactions were
monitored by TLC (Merck A.sub.254) under short and long wavelength UV
light. Flash chromatography was performed on 220-440 mesh silica gel 60
from Fluka. Intermediate products which gave single spots on TLC were
identified by their .sup.1 H-NMR spectra measured with an AMX-300
instrument. UV/VIS absorption spectra were measured with a Perkin Elmer
Lambda 6 spectrophotometer and fluorescence emission spectra with a Perkin
Elmer MFP 44B spectrofluorometer.
Synthesis of Heterodimers. As outlined in FIG. 1, the N-3-iodopropyl
derivatized lepidine (1), reacted in under 15 minutes with the N-methyl
2-methylthio benzothiazole (2) or N-methyl 2-(N'-phenyl, N'-acetyl,
3-azopropylidene benzothiazole (4) to yield the iodopropyl thiazole orange
intermediate (3), or iodopropyl thiazole blue intermediate (5) (TB), in
good yields following the method of Brooker et al. ((1942) JACS
64:199-210; (1941) JACS 63:3192-3203). Compound (1) was produced in high
yield by alkylation of lepidine with 5 equivalents of 1,3-diiodopropane in
refluxing dioxane, while compound (2) was formed quantitatively when 3
equivalents of iodomethane were reacted with
3-methylbenzothiazole-2-thione in refluxing 200 proof ethanol and
precipitated with ether. Compound (2) was also used as an intermediate in
the synthesis of (3) (Brooker et al., supra).
FIG. 1. Synthesis of thiazole monomer intermediates. a) Suspend in absolute
EtOH, add 1 equivalent TEA, stir at room temperature for 15 minutes,
precipitate with ether, recrystallize from acetone/ether; yield 80% 3, 60%
5.
To synthesize the thiazole orange-thiazole blue heterodimer, the
iodopropropyl thiazole orange derivative (4) was reacted with excess
tetramethyl-1,3-diaminopropane (Scheme 2) to produce the intermediate
tetramethyldiaminopropyl thiazole orange derivative (6). Compound (6)
(TO6), after purification by recrystallization, reacted with the thiazole
blue derivative (5) to produce a good yield of the thiazole orange linked
thiazole blue heterodimer TOTAB (7). Dimerization of (4) (as described in
Rye et al., Nucleic Acids Res. (1992) 20:2803-2812) was used to synthesize
nearly quantitatively the homodimer TOTO (8) employing 0.5 equivalents of
tetramethyl-1,3-diaminopropane.
FIG. 2. Synthesis of the thiazole dimers. a) Suspend in anhydrous MeOH, add
6 equivalents 5 tetramethyl-1,3-propanediamine, reflux 6 hours,
precipitate acetone/ether, recrystallize MeOH:CH.sub.2 Cl.sub.2
(1:10)/acetone; yield 80% 6. b) Suspend in anhydrous MeOH, add 1
equivalent (5), reflux 10 hours, precipitate with ether, triturate solid
with MeOH:CH.sub.2 Cl.sub.2 (1:10), flash column EtOAc:AcOH:H.sub.2 O
(1:2:2); yield 70% 7. c) Suspend in anhydrous MeOH, add 0.5 equivalents
tetramethyl-1,3-propanediamine, reflux 18 hours, precipitate with acetone,
titurate solid with MeOH:CH.sub.2 Cl.sub.2 (1:10), recrystallize from
MeOH/acetone; yield 95% 8.
The thiazole orange-ethidium heterodimer was obtained as outlined in FIG. 3
from 3,8-diamino-9-phenylphenanthridine (9) via intermediate (10). In
general, the synthesis of the thiazole orange-ethidium heterodimer (11)
followed the method of Gaugain et al. ((1978) Biochemistry 17:5071-5078)
except that carbobenzyloxy groups were used to protect the amino
substituents in place of acetate or carboethoxy groups. After coupling
(10) | | |
