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Claims  |
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We claim:
1. The spectroscopy method for detecting a target single-strand
polynucleotide sequence in a polynucleotide sample in which either (i) a
fluorescent polynucleotide single probe sequence complementary to said
target sequence is hybridized thereto, or (ii) a plurality of probes of
such sequences complementary to sequentially adjacent portions of said
target sequences are hybridized thereto, wherein the improvement comprises
having present in said single probe or in said plurality of probes at
least one pair of fluorescent moieties connected by linker arms to nucleic
acid base units, said fluorescent moieties comprising respectively donor
and acceptor moieties selected so that the emission spectrum of the donor
moiety overlaps the excitation spectrum of the acceptor moiety to permit
non-radioactive energy transfer with efficient fluorescent emission by the
acceptor fluorophores, the wavelength maximum of the emission spectrum of
the acceptor moiety being at least 100 nm greater than the wavelength
maximum of the excitation spectrum of the donor moiety, said linker arms
having lengths of 4 to 30 Angstroms, the donor and acceptor moieties being
connected to non-contiguous base units in said single probe or to base
units in said plural probes other than 3' and 5' and units thereof, when
said single probe or said plural probes are hybridized to the target
sample the base units to which said donor and acceptor moieties are
connected being paired through hybridization to base units of said target
sequence which are separated by 2 to 7 intervening nucleotide base units
of the target sequence.
2. The method improvement of claim 1 in which said donor and acceptor
moieties are on a single probe.
3. The method improvement of claim 1 in which said donor and acceptor
moieties are on a pair of probes.
4. The method of claim 1, 2, or 3 in which said donor moiety is fluorescein
and said acceptor moiety is Texas Red.
5. The method of claim 1 in which said linker arms have lengths of from 10
to 25 Angstroms.
6. The method of claim 1 in which when said single probes or said dual
probe are hybridized to the target sequence the base units connected to
said donor and acceptor moieties are paired with nucleotide base units of
the target sequence which are separated by 3 to 6 intervening base units.
7. The spectroscopy method for detecting a target single-strand
polynucleotide sequence in a polynucleotide sample immobilized on a
support in which either (i) a fluorescent polynucleotide single probe
sequence complementary to said target sequence is hybridized thereto, or
(ii) a plurality of probes of such sequences complementary to sequentially
adjacent portions of said target sequences are hybridized thereto, said
probes being synthetic polynucleotides of from 10 to 100 base units in
length, wherein the improvement comprises having present in said single
probe or in said plurality of probes at least one pair of fluorescent
moieties connected by linker arms to nucleic acid base units, said
fluorescent moieties comprising respectively donor and acceptor moieties
selected so that the emission spectrum of the donor moiety overlaps the
excitation spectrum of the acceptor moiety to permit non-radiative energy
transfer with efficient fluorescent emission by the acceptor fluorophore,
the wavelength maximum of the emission spectrum of the acceptor moiety
being at least 100 nm greater than the wavelength maximum of the
excitation spectrum of the donor moiety, said linker arms having lengths
of 10 to 25 Angstroms, the donor and acceptor moieties being connected to
non-contiguous base units in said single probe and to base units in said
plural probes other than the 3' and 5' end units, when said single probe
or plural probes are hybridized to the target polynucleotide the base
units to which said donor and acceptor moieties are connected being paired
with hybridized base units of said target sequence which are separated by
2 to 7 intervening nucleotide base units of the target sequence.
8. The method of claim 7 in which said donor and acceptor moieties are on a
single probe.
9. The method of claim 7 in which said donor and acceptor moieties are on a
pair of probes.
10. The method of claim 7, 8, of 9 in which said donor moiety is
fluorescein and said acceptor moiety is Texas Red.
11. The method of claim 1 or 7 in which said donor and acceptor moieties
are on a single probe and are connected to base units thereof other than
the 3' and 5' end units and which are separated by 4 to 6 intervening base
units.
12. The method of claim 1 or 7 in which said moieties are on a pair of
probes respectively having 3' and 5' ends hybridizing to adjacent base
units of the target polynucleotide and when so hybridized the donor and
acceptor moieties being separated by 4 to 6 intervening base units.
13. The methods of claim 11 or 12 in which said donor moiety is fluorescein
and said acceptor moiety is Texas Red.
14. A polynucleotide probe for fluorescent spectroscopy assaying of a
target single-stranded polynucleotide, comprising a synthetic
single-stranded polynucleotide containing from 10 to 100 nucleic acid base
units, two of said base units having respectively attached thereto by
separate linker arms a donor fluorescent moiety and an acceptor
fluorescent moiety, said fluorescent moieties being selected so that the
emission spectrum of the donor moiety overlaps the excitation spectrum of
the acceptor moiety to permit non-radiative energy transfer with efficient
fluroescent emission by the acceptor fluorophore, the wavelength maximum
of the emission spectrum of the acceptor moiety being at least 100 nm
greater than the wavelength maximum of the excitation spectrum of the
donor moiety, said linker arms having lengths of 4 to 30 Angstroms, said
donor and acceptor moieties being connected to base units other than the
3' and 5' end units and which units are separated by 2 to 7 intervening
base units.
15. The probe of claim 14 in which said linker arms have lengths of from 10
to 25 Angstroms.
16. The probe of claim 14 in which said donor moiety is fluorescein and
said acceptor moiety in Texas Red.
17. A polynucleotide probe for fluorescent spectroscopy assaying of a
target single-stranded polynucleotide, comprising a synthetic
single-stranded polynucleotide containing from 10 to 100 nucleic acid base
units, two of said base units having respectively attached thereto by
linker side chains a donor fluorescent moiety and an acceptor fluorescent
moiety, said fluorescent moieties being selected so that the emission
spectrum of the donor moiety overlaps the excitation spectrum of the
acceptor moiety to permit non-radiative energy transfer with efficient
fluorescent emission by the acceptor fluorophore, the wavelength maximum
of the emission spectrum of the acceptor moiety being at least 100 nm
greater than the wavelength maximum of the excitation spectrum of the
donor moiety, said linker side chains having lengths of 10 to 25
Angstroms, said donor and acceptor moieties being connected to base units
other than the 3' and 5' end units and which units are separated by 3 to 6
intervening base units.
18. The probe of claim 17 in which said donor moiety is fluorescein and
said acceptor moiety is Texas Red.
19. A pair of polynucleotide probes for fluorescent spectroscopy assaying
of a target single-stranded polynucleotide, comprising first and second
synthetic single-stranded polynucleotide probes each containing from 10 to
100 nucleic acid base units, said probes hybridizing to adjacent sequences
of said target polynucleotide with the 3' end of the first probe adjacent
to the 5' end of the second probe, said probes having fluorescent moieties
attached to base units thereof other than said adjacent 3' and 5' end
units by linker arms having lengths of 4 to 30 Angstroms, one of said arms
being connected to a donor moiety and other to an acceptor moiety, said
moieties being selected so that the emission spectrum of the donor moiety
overlaps the excitation spectrum of the acceptor moiety to permit
non-radiative energy transfer with efficient fluorescent emission by the
acceptor fluorophore, the wavelength maximum of the emission spectrum of
the acceptor moiety being at least 100 nm greater than the wavelength
maximum of the excitation spectra of the donor moiety, said linker arms
having lengths of from 4 to 30 Angstroms, and being connected to base
units other than the 3' and 5' end units, when said probes are hybridized
to the target polynucleotide sequences the base units to which said donor
and acceptor moieties are connected being paired with hybridized base
units of said target sequences which are separated by 2 to 7 intervening
base units.
20. The pair of probes of claim 19 in which said linker arms have lengths
of from 10 to 25 Angstroms.
21. The pair of probes of claim 19 in which said donor moiety is
fluorescein and acceptor moiety is Texas Red.
22. A pair of polynucleotide probes for fluorescent spectroscopy assaying
of a target single-stranded polynucleotide, comprising first and second
synthetic single-stranded polynucleotide probes hybridizing to adjacent
sequences of said target polynucleotide with the 3' end of the first
probes adjacent to the 5' end of the second probe, said probes having
fluorescent moieties attached to base units other than said adjacent 3'
and 5' end units by linker arms having lengths of 10 to 25 Angstroms, one
of said side chains being connected to a donor moiety and the other to an
acceptor moiety, said moieties being selected so that the emission
spectrum of the donor moiety overlaps the excitation spectrum of the
acceptor moiety to permit non-radiative energy transfer with efficient
fluorescent emission by the acceptor fluorophore, the wavelength maximum
of the emission spectrum of the acceptor moiety being at least 100 nm
greater than the wavelength maximum of the excitation spectrum of the
donor moiety, said linker arms having lengths of from 10 to 25 Angstroms,
and being connected to base units other than the 3' and 5' end units when
said probes are hybridized to the target polynucleotide sequences the base
units to which said donor and acceptor moieties are connected being paired
with base units of the target sequences which are separated by 4 to 6
intervening base units.
23. The pair of probes of claim 22 in which said donor moiety is
fluorescein and said acceptor moiety is Texas Red.
24. A method of detecting a target nucleic acid comprising hybridizing a
poly- or oligonucleotide probe or proves to said nucleic acid, wherein
said probe or probes have or hybridize to form a pair of fluorescent
moieties connected to separate nucleotides and wherein the nucleotides
connected to the fluorescent moieties are separated by as least two but
not more than seven nucleotides, and detecting the hybridization by
fluorescent means based on the interaction between the moieties.
25. The method of claim 24, wherein the fluorescent moieties are separated
by at least three but not more than six nucleotides.
26. The method of claim 24, wherein the fluroescent moieties are separated
by at least four but not more than six nucleotides.
27. A poly- or oligonucleotide probe comprising a pair of fluorescent
moieties connected to separate nucleotides and wherein the nucleotides
connected to the fluorescent moieties are separated by at least two but
not more than seven nucleotides.
28. The nucleotide probe of claim 27, wherein the fluorescent moieties are
separated by at least three but not more than six nucleotides.
29. The nucleotide probe of claim 27, wherein the fluorescent moieties are
separated by at least four but not more than six nucleotides. |
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Claims |
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Description |
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FIELD OF INVENTION
The field of this invention is fluorescent labelled polynucleotide probes
for use in polynucleotide (DNA or RNA) hybridization assays. In general,
this invention is concerned with improving the properties of fluorescent
labelled probes for more sensitive detection in hybridization assay
systems. In particular it is concerned with the selection and unique
positioning of two or more fluorophores which through an efficient energy
transfer mechanism produce a fluorescent probe or probes with
significantly improved detection properties.
BACKGROUND OF INVENTION
Hybridization assays may be employed for the detection and identification
of DNA or RNA sequences. Published methods as used particularly in
recombinant DNA research are described in Methods of Enzymology, Vol. 68,
pp. 379-469 (1979); and Vol. 65, Part 1, pp. 468-478 (1968). One such
method involving a preliminary separation of the nucleic acid fragments by
electrophoresis is known as the "Southern Blot Filter Hybridization
Method." See Southern E., J. Mol. Biol. 98, p. 503 (1975). A recent and
more complete review of nucleic acid hybridization methods and procedures
can be found in Meinkoth, J. and Wahl, G., Analytical Biochemistry, 138,
pp. 267-284 (1984).
Fluorescent labeled synthetic polynucleotide probes are commercially
available in the United States. Chemical methods for incorporating
modified nucleotides into synthetic polynucleotides are described in the
published PCT Application WO 84/03285, dated Aug. 30, 1985. The synthetic
polynucleotide containing the modified nucleotide (usually referred to as
a "linker arm nucleotide") can subsequently be derivatized with a
fluorescent moiety. As described in the cited PCT application, only a
single fluorescent moiety is attached to the probe.
Certain problems are encountered when using polynucleotide probes labelled
with commonly available fluorophores such as fluorescein, rhodamine,
pyrenes, etc. The most serious problem involves limited sensitivity for
direct detection of the probe in the assay system. For most hybridization
assays a sensitivity or detection level of at least 10.sup.-18 mole of
labelled probe (10.sup.6 target molecules) is required. While many
fluorophores inherently have this level of sensitivity, secondary
interferences from the sample and components in the assay system prevent
these levels of detection from being reached. At a level of 10.sup.-18
mole of fluorescent probe, fluorescence from the sample itself, Rayleigh
scatter, reflection from support materials (nitrocellulose filters, etc.)
and in particular Raman (water) scatter can produce background signals
many orders of magnitude higher than the signal from the fluorescent
probe.
Ideally, improvement in detecting fluorescent probes in such assay systems
could be obtained by selecting a fluorophore which has: (1) a large Stokes
shift, that is, a large separation between the wavelengths for maximum
excitation (EX) and the wavelength for maximum emission (EM); (2) a high
quantum yield (QY>0.5); (3) a high extinction coefficient (EC>30,000); (4)
an emission beyond 600 nm (red fluorescence); and (5) an excitation
maximum close to a laser line (442 nm Helium-Cadmium or 448 nm Argon).
Unfortunately, there are no common fluorophores which fully satisfy these
criteria. For example, fluorescein (EX: 495 nm, EM: 525 nm, QY=0.5) is a
highly fluorescent label with an excitation maximum near a laser line, but
has a Stokes shift of only .about.30 nm.
It is known that a larger Stokes shift can be obtained by employing a pair
of donor/acceptor fluorophores which have overlapping spectra and which
are arranged in close proximity for non-radiative energy transfer between
the donor and acceptor fluorophores. This form of energy transfer was
proposed by Forster, who developed equations of transfer efficiency in
relation to separation distances between the fluorophores. See, for
example, Forster, Th., Ann. Phys. (Leipzig) 2:55-75 (1948). A recent
summary of Forster's non-radiative energy transfer is given in "Principles
of Fluorescent Spectroscopy," J. R. Lakowicz, Chapt. 10 (1983). The
Forster mathematical analysis predicates that the closer the spacing of
the fluorescent moieties the greater the efficiency of energy transfer.
Prior experimental evidence confirmed this prediction.
Stryer and Haugland (Proc. Natl. Acad. Sci. 58, 719-729, 1967) reported
experiments with variable spacing for an energy donor and acceptor pair
attached to oligopeptides. An energy donor group and an energy acceptor
group were attached to the ends of proline oligomers which served as
spacers of defined lengths. Spacings of 1 to 12 units were tested, with a
separation range of 12 to 46 Angstroms (.ANG.). The longer oligomers were
found to be in helical conformation. The energy transfer efficiency
decreased from 100% at a distance of 12.ANG. to 16% at 46.ANG.. It was
concluded that the dependence of the transfer efficiency on distance was
in excellent agreement with the dependence predicted by the Forster
equations. The results were so close to theoretical predictions that the
authors proposed use of non-radiative energy transfer as a spectrocopic
ruler. Related experiments with model systems reported by other
researchers are confirmatory. See, for example, Gabor, Biopolymers
6:809-816 (1968); and Katchalski-Katzir, et al., Ann. N. Y. Acad. Sci.
366: 44-61 (1981). The use of the Forster energy transfer effect has been
described in the following immunofluorescent assay patents. (See U. S.
Pat. Nos. 3,996,345; 3,998,943; 4,160,016; 4,174,384; and 4,199,599). The
energy transfer immunofluorescent assays described in these patents are
based on the decrease or quenching of the donor fluorescence rather than
fluorescent re-emission by the acceptor [Ullman, E. F., et al., J. Biol.
Chem., Vol. 251, 14, pp. 4172-4178 (1976)].
Homogeneous immunoassay procedures based on chemiluminescent labels or
bioluminescent proteins have been reported which involve non-radiative
energy transfer, see Patel, et al., Clin. Chem. 29 (9):1604-1608 (1983);
and European Patent Application No. 0 137 515, published Apr. 17, 1985. By
close spacing of the donor-acceptor group according to the principles of
nonradiative energy transfer for high transfer efficiency it was proposed
that homogeneous assays could be made practical. Homogeneous assays are
inherently simpler to carry out but their use had been subject to the
limitation that unbound labelled probe remains in solution and causes
interfering background signal. European Patent Application No. 0 137 515
published Apr. 17, 1985 refers to various ligand-ligand interactions which
can be used with the bioluminescent proteins including nucleic acidnucleic
acid interactions. The examples, however, are directed to protein ligands
rather than nucleic acids.
European Patent Application No.,0 070 685, published Jan. 26, 1983, relates
to homogeneous nucleic acid hybridization assays employing non-radioative
energy transfer between absorber/emitter moieties positioned within 100
Angstroms of each other. As described, the hybridization probes are
prepared by attaching the absorber-emitter moieties to the 3' and 5' end
units of pairs of single-stranded polynucleotide fragments derived from
DNA or RNA by restriction enzyme fragmentation. The pairs of
polynucleotide fragments are selected to hybridize to adjacent
complementary sequences of the target polynucleotide with the labelled
ends with no overlap and with few or no base-pairing spaces left between
them. The preferred donor moiety is a chemiluminescent catalyst and the
absorber moiety is a fluorophore or phosphore.
THE DRAWINGS
FIGS. 1 to 5 illustrate preferred embodiments of Stokes shift probes for
use in practicing the invention.
SUMMARY OF INVENTION
This invention is based in part on the discovery that polynucleotides (DNA
or RNA) provide an environment which strongly influences non-radiative
energy transfer between donor-acceptor fluorescent moieties attached to
polynucleotide probes. Prior to the present invention, it was not known
how to design fluorophore-labelled probes with donor-acceptor moieties for
practical and effective use with polynucleotides, particularly with regard
to efficient emission by the acceptor fluorophore. It has been found that
a novel spacing of the fluorescent moieties is critical for maximizing
energy transfer and producing highly efficient fluorescent emission by the
acceptor. Surprisingly, the optimum spacing requires intervening base pair
units between the nucleotides to which the fluorescent moieties are
attached. In particular, contrary to prior knowledge about Forster
nonradiative energy transfer attachment of the fluorescent moieties to
immediately adjacent nucleotide units (donor/acceptor distance 10-15.ANG.)
or with only a single intervening unit results in an unacceptably low
transfer efficiency. The theoretical explanation for this new phenomenon
is not known. However, it apparently relates to the formation of
excitation traps when the fluorescent probe(s) is hybridized to the target
polynucleotide. This "microenvironment" of the helical double-stranded
polynucleotides has a marked effect on the optimum spacing for
non-radiative energy transfer and efficient fluorescent emission by the
acceptor.
More specifically, it has been found that for efficient acceptor emission
the donor-acceptor fluorescent moieties should be separated when
hybridized by at least two intervening base units but not over seven
units. For optimum efficiency with either single probe or dual probe
embodiments a separation range of from 3 to 6 base units is preferred. To
maximize the benefits of this invention, the linker arm side chains which
connect the fluorescent moieties to the nucleic acid (pyrimidine or
purine) base units should have lengths within the range from 4 to 30
Angstroms (.ANG.) and preferably from about 10 to 25.ANG.. With either the
single or the double probe embodiments, neither of the fluorescent
moieties should be attached to end units of the probes.
The probes of this invention are believed unique for the following reasons:
(1) Unexpectedly, those donor and acceptor positions (e.g., distances of
20.ANG. or less) predicted by the Forster equations and confirmed
experimentally in model systems by prior investigators to provide maximum
energy transfer efficiency were found to have minimal observed efficiency
for hybridized polynucleotide probes. (2) Maximum observed energy transfer
efficiency, "in terms of fluorescent emission by the acceptors" was found
only for a relatively restricted number of positions requiring more
nucleotide spacing between the fluorophores. (3) Maximum observed acceptor
fluorophore emission was also found to be dependent upon hybridization of
the probe to its complementary target sequence, viz., in the single probe
embodiment efficiency was increased by hybridization. (4) With proper
spacing an exceptionally high value (viz. 80%) for fluorescent emission by
the acceptor fluorophore can be obtained.
DESCRIPTION OF PREFERRED EMBODIMENTS
This invention is applicable to synthetic polynucleotide probes containing
from 10 to 100 base units, and in particular 15 to 35 base units. With
synthetically-prepared probes, precise attachment of the fluorophores can
be obtained by the methods described in PCT Application WO 84/03285,
published Aug. 30, 1984. This greatly simplifies the practice of the
present invention with respect to preparation of the required probes.
Preferred embodiments of this invention utilize selected donor and acceptor
fluorophore pairs appropriately positioned in single or dual
polynucleotides. Such probes may be designed to produce a large Stokes
shift and highly efficient acceptor fluorescent emission at wavelengths
greater than 600 nm. In both the single and dual probe embodiments the
fluorophores are attached to the polynucleotide with intervening base
units as hybridized. The invention is generally applicable to plural
probes, including triple and quadruple probes as well as dual probes.
One preferred embodiment utilizes fluorophores positioned near the end base
units of dual polynucleotide probes. By avoiding attachment of either
fluorophore to the terminal base units appropriate intervening base units
are provided. Hybridization of the dual fluorophore probes to
complementary target sequences can accurately position the donor and
acceptor fluorophores according to the spacing requirements of this
invention.
In the single probe embodiment, the donor and acceptor fluorophores should
be attached to the polynucleotide probe at positions which give them a
relative separation of two to seven intervening base units. In the dual
probe embodiment, after both probes are hybridized to the target sequence,
the donor fluorophore on one probe and the acceptor fluorophore on the
other probe should also be attached to give a relative separation of two
to seven base units. The preferred separation for both the single and dual
probe embodiments is from 3 to 6 intervening base units. The optimized
spacing is believed to be 4 to 5 units. In both single and dual probe
embodiments when the probes are hybridized to the target polynucleotide
the base units to which the donor and acceptor moieties are connected
should be paired with base units of the target sample which are separated
by 2 to 7 intervening base units.
Selection of Fluorophores
Selection of the donor and acceptor fluorophores is of importance to obtain
the advantages of this invention. In general, the fluorescent moiety
should comprise respectively donor and acceptor moieties selected so that
the emission spectrum of the donor moiety overlaps the excitation spectrum
of the acceptor moiety to produce efficient non-radiative energy transfer
therebetween. Wavelength maximum of the emission spectrum of the acceptor
moiety should be at least 100 nm greater than the wavelength maximum of
the excitation spectrum of the donor moiety.
In addition, the fluorescent donor and acceptor pairs are preferably chosen
for (1) high efficiency Forster energy transfer; (2) a large final Stokes
shift (>100 nm); (3) shift of the emission as far as possible into the red
portion of the visible spectrum (>600 nm); and (4) shift of the emission
to a higher wavelength than the Raman water fluorescent emission produced
by excitation at the donor excitation wavelength. For example, a donor
fluorophore may be chosen which has its excitation maximum near a laser
line (in particular Helium-Cadmium 442 nm or Argon 488 nm), a high
extinction coefficient, a high quantum yield, and a good overlap of its
fluorescent emission with the excitation spectrum of the acceptor
fluorophore. In general, an acceptor fluorophore is preferably chosen
which has a high extinction coefficient, a high quantum yield, a good
overlap of its excitation with the emission of the donor, and emission in
the red part of the visible spectrum (>600 nm).
Fluorescein is a particularly desirable donor moiety. Lucifer Yellow can
also be employed as a donor moiety, particularly in combination with Texas
Red as an acceptor moiety. The emission spectra of fluorescein
(EX.about.492 nm, EM.about.520 nm, EC.about.70,000, QY high) and of
Lucifer Yellow (EX.about.428 nm, EM.about.540 nm, EC.about.12,000, QY
medium) both sufficiently overlap the excitation spectrum of Texas Red
(EX.about.590 nm, EM.about.615 nm, EC.about.70,000, QY high).
Fluorescein's excitation maximum (.about.492 nm) comes very close to the
488 nm Argon laser line and Lucifer Yellow's excitation maximum
(.about.428 nm) comes very close to the 442 nm Helium-Cadmium laser line.
In addition the fluorescein/Texas Red and Lucifer Yellow/Texas Red
combinations provide large Stokes shifts of .about.130 nm and .about.170
nm respectively. In both cases the 615 nm to 620 nm Texas Red emission is
at significantly higher wavelengths than the Raman water lines (.about.585
nm for 448 nm excitation and .about.520 nm for 442 nm excitation). As
compared with the use of a fluorescein reporter group alone, the
combination with a Texas Red acceptor provides a ten to twenty fold
increase in the relative detection sensitivity in the 615 nm to 620 nm
emission region for excitation at .about.490 nm. As compared with the use
of Lucifer Yellow group alone, the combination with a Texas Red acceptor
provides two to three fold increase in relative detection sensitivity in
the 615 nm to 620 nm emission region.
Fluorescein fluorophores can be incorporated in the polynucleotide probe as
a fluorescein isothiocyanate derivative obtainable from Molecular Probes,
Inc., Junction City, Oreg., or Sigma Chemical Co., St. Louis, Mo. Texas
Red sulfonyl chloride derivative of sulforhodamine 101 is obtainable from
Molecular Probes, Inc. Texas Red can also be prepared from sulforhodamine
101 by reaction with phosphorous oxychloride, as described in Titus, et
al., J. Immunol. Meth., 50, pp. 193-204, 1982. Lucifer Yellow is
obtainable from Aldrich Chemical Co., Milwaukee, Wis., as the vinyl
sulfone derivative (Lucifer Yellow VS). Lucifer Yellow VS is a
4-amino-N-[3-vinylsulfonyl) phenyl]naphthalimide-3, 5-disulfonate
fluorescent dye. For a description of its use, see Stewart, W., Nature,
Vol. 292, pp. 17-21 (1981).
The foregoing description should not be understood as limiting the present
invention to combinations of fluorescein with Texas Red or Lucifer Yellow
with Texas Red. Those combinations preferred by the principles of the
invention are more broadly applicable. The spacing feature of this
invention can be utilized with other donor-acceptor pairs of fluorophores.
For example, with fluorescein and Lucifer Yellow as donors, the acceptor
fluorophore moieties prepared from the following fluorescent reagents are
acceptable: Lissamine rhodamine B sulfonyl chloride; tetramethyl rhodamine
isothiocyanate; rhodamine x isothiocyanate; and erythrosin isothiocyanate.
Other suitable donors to the acceptors listed above (including Texas Red)
are B-phycoerythrin and 9-acridineisothiocyanate derivatives.
When fluorescein is used as the acceptor moiety then suitable donors can be
obtained from Lucifer Yellow VS; 9-acridineisothiocyanate;
4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid;
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin.
When diethylenetriamine pentaacetate or other chelates of Lanthanide ions
(Europium and Terbium) are used as acceptors, then suitable donors can be
obtained from succinimdyl 1-pyrenebutyrate; and
4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives.
Linker Arms
The length of the linker arms connecting the fluorescent moieties to the
base units of the probes is also an important parameter for obtaining the
full benefit of the present invention. The length of the linker arms for
the purpose of the present invention is defined as the distance in
Angstroms from the purine or pyrimidine base to which the inner end is
connected to the fluorophore at its outer end. In general, the arm should
have lengths of not less than 4 nor more than 30.ANG.. The preferred
length of the linker arms is from 10 to 25.ANG.. The linker arms may be of
the kind described in PCT application No. WO 84/03285. That application
discloses a method for attaching the linker arms to the selected purine or
pyrimidine base and also for attaching the fluorophore to the linker arm.
The linker arm represented below is illustrative of the linker arms which
may be employed for the purposes of the present invention as further
described in the cited PCT application.
##STR1##
The linker arm as represented above contains 12 units in the chain and has
a length of approximately 14.ANG.. Use of this linker arm in preparing
probes in accordance with the present invention is further illustrated in
the experimental examples.
THE DRAWINGS
The drawing herein comprising FIGS. 1 and 2 provide diagrammatic
illustrations of preferred embodiments. Referring first to FIG. 1, there
is represented a single probe with 5 nucleotide base (n=5) spacing of the
fluorophores. The polynucleotide probe may contain from 10 to 100
nucleotide bases. Intermediate the 5' and 3' ends of the probe, the donor
fluorophore (D) and the acceptor fluorophore (A) are attached to base
units through linker arms of 4 to 30.ANG.. Units to which the linker arms
connect the fluorophores are separated by 5 nucleotide based units (+5).
The letters represent the bases of DNA: G for guanine, T for thymine, A
for adenine, and C for cytosine. As indicated by the arrows, excitation
light directed on the probe is absorbed by the donor fluorophore,
transferred by the non-radiative energy process to the acceptor
fluorophore, and emitted as fluorescent light by the acceptor fluorophore.
FIG. 2 illustrates dual probes hybridized to a target polynucleotide
sequences of a nucleic acid sample. Both the sample and the probes contain
the bases of DNA (G, T, A and C). In the hybridized condition, the
nucleotide bases pair in the manner of double-stranded DNA (G-C and T-A).
In the illustration given, each contains 25 nucleotide bases. Linker arms
are attached to base units spaced from the adjacent 3', 5' ends of the
probes as hybridized. Specifically, a Texas Red fluorophore is linked to a
thymine unit (T) of probe 1, which is the third unit from the 3' end.
Fluorescein is linked to a thymine (T) of probe 2, which is the fifth unit
from the 5' end. When these dual probes are in hybridized relation, as
shown, separation between the base units to which the donor and acceptor
fluorophores are attached is 6 units (n=6). The linker arms have a length
of approximately 14.ANG. and may comprise the linker arm illustrated
above.
FIGS. 3 and 4 represent modifications of the probes in which single
acceptor fluorophores arranged in space relation with a plurality of donor
fluorophores. These embodiments employ the same spacing requirements
discussed above with respect to the base unit separations and the linker
arm lengths. FIG. 3 illustrates a single-probe embodiment in which an
acceptor flurophore is linked to an intermediate base positioned between
two donor fluorophores linked to bases spaced from the base of the
acceptor fluorophore by 4 base units (n=4). FIG. 4 illustrates a dual
probe embodiment in which a donor fluorophore is linked to one probe and a
donor and acceptor fluorophore to the other probe. As illustrated probe 1
has the donor fluorophore linked to the third base from its 3' end. Probe
2 has the acceptor fluorophore linked to the third base from its 5' end,
and also has a donor fluorophore linked to the eighth base unit from the
5' end. This provides a spacing of four base units between the donor and
acceptor fluorophores of probe 2 (n=4) when these probes are in hybridized
relation to the target sequences as illustrated with respect to FIG. 2.
Similar spacing (n=4) will be provided between the donor fluorophore of
probe 1 and the acceptor fluorophore of probe 2. With the donor/acceptor
fluorophore arrangements of FIGS. 3 and 4, the amount of nonradiative
energy transferred to the acceptor fluorophore can be increased.
FIG. 5 shows a three probe embodiment which includes two pairs of donor and
absorber fluorophores. Probe 1 has a donor moiety attached to the third
base from its 5' end which as hybridized to the target polynucleotide
pairs with the absorber moiety on probe 2 which is attached to the third
base from its 3' end, giving a separation of 4 base units (n=4). The 5'
end of probe 2 has an acceptor moiety attached to the third base which
when hybridized pairs with a donor moiety on probe 3 attached to the third
base to also provide an n=4 spacing.
Assay Procedures
The probes of this invention may be employed in either heterogeneous or
homogeneous assays of the kind heretofore used for DNA or RNA
hybridization assays. To obtain the maximum benefit of the invention,
however, it is preferred to employ the probes in conjunction with
heterogeneous assays in which the target DNA or RNA is hybridized to a
support. The test samples containing the target sequences may be prepared
by any one of a number of known procedures and attached to suitable
immobilization support matrices. Such procedures are described in Methods
in Enzymology, Vol. 66, pp. 379-469 (1979), Vol. 65, Part 1, pp. 468-478
(1980, and Analytical Biochemistry. 138, pp. 267-284 (1984). See also U.S.
Pat. No. 4,358,539 and published European Patent Applications Nos. 0 070
685 and 0 070 687. The usual supports used in hybridization assays include
nitrocellulose filters, nylon (Zetabind) filters, polystyrene beads, and
Agarose beads to name a few. Further details of a typical heterogeneous
assay procedure are set out in one of the following examples. The probes
of this invention, their method of use, and the results obtained are
further illustrated by the following examples.
EXAMPLE I
By way of specific illustration, the preparation of a polynucleotide probe
containing a fluorescein and a Texas Red moiety with an n=5 spacing can be
carried out as follows. The starting material is approximately 300.mu.g of
the appropriate synthetic (25mer) polynucleotide probe containing two
primary amine functionalized linker arm nucleotides separated by five
nucleotides within the sequence. The 300.mu.g of polynucleotide is taken
up in about 20.mu.l of 0.5M sodium bicarbonate buffer at pH 8.8. About
100.mu.g of Texas Red dissolved in 10.mu.l of water is added to the
polynucleotide solution. A limited reaction is carried out at
0.degree.-5.degree. C. for approximately 15 minutes. At this point about
10.mu.l of a 7M urea solution is added and the reaction mixture is
separated over a 0.7cm.times.3.0cm G-25 Sephadex Column. The initial
fractions (excluded volume) contain the unreacted polynucleotide,
mono-substituted Texas Red polynucleotide probe, and di-substituted Texas
Red polynucleotide probe. The final fractions (included volume) contain
the unreacted Texas Red. The inital fractions are pooled and lyophilized,
and the final fractions are discarded. The lyophilized pooled fractions
are brought up in a small volume (5-10.mu.l) of 3.5M urea for separation
by gel electrophoresis.
Electrophoresis on a 20% polyacrylamide gel (7-8M urea) separates the
sample into three distinct bands, the lower is the unreacted
polynucleotide, the middle band is the mono-substituted Texas Red
polynucleotide, and the upper band is the di-substituted Texas Red
polynucleotide. Reaction conditions were originally controlled in order to
prevent total conversion of the polynucleotide to the di-substituted Texas
Red polynucleotide derivative. At this point the band containing the
mono-substituted Texas Red polynucleotide derivative is carefully excised
from the gel and the derivative is extracted with water, and the resulting
solution lyophilized to dryness. The lyophilized sample is now taken up in
a small volume of water and desalted on a G-25 Sephadex column. The
fractions containing the mono-substituted Texas Red polynucleotide probe
are pooled and lyophilized.
The sample is now ready for the second reaction to incorporate the
fluorescein moiety into mono-substituted Texas Red polynucleotide probe.
The sample is again taken up in about 20.mu.l of 0.5M sodium bicarbonate
buffer at pH 8.8. About 500.mu.g of fluorescein isthiocyanate (FITC) in
10.mu.l water is added to the buffered solution containing the
mono-substituted Texas Red polynucleotide probe. The reaction is carried
out at 0.degree.-5.degree. C. for about two hours. About 10.mu.l of a 7M
urea solution is added, and the sample is run over another G-25 Sephadex
column, as described previously, to separate reacted polynucleotide probe
from FITC. Again appropriate fractions are pooled and lyophilized. The
sample is again electrophoresed on a 20% polyacrylamide gel, separating
the sample into two bands: the lower being unreacted monosubstituted Texas
Red polynucleotide probe and the upper band being the fluorescein and
Texas Red substituted polynucleotide probe. The upper band is carefully
excised, extracted, lyophilized, and desalted on a G-25 Sephadex column as
was described above. The final purified fluorescein-Texas Red
polynucleotide probe is then analyzed by UV/Visible spectroscopy. The
ratio of adsorption (O.D.) at 260 nm, 492 nm, and 592 nm can be used to
determine proper stoichiometry for the probe; the 25mer polynucleotide
probe contains one fluorescein and one Texas Red moiety.
The synthesis and purification of probes containing a single fluorophore is
straightforward. The starting material is a 25mer polynucleotide probe
containing only one amine functionalized linker arm nucleotide
incorporated at the appropriate position within the probe. In the case of
both Texas Red and FITC, the reactions are carried out for a longer time
(about two hours) in order to increase yield of the fluorophore
substituted probe. Subsequent steps for purification are the same as those
described above.
EXAMPLE II
A series of fluorescein-Texas Red 25mer polynucleotide probes (F&TR probes)
were prepared in which the separation between the fluorophore moieties was
n=0, n=1, n=5, n=6, n=9, and n=12. The probes were designed to hybridize
to Herpes Simplex Virus (type 1) target DNA. The procedure was as
described in Example I using the 14.ANG. linker arm previously
illustrated. The actual sequence and relative position of fluorophores in
the n=5, F&TR probe is shown below.
##STR2##
It should be pointed out that the fluorophores can occupy either linker arm
position on the probe. But each probe contains only one fluorescein and
one Texas Red. Fluorescent analysis was carried out on samples containing
from 200ng to 2.mu.g of the F&TR probe in 250.mu.l of 0.01M sodium
phosphate (pH7.6), 0.1M sodium chloride buffer.
Fluorescent emission spectra were obtained for samples at 490 nm, the
approximate excitation maximum for the donor (fluorescein) and at 590 nm,
the approximate excitation maximum for the acceptor (Texas Red). All
values were corroborated by also obtaining the fluorescent excitation
spectra for each of the F&TR probes in the series. Observed energy
transfer efficiency, in terms of fluorescent emission of the acceptor, was
determined by taking the ratio for fluorescent emission at 615 nm for the
F&TR probe excited at 490 nm (excitation of donor, fluorescein) to the
fluorescent emission at 615 nm for a single labelled Texas Red probe (TR
probe) excited at 590 nm (excitation for the acceptor, Texas Red)
multiplied by 100.
##EQU1##
Thus, a value of "75" means that the F&TR probe when excited at 490 nm
produces 75% of the fluorescent emission (615 nm) of an equal amount (in
terms of Texas Red) of a TR probe excited at 590 nm. Observed energy
transfer efficiencies were determined for the complete F&TR probe series
both hybridized to a complementary target polynucleotide and unhybridized.
Results for the F&TR probe series is given in Table A.
TABLE A
______________________________________
Observed Energy Transfer Efficiencies
for Fluorescein-Texas Red Probes
F&TR Probe (Emission 615 nm, Excitation 490 nm)
(N) Unhybridized
Hybridized
______________________________________
0 25 17
1 29 31
5 35 82
6 39 59
9 50 30
12 17 13
TR Probe 100 100
(EX 590 nm)
______________________________________
The results in Table A show that the observed energy transfer efficiency is
highest (82) for the n=5 F&TR probe in the hybridized series. In the
hybridized series, energy transfer is observed to decrease for the probes
with the longer donor-acceptor distances (n=6, 9, 12) as would be expected
from the Forster equation. However, unexpectedly the efficiencies also
drop off for the n=0 and n=1 probes, with the closer donor-acceptor
distances.
The F&TR probe series does not follow the expected type of behavior. The
high energy transfer efficiency is only found for a relatively restricted
number of positions approximated to be between 20 to 30.ANG.. These high
efficiency positions are believed to include the n=3 and n=4 positions as
well as the experimentally determined n=5 and n=6, and also the n=7
position.
Table A also shows that the results for the unhybridized F&TR probe series
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