U.S. patent application number 11/083210 was filed with the patent office on 2005-10-20 for nucleic acid probes and methods to detect and/or quantify nucleic acid analytes.
This patent application is currently assigned to PROLIGO, LLC. Invention is credited to Bruce, Ian, Davies, Martin, Wolter, Andreas.
Application Number | 20050233360 11/083210 |
Document ID | / |
Family ID | 23316069 |
Filed Date | 2005-10-20 |
United States Patent
Application |
20050233360 |
Kind Code |
A1 |
Davies, Martin ; et
al. |
October 20, 2005 |
Nucleic acid probes and methods to detect and/or quantify nucleic
acid analytes
Abstract
The invention comprises novel methods and strategies to detect
and/or quantify nucleic acid analytes. The methods involve nucleic
acid probes with covalently conjugated dyes, which are attached
either at adjacent nucleotides or at the same nucleotide of the
probe and novel linker molecules to attach the dyes to the probes.
The nucleic acid probes generate a fluorescent signal upon
hybridization to complementary nucleic acids based on the
interaction of one of the attached dyes, which is either an
intercalator or a DNA groove binder, with the formed double
stranded DNA. The methods can be applied to a variety of
applications including homogeneous assays, real-time PCR
monitoring, transcription assays, expression analysis on nucleic
acid microarrays and other microarray applications such as
genotyping (SNP analysis). The methods further include pH-sensitive
nucleic acid probes that provide switchable fluorescence signals
that are triggered by a change in the pH of the medium.
Inventors: |
Davies, Martin; (Kent,
GB) ; Bruce, Ian; (East Sussex, GB) ; Wolter,
Andreas; (Esmarchstrasse, DE) |
Correspondence
Address: |
SWANSON & BRATSCHUN L.L.C.
1745 SHEA CENTER DRIVE
SUITE 330
HIGHLANDS RANCH
CO
80129
US
|
Assignee: |
PROLIGO, LLC
Boulder
CO
|
Family ID: |
23316069 |
Appl. No.: |
11/083210 |
Filed: |
March 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11083210 |
Mar 16, 2005 |
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10278047 |
Oct 21, 2002 |
|
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6902900 |
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60336432 |
Oct 19, 2001 |
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Current U.S.
Class: |
435/6.18 ;
435/6.1; 536/24.3; 536/25.32 |
Current CPC
Class: |
C12Q 1/6818 20130101;
C12Q 2563/173 20130101; C12Q 2565/1015 20130101; C12Q 2565/1015
20130101; C12Q 2525/113 20130101; C12Q 2561/113 20130101; C12Q
2525/313 20130101; C12Q 2565/1015 20130101; C12Q 2565/107 20130101;
C12Q 2525/313 20130101; C12Q 2563/173 20130101; C12Q 2527/119
20130101; C12Q 1/6818 20130101; C12Q 1/6818 20130101; C12Q 1/6818
20130101; C12Q 1/686 20130101; C12Q 1/6818 20130101; C07H 21/00
20130101 |
Class at
Publication: |
435/006 ;
536/024.3; 536/025.32 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
1. A nucleic acid probe, wherein said nucleic acid probe is
comprised of a nucleic acid that is derivatized with two or more
non-identical covalently attached dyes, wherein at least one dye is
fluorescent, and wherein at least one dye has a high affinity to
double stranded nucleic acids, wherein the dyes are attached at
either the same or at adjacent nucleotides of the nucleic acid
probe.
2. The nucleic acid probe of claim 1 wherein the dye that has a
high affinity to double stranded nucleic acids is a fluorescent
intercalator.
3. The nucleic acid probe of claim 1 wherein the dye that has a
high affinity to double stranded nucleic acids is a fluorescent
groove binder.
4. The nucleic acid probe of claim 1 wherein the nucleic acid probe
comprises a fluorescent intercalator and a non-fluorescent
quencher.
5. The nucleic acid probe of claim 1 wherein the nucleic acid probe
comprises a fluorescent intercalator and a second fluorescent dye
wherein the second fluorescent dye functions as the donor of a FRET
system formed between the intercalator and the second dye.
6. The nucleic acid probe of claim 1 wherein the nucleic acid probe
comprises a fluorescent intercalator and a second fluorescent dye
wherein the second fluorescent dye functions as the acceptor of a
FRET system formed between the intercalator and the second dye.
7. The nucleic acid probe of claim 1 wherein the nucleic acid probe
comprises a fluorescent intercalator and two dyes that form an
excimer pair.
8. The nucleic acid probe of claim 1 wherein the nucleic acid probe
comprises a fluorescent intercalator and two dyes that form an
exciplex pair.
9. The nucleic acid probe of claim 1 wherein the nucleic acid probe
comprises a fluorescent groove bonder and a non-fluorescent
quencher.
10. The nucleic acid probe of claim 1 wherein the nucleic acid
probe comprises a fluorescent groove binder and a second
fluorescent dye wherein the second fluorescent dye functions as the
donor of a FRET system formed between the intercalator and the
second dye.
11. The nucleic acid probe of claim 1 wherein the nucleic acid
probe comprises a fluorescent groove binder and a second
fluorescent dye wherein the second fluorescent dye functions as the
acceptor of a FRET system formed between the intercalator and the
second dye.
12. The nucleic acid probe of claim 1 wherein the nucleic acid
probe comprises a fluorescent groove binder and two dyes that form
an excimer pair.
13. The nucleic acid probe of claim 1 wherein the nucleic acid
probe comprises a fluorescent groove binder and two dyes that form
an exciplex pair.
14. The nucleic acid probe of claim 2 wherein the fluorescent
intercalator is selected from thiazole orange or ethidium.
15. The nucleic acid probe of claim 3 wherein the fluorescent
groove binder is selected from thiazole orange or ethidium.
16. The nucleic acid probe of claim 4 wherein the fluorescent
intercalator is selected from thiazole orange or ethidium.
17. The nucleic acid probe of claim 5 wherein the fluorescent
intercalator is selected from thiazole orange or ethidium.
18. The nucleic acid probe of claim 6 wherein the fluorescent
intercalator is selected from thiazole orange or ethidium.
19. The nucleic acid probe of claim 7 wherein the fluorescent
intercalator is selected from thiazole orange or ethidium.
20. The nucleic acid probe of claim 8 wherein the fluorescent
intercalator is selected from thiazole orange or ethidium.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 10/278,047, filed Oct. 21, 2002, which is a
non-provisional of Provisional Application Ser. No. 60/336,432,
filed Oct. 19, 2001, both of which are entitled "Nucleic Acid
Probes And Methods To Detect And/Or Quantify Nucleic Acid Analytes"
and each of which is incorporated herein by reference in its
entirety.
FIELD OF INVENTION
[0002] The present invention relates to the field of molecular
biology. More specifically, the present invention relates to the
field of assays that utilize nucleic acid probes to detect and/or
quantify nucleic acid analytes.
BACKGROUND OF THE INVENTION
[0003] Advances in DNA technology and sequencing, specifically the
sequencing of whole genomes including the human genome, have
resulted in a significantly increased need to detect and/or
quantify specific nucleic acid sequences. Applications include the
fields of in vitro diagnostics, including clinical diagnostics,
research in the fields of molecular biology, high throughput drug
screening, veterinary diagnostics, agricultural-genetics testing,
environmental testing, food testing, industrial process monitoring
and insurance testing. In vitro diagnostics and clinical
diagnostics is related to the analysis of nucleic acid samples
drawn from the body to detect the existence of a disease or
condition, its stage of development and/or severity, and the
patient's response to treatment. In high throughput drug screening
and development nucleic acids are used similarly to other agents,
such as, antigens, antibodies, receptors, etc, to analyze the
response of biological systems upon exposure to libraries of
compounds in a high sample number setting to identify drug leads.
Veterinary diagnostics and agricultural genetics testing involve
samples from a non-human animal or a plant species similar to in
vitro diagnostics and to provide means of quality control for
agricultural genetic products and processes. In environmental
testing, organisms and their toxins that indicate the pollution of
an environmental medium, e.g. soil, water, air, etc., are analyzed.
Food testing includes the quantitation of organisms, e.g. bacteria,
fungi, etc., as a means of quality control. In industrial process
monitoring, nucleic acids are detected and/or quantified to
indicate proper control of a production process and/or to generate
a signal if such processes are out of control. In insurance testing
organisms and/or their toxins are identified in screening tests to
determine the risk category of a client or to help approve
candidates. There are various other applications of the detection
and/or quantitation of nucleic acids and new applications are being
developed constantly.
[0004] The most common techniques to detect and measure nucleic
acid analytes are based on the sequence-specific hybridization of
the analyte with a complimentary nucleotide sequence probe which is
marked with a detectable label, typically a fluorescent label, a
radioactive label or another chemical label that can be detected in
a secondary reaction. The probe is combined with the nucleic acid
analyte, either in situ as part of a biological system or as
isolated DNA or RNA fragments. The hybridization conditions are
those that allow the probe to form a specific hybrid with the
analyte, while not becoming bound to non-complementary nucleic acid
molecules. The analyte can be either free in solution or
immobilized on a solid substrate. The probe's detectable label
provides a means for determining whether hybridization has occurred
and thus, for detecting the nucleic acid analyte. The signal that
is generated via the detectable sample can in some instances be
measured quantitatively to back-calculate the quantity and the
concentration of the analyte.
[0005] Current methods used to detect and measure nucleic acid
analytes are briefly described below.
[0006] PCR Methods
[0007] The polymerase chain reaction (PCR) amplification of nucleic
acids is regularly performed using fluorescently labeled
oligonucleotide primers to produce an amplified DNA product that
can be detected and quantified absolutely. A wide range of
fluorochromes are now commercially available with spectral
characteristics (.lambda..sub.max excitation and .lambda..sub.max
emission) covering the wavelength range 350 to 700 nm, and into the
near infra-red region of the electromagnetic spectrum. Thus,
simultaneous, multiple detection of labeled molecules can be
performed on the same sample, for example, following `multiplex`
PCR amplification of several nucleic acid sequences using pairs of
oligonucleotide primers labeled with different fluorophores. Each
pair gives rise to a separate amplified product that can be
unambiguously identified due to its fluorescent label.
[0008] FISH Methods
[0009] Fluorescent in situ hybridization (FISH) is an important
tool for clinical diagnosis and gene mapping. Labeled nucleic acid
probes are used with multiple, simultaneous fluorescent detection
to locate specific nucleic acid sequences in cells and tissues, and
with the number of fluorochromes available there is the potential
to visualize several fluorescent signals relating to different
genetic sequences within the same sample.
[0010] Nucleic Acid Microarrays
[0011] Microarrays of nucleic acids that are prepared by
combinatorial chemistry methods provide a convenient means to assay
multiple, up to tens of thousands, analytes simultaneously.
Typically, microarrays are probed with fluorescently labeled
nucleic acid species, for example, from a clinical sample, and the
position of any hybridized, labeled nucleic acid identified using
fluorescence microscopy. The position relates to a known nucleic
acid sequence immobilized at that part of the microarray.
[0012] Fluorescence Energy-Transfer (FRET) Methods
[0013] FRET relies upon the interaction of a fluorescent donor dye
and a fluorescent acceptor dye, both of which are attached to the
same molecule. If the donor and acceptor dyes are in proximity to
one another, the acceptor dye quenches the fluorescent signal of
the donor dye upon excitation. However, when the two dyes are held
apart from one another, the fluorescence of the donor dye can be
detected.
[0014] Molecular Beacon Methods
[0015] Molecular beacons are nucleic acid probes that contain both
a covalently attached fluorescent dye and a non-fluorescent
quencher moiety. Molecular beacons allow the diagnostic detection
of specific nucleic acid sequences through their structural
characteristics. The probes possess hairpin-forming regions, and in
the absence of a complementary nucleic acid strand, the fluorescent
dye and the quencher are in close proximity to one another and
quenching of the fluorescent signal results. When incubated with a
target nucleic acid analyte that possesses a complementary
sequence, the probe anneals to the target, such that the
fluorescent dye and the probe are held apart from one another, and
fluorescence can be detected signifying the presence of a
particular nucleic acid sequence.
[0016] Preferably, methods to detect and/or quantify nucleic acid
analytes are carried out as homogeneous assays, which require no
separate analyte manipulation or post-assay processing.
Classically, agarose gel electrophoresis, possibly followed by
Southern-blot hybridization or enzyme-linked immunoassays was used
to detect and quantitate nucleic acid. Maniatis et al. (1982)
"Molecular Cloning: A Laboratory Manual," Cold Spring Harbor
Laboratory Press, NY, which is incorporated herein by reference in
its entirety. Such procedures, and other methods that similarly
rely on end-point analysis are generally labor intensive, require
several separate and distinct handling processes and skilled
personnel, are relatively slow to produce a result, and are prone
to contamination and false positives due to the open system. In
comparison, the advantages of a homogeneous assay, which represents
a fully enclosed homogenous real-time detection system, include a
faster turn-around time, especially when using microvolumes, higher
throughput, better options for automation and multi-parallel
analysis, and the use of a fully enclosed test system, which
reduces the likelihood of contamination.
[0017] Homogeneous assays are particularly desirable with PCR
methods and other amplification methods, because the amplification
and the detection of the nucleic acid analyte can be carried out in
one step without any risk of cross-contamination, which is a severe
problem with all methods that rely on extensive amplification,
especially in high-throughput analysis labs.
[0018] Prior art homogeneous detection and quantification methods
for nucleic acid analytes involve a variety of chemistries and
formats, which are exemplified below. Each of these methods is
associated with certain disadvantages that create a need for
improved detection/quantification strategies.
[0019] Hydrolysis Probes
[0020] Hydrolysis probes are described in Holland and Gelfand
(1991) Proc. Natl. Acad. Sci. USA 88:7376-80 and U.S. Pat. No.
5,210,015, each of which is incorporated herein by reference in its
entirety. This method takes advantage of the 5'-exonuclease
activity present in the thermostable Taq DNA polymerase enzyme used
in PCR (TAQMAN.TM. probe technology, Perkin-Elmer Applied
Biosystems, Foster City, Calif., USA) and is applied to homogeneous
detection in PCR, as described by Heid et al. (1996) Genome Methods
6:986-94, which is incorporated herein by reference in its
entirety. This method involves the use of a nucleic acid probe is
used which is labeled with a fluorescent detector dye and an
acceptor dye. Typically, the 2 dyes are attached to the 5'- and
3'-termini of the probe and when the probe is intact, the
fluorescence of the detector dye is quenched by fluorescence
resonance energy transfer (FRET). The probe anneals downstream of
the amplification target site on the template DNA in PCR reactions.
Amplification is directed by standard primers upstream of the
probe, using the polymerase activity of the Taq enzyme. FRET
quenching continues until the Taq polymerase reaches the region
where the labeled probe is annealed. Taq polymerase recognizes the
probe-template hybrid as a substrate, hydrolyzing the 5' detector
dye during primer-directed DNA amplification. The hydrolysis
reaction releases the quenching effect of the quencher dye on the
reporter dye, thus resulting in increasing detector fluorescence
with each successive PCR cycle.
[0021] Mixed RNA/DNA sequence probes can also serve as hydrolysis
probes to monitor PCR reactions, as described by Winger et al.,
U.S. Pat. No. 6,251,600 B1, which is hereby incorporated herein by
reference in its entirety. The mixed RNA/DNA probes contain blocks
of DNA nucleotides at either end of the probe and a stretch of RNA
nucleotide sequence between the flanking DNA blocks. This type of
probe also contains a detector and an acceptor dye, which are
attached to the respective DNA blocks of the probe. Upon
hybridization to a nucleic acid analyte, the resulting hybrid
contains two stretches of DNA:DNA duplex structure, flanking a
stretch of DNA:RNA duplex structure. In the presence of the enzyme
RNAse H, the DNA:RNA duplex structure is subject to cleavage,
because RNAse H specifically recognizes DNA:RNA duplexes and
cleaves the RNA portion of these duplexes. As a result the two
blocks of DNA nucleotide sequence of the probe are separated, which
in turn results in an increased fluorescence of the detector dye,
which is no longer quenched by the acceptor.
[0022] The efficiency of hydrolysis probes in homogeneous assays is
generally limited by their inherent fluorescence background, which
is caused by incomplete quenching. Fluorescence quenching in these
probes is caused by fluorescence energy transfer (FRET), which
decreases with the inverse sixth power of the distance between the
donor and the acceptor. Since the two dyes of the FRET pair are not
in close molecular proximity, the quenching in hydrolysis probes is
inherently incomplete resulting in an observable fluorescence
background and therefore in a low signal to noise ratio.
Additionally, the efficiency of hydrolysis probes is highly
dependent on the purity of the probes, because contamination with
singly labeled probes results in unquenched fluorescence and
therefore a high background.
[0023] Hairpin Probes
[0024] Hairpin probes or molecular beacons are described by Tyagi
et al. (1996) Nat. Biotechnol. 14:303-308, and are applied to
homogeneous detection in PCR, as described by Marras et al. (1999)
Genetic Analysis 14:151-156, each of which is incorporated herein
by reference in its entirety. Molecular beacons are nucleic acid
probes that are able to form a hairpin substructure due to the
presence of two inverted repeat sequences. They carry covalently
attached detector and quencher dyes at the end of both arms of the
hairpin substructure of the probe. This design allows for
self-complementary hybridization of the two inverted repeat
sequences to form a stable, hairpin structure in the absence of a
specific target. The detector and quencher dyes are in close
proximity to one another in this conformation, which results in
quenching of the detector fluorescence. The stretch of nucleotide
sequence between the inverted repeat sequences of a molecular
beacon is complementary to its target, thus directing specific
probe-target hybridization, which results in efficient separation
of the quencher dye from the detector dye with subsequent light
emission. Thus, in the presence of a complementary target sequence,
the hairpin structure is eliminated and the separated dye
fluoresces. No overlap is required between the emission spectrum of
the fluorophore and the absorption spectrum of the quencher. This
allows for a wider range of fluorophores to be used in molecular
beacons as compared with hydrolysis probes (TAQMAN.TM. probes).
Hairpin probes are most commonly used as "free-floating" probes to
detect amplicons as they are produced by standard PCR
amplification, but can also be attached to one of the primers to
act as a self-probing beacon as described by Whitcombe et al.
(1999) Nat. Biotechnol. 17:804-807, which is incorporated herein by
reference in its entirety.
[0025] Hairpin probes are particularly difficult to design because
their successful application requires several design conditions to
be fulfilled simultaneously. Firstly, the two inverted repeats of
the hairpin structure must have complementary counterparts in the
target nucleic acid, which in turn requires the presence of
inverted repeats in the target as well, a condition that is not
generally met. Secondly, the T.sub.m of the loop portion of the
hairpin structure with a complementary nucleic acid sequence and
the T.sub.m of the stem portion need to be carefully balanced with
respect to the temperature of the assay to allow the specific
unfolding of the hairpin probe in the presence of the target
without unspecific unfolding. Improper design of hairpin probes
results in high fluorescence background and therefore a low signal
to noise ratio. The efficiency of hairpin probes is particularly
sensitive to the purity of the probes, because even minimal amounts
of singly labeled impurities result in a high background in the
assay.
[0026] Hybridization Probes
[0027] Hybridization probe design entails the use of two
sequence-specific nucleic acid probes, each labeled with a
fluorescent dye, one dye being a donor dye, the other dye being an
acceptor dye. Typically, the two probes are designed to hybridize
to a nucleic acid analyte close to each other in a head-to-tail
arrangement that brings the two dyes into close proximity. In this
arrangement, as demonstrated by Cardullo et al. (1988) Proc. Natl.
Acad. Sci. USA 85:8790-04, which is incorporated herein by
reference in its entirety, the fluorescence of the acceptor dye is
enhanced if the donor is excited due to the radiationless uptake of
energy from the donor. This method is applicable to PCR reactions
(LIGHTCYCLER.TM. PCR technology, Roche Diagnostics, Indianapolis,
Ind., USA), as demonstrated by e.g. Espy et al. (2000) J. Clin.
Microbiol. 38:795-799, which is incorporated herein by reference in
its entirety. For use with the LIGHTCYCLER.TM. instrument of Roche
Diagnostics the 3'-end of one probe is labeled with fluorescein as
a donor and the 5'-end of the other probe can be labeled with LC
Red 640 or LC Red 705 as an acceptor. Upon the occurrence of FRET
between the donor and the acceptor, the fluorescence of the
acceptor is detected. The transfer of fluorescent resonance energy
only occurs when both probes hybridize to the target in very close
proximity, the optimal distance being one to five intervening bases
between probes. Hybridization probes are used in conjunction with
standard primers to direct the PCR amplification.
[0028] Assays based on hybridization probes require the design of
two oligonucleotide probes and their synthesis and purification,
which adds cost and increases the complexity of assays. The use of
two different probes in each analysis is particularly
disadvantageous in high-throughput settings where a multitude of
samples need to be analyzed due to the linear relationship of the
number of involved probes and the number of analyses to be
performed. Additionally, assays based on hybridization probes are
more difficult to multiplex due to the presence of a higher number
of probes, each of which could potentially generate artifacts, such
as false positives in a multiplexed analysis.
[0029] Probeless Detection
[0030] Probeless detection of nucleic acids takes advantage of the
affinity of certain dyes for double stranded DNA. Ideally, a dye
that is suitable for a probeless detection displays low or no
fluorescence at all when not bound to double stranded DNA, but a
bright fluorescence when attached to the DNA. Thus, upon binding of
the dye to DNA, a fluorescent signal is generated that indicates
the presence of the DNA. The binding of the dye occurs in a
non-covalent manner and is not specific for the sequences of the
DNA analyte. The method is applicable to PCR reactions where the
presence or absence of amplicons can be monitored as the PCR
reactions progress. Examples of probeless detection strategies for
PCR reactions are exemplified by Higuchi et al. (1992)
Biotechnology 10:412-417 and Wittwer et al. (1997) BioTechniques
22:130-138, each which is incorporated herein by reference in its
entirety. Probeless detection strategies also involve the use of
covalently linked dye pairs, wherein one of the dyes is a
fluorescent intercalator, as described by Makino et al., U.S. Pat.
Application Serial No. 2001/0014452A1. In this technique, the
fluorescence of the intercalator is quenched by the second dye, the
efficiency of the quenching being dependent on the presence of
double stranded DNA. Upon the interaction of the covalently linked
dye pair with double stranded DNA the quenching becomes less
efficient and a fluorescence signal can be detected. Probeless
detection and quantitation strategies are inherently
disadvantageous due to their non-specific nature. In general, these
methods detect any kind of double stranded DNA regardless of the
presence or absence of specific sequences. Therefore, probeless
detection methods are prone to generate "false positives," caused
by e.g. the formation of primer dimers or non-specific
amplification products in PCR reactions.
[0031] The detection of nucleic acid targets has also been
described with a variety of other strategies that involve
fluorescent detection. For example, Cardullo et al. (1998) Proc.
Natl. Acad. Sci. USA 85:8790-04, describe the use of competitive
hybridization probes, i.e. a pair of complementary
oligodeoxynucleotides, each member of the pair being labeled with a
covalently attached fluorescent dye at the 5'-terminus, which form
a short stretch of double stranded DNA in the assay. The two dyes
of the oligo-deoxynucleotide pair form a FRET system in which the
fluorescence of the donor dye is quenched while the
oligodeoxynucleotides are hybridized to each other. In the presence
of a target nucleic acid analyte, the probes competitively
hybridize with the target, which separates the two components of
the FRET system resulting in observable fluorescence of the donor
component. This method suffers from the disadvantage of being
dependent on a FRET mechanism with the associated high fluorescence
background. In addition, two probes are required per assay, which
increases the complexity and the cost of the assay.
[0032] None of the described fluorescence based methods combines
the desired features of homogeneous methods to detect and/or
quantify nucleic acid analytes, i.e. high specificity, low
fluorescence background and therefore a high signal to noise ratio,
ease of probe design without restrictions caused by the sequence of
the target, and low complexity associated with low cost.
[0033] The instant invention includes novel fluorescence based
methods to detect and/or quantify nucleic acid analytes and novel
nucleic acid probes that combine the desired features of
homogeneous assays. The methods and probes of this invention have
significant advantages and do not suffer from the limitations
inherent to the prior art methods and probes. The nucleic acid
probes described in this invention carry a multitude of covalently
attached dyes in close molecular proximity and therefore have a
very low intrinsic fluorescence background. They are highly
sequence specific and not limited by complex design criteria, as
for example hairpin probes, and are applied as a single probe per
assay. They can easily be adopted in homogeneous assays, in
particular in PCR based assays, and provide the results of the
assays in real time. They are amendable to multiplexing in such
assays and can be used as primers of a PCR reaction, which further
simplifies PCR based assays. The probes are also applicable in
assays conducted on nucleic acid microarrays. Furthermore, in one
embodiment of the invention the probes provide switchable labels
that can be activated and deactivated by an adjustment of the pH of
the assay.
[0034] Probes that carry two covalently attached dyes in close
molecular proximity have been described by Shinozuka et al. (1994)
J. Chem. Soc. Chem. Comm. 1377-1378, which is incorporated herein
by reference in its entirety. However, the probes disclosed by
Shinozuka display a high fluorescence that is reduced upon the
interaction with a complementary nucleic acid target. These probes,
despite their usefulness in general studies of nucleic acid
association and hybridization, cannot be applied effectively in
homogeneous assays because of their intrinsic high fluorescence.
The probes of this invention have a very low intrinsic fluorescence
and are therefore superior to the prior art probes.
SUMMARY OF THE INVENTION
[0035] The present invention includes novel methods for detecting
nucleic acid analytes through their interactions with a nucleic
acid probe. The nucleic acid probes of the invention are comprised
of a nucleic acid that is derivatized with two or more
non-identical covalently attached dyes, at least one of the dyes
being a detector dye, which is fluorescent. The nucleic acid probes
are further characterized in that the attached dyes are in close
molecular proximity, as defined by being attached through linkers
at either the same or at adjacent nucleotides of the nucleic acid
probe. The methods provided by the invention are based on the
specific interaction of one of the dyes of a nucleic acid probe
with the analyte. The specific interaction of the dye with the
analyte results in a change of fluorescence of the detector dye
which can be measured to detect or quantify the analyte.
[0036] In the preferred embodiments of the invention the nucleic
acid probes, as defined herein, are comprised of the following
combinations of covalent dyes:
[0037] I) a fluorescent intercalator and a non-fluorescent
quencher;
[0038] II) a fluorescent intercalator and a donor dye of a FRET
system;
[0039] III) a fluorescent intercalator and an acceptor dye of a
FRET system;
[0040] IV) an intercalator and two dyes forming an excimer
pair;
[0041] V) an intercalator and two dyes forming an exciplex
pair;
[0042] VI) a fluorescent groove binder and a non-fluorescent
quencher;
[0043] VII) a fluorescent groove binder and a donor dye of a FRET
system;
[0044] VIII) a fluorescent groove binder and an acceptor dye of a
FRET system;
[0045] IX) a groove binder and two dyes forming an excimer pair;
and
[0046] X) a groove binder and two dyes forming an exciplex
pair.
[0047] The probes described herein generally provide low
fluorescent backgrounds, display enhanced binding affinities to
complementary nucleic acid analytes, do not rely on changes in
their secondary structure upon hybridization and do not require
secondary reactions, such as enzymatic reactions, to generate a
fluorescent signal.
[0048] In other embodiments, the present invention discloses novel
synthetic methods to covalently attach multiple dyes to nucleic
acids via multi-functional linker molecules. In addition
"universal" quencher molecules are introduced to attach two dyes,
typically a non-fluorescent quencher and a fluorescent
intercalator, to nucleic acids.
[0049] The nucleic acid probes described herein are particularly
useful in homogeneous assays, which do not require post-assay
manipulations of the reagents and assay products, can be carried
out in closed tubes to avoid cross-contamination, do not require
particularly trained personnel to conduct the assays, and provide
the analysis of samples in real time. The present invention
includes applications for PCR reactions in order to provide the
detection and/or quantification of PCR products in real time, and
transcription assays that provide the analysis of mRNA transcripts
in real time. Further applications of the novel nucleic acid probes
include assays that are conducted on nucleic acid microarrays, in
particular expression analysis and genotyping, especially the
detection of single nucleotide polymorphisms on genomic DNA.
[0050] In yet another embodiment, the invention discloses nucleic
acid probes comprised of a covalently attached pH-sensitive dye and
second fluorescent dye in close molecular proximity. These probes
are useful as pH sensitive probes and provide switchable labels
that allow the fluorescent signal of an assay to be turned on and
off depending on pH.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0051] FIG. 1 is a schematic representation of a method for
detecting and quantifying nucleic acid analytes employing specific
nucleic acid probes, which carry an intercalator serving as
detector dye and a non-fluorescent quencher (type I nucleic acid
probes).
[0052] FIG. 2 is a schematic representation of a method for
detecting and quantifying nucleic acid analytes employing specific
nucleic acid probes, which are attached to a FRET system composed
of a donor dye and a fluorescent intercalator serving as detector
dye (type II nucleic acid probes).
[0053] FIG. 3 is a schematic representation of a method for
detecting and quantifying nucleic acid analytes employing specific
nucleic acid probes, which are attached to a FRET system composed
of an acceptor dye and a fluorescent intercalator serving as
detector dye (type III nucleic acid probes).
[0054] FIG. 4 is a schematic representation of a method for
detecting and quantifying nucleic acid analytes employing specific
nucleic acid probes, which carry an intercalator and two dyes
forming an excimer pair (type IV nucleic acid probes). Upon
hybridization of the probe and subsequent incorporation of the
intercalator into the double stranded region the distance of the
paired dyes increases leading to a reduced fluorescence.
[0055] FIG. 5 is a schematic representation of a method for
detecting and quantifying nucleic acid analytes employing specific
nucleic acid probes, which carry an intercalator and two dyes
forming an exciplex pair (type V nucleic acid probes). Upon
hybridization of the probe and subsequent incorporation of the
intercalator into the double stranded region the distance of the
paired dyes increases leading to a reduced fluorescence.
[0056] FIG. 6 depicts a schematic representation of a method for
detecting and quantifying nucleic acid analytes employing specific
nucleic acid probes, which carry both a groove binder serving as
detector dye and a non-fluorescent quencher (type VI nucleic acid
probes).
[0057] FIG. 7 depicts a schematic representation of a method for
detecting and quantifying nucleic acid analytes employing specific
nucleic acid probes, which are attached to a FRET system composed
of a donor dye and a fluorescent groove binder serving as detector
dye (type VII nucleic acid probes).
[0058] FIG. 8 depicts is a schematic representation of a method for
detecting and quantifying nucleic acid analytes employing specific
nucleic acid probes, which are attached to a FRET system composed
of an acceptor dye and a fluorescent groove binder serving as
detector dye (type VIII nucleic acid probes).
[0059] FIG. 9 is a schematic representation of a method for
detecting and quantifying nucleic acid analytes employing specific
nucleic acid probes, which carry a groove binder and two dyes
forming an excimer pair (type IX nucleic acid probes). Upon
hybridization of the probe and subsequent interaction of the groove
binder with the double stranded region the distance of the paired
dyes increases leading to a reduced fluorescence.
[0060] FIG. 10 is a schematic representation of a method for
detecting and quantifying nucleic acid analytes employing specific
nucleic acid probes, which carry a groove binder and two dyes
forming an exciplex pair (type IX nucleic acid probes). Upon
hybridization of the probe and subsequent interaction of the groove
binder with the double stranded region the distance of the paired
dyes increases leading to a reduced fluorescence.
[0061] FIG. 11 is a schematic representation of the principle of
nucleic acid probes that carry both a pH sensitive dye, which is
represented as a trityl group, e.g. a DMT-group, and another
fluorescent dye (type XI nucleic acid probes). At high pH, e.g. pH
7-9, the pH-sensitive trityl group exists in its protonated form
and has essentially no absorbance. Under these conditions of pH the
fluorescence of the second dye is observable. Upon a change in the
pH towards more acidic conditions, e.g. pH 3-6, a trityl cation is
formed that absorbs strongly at approximately 500 nm and the
fluorescence of the second dye is efficiently quenched leading to a
reduced fluorescence.
[0062] FIG. 12 is a schematic representation of a method for
detecting and quantifying nucleic acid analytes employing specific
nucleic acid probes, which carry an intercalator serving as
detector dye and a non-fluorescent quencher (type I nucleic acid
probes), in homogenous assays simultaneously with a PCR reaction. A
fluorescence signal is generated in the annealing step of each
cycle of the PCR that is proportional to the amount of amplicon
formed in the reaction.
[0063] FIG. 13 is a schematic representation of a method for
detecting and quantifying nucleic acid analytes employing specific
nucleic acid probes, which carry an intercalator serving as
detector dye and a non-fluorescent quencher (type I nucleic acid
probes), as sequence specific primers of a PCR reaction in
homogenous assays. A fluorescence signal is generated that is
proportional to the amount of amplicon formed in the reaction.
[0064] FIG. 14 displays the temperature dependence of the relative
fluorescence intensity of nucleic acid probe (17.2) in the absence
and in the presence of a complementary sequence monitored at
emission wavelengths of 530 nm and 645 nm upon excitation at 470
nm, as described in Example 10.
[0065] FIG. 15 displays the temperature dependence of the
fluorescence intensity of the nucleic acid probe (17.11) in the
absence and in the presence of a complementary sequence monitored
at an emission wavelength of 528 nm upon excitation at 510 nm, as
described in Example 11.
[0066] FIG. 16 displays the temperature dependence of the
fluorescence intensity of the nucleic acid probe (17.20) in the
absence and in the presence of a complementary sequence monitored
at an emission wavelength of 625 nm upon excitation at 510 nm, as
described in Example 11.
[0067] FIG. 17 displays the temperature dependence of the
fluorescence intensity of the nucleic acid probe (17.21) in the
absence and in the presence of a complementary sequence monitored
at an emission wavelength of 528 nm upon excitation at 510 nm, as
described in Example 11.
[0068] FIG. 18 displays the temperature dependence of the
fluorescence intensity of the nucleic acid probe (17.21) in the
absence and in the presence of a complementary sequence monitored
at an emission wavelength of 528 nm upon excitation at 420 nm, as
described in Example 11.
[0069] FIG. 19 displays the observed fluorescence in a real-time
PCR experiment with the nucleic acid probe (17.21) and a target
nucleic acid sequence related to the human adenine deaminase gene
as a function of the PCR cycle number, as described in Example
14.
[0070] FIG. 20 displays the observed fluorescence in real-time PCR
experiments with the nucleic acid probe (17.21) as a function of
the PCR cycle number, wherein a serial dilution of the target
nucleic acid is employed, as described in Example 15.
[0071] FIG. 21 displays the observed fluorescence in a real-time
PCR experiment with the nucleic acid probe (17.21) and a target
nucleic acid sequence related to the human prothrombin gene as a
function of the PCR cycle number, as described in Example 14.
DETAILED DESCRIPTION OF THE INVENTION
[0072] The present invention includes novel methods for detecting
nucleic acid analytes through their interactions with a nucleic
acid probe. The nucleic acid probes of the invention are comprised
of a nucleic acid that is derivatized with two or more
non-identical covalently attached dyes, at least one of the dyes
being a detector dye, which is fluorescent. The nucleic acid probes
are further characterized in that the attached dyes are in close
molecular proximity, as defined by being attached through linkers
at either the same or at adjacent nucleotides of the nucleic acid
probe. The methods provided by the invention are based on the
specific interaction of one of the dyes of a nucleic acid probe
with the analyte. The specific interaction of the dye with the
analyte results in a change of fluorescence of the detector dye
which can be measured to detect or quantify the analyte.
[0073] Various terms are used herein to refer to aspects of the
present invention. To aid in the clarification of the description
of the components of the invention, the following descriptions are
provided.
[0074] It is to be noted that the term "a" or "an" entity refers to
one or more of that entity; for example, a nucleic acid that
carries a multitude of dyes refers to one or more nucleic acids
that carry a multitude of dyes. As such, the terms "a" or "an,"
"one or more" and "at least one" are used interchangeably
herein.
[0075] The term "analyte" refers to a nucleic acid molecule or a
mixture of nucleic acid molecules, as defined below, that is to be
detected or quantified using the method of this invention. The
terms "target nucleic acid analyte" and "nucleic acid analyte" are
used interchangeably with the term analyte in the context of this
invention.
[0076] As used herein, "nucleic acid" means either DNA, RNA,
single-stranded or double-stranded and any chemical modifications
thereof, such as PNA and LNA. Nucleic acids can be of any size and
are preferably oligonucleotides. Modifications include, but are not
limited to, those that provide other chemical groups that
incorporate additional charge, polarizability, hydrogen bonding,
electrostatic interaction, and functionality to the individual
nucleic acid bases or to the nucleic acid as a whole. Such
modifications include, but are not limited to, modified bases such
as 2'-position sugar modifications, 5-position pyrimidine
modifications, 8-position purine modifications, modifications at
cytosine exocyclic amines, substitution of 5-bromo-uracil; backbone
modifications, methylations, unusual base-pairing combinations such
as the isobases isocytidine and isoguanidine and the like.
Modifications can also include 3' and 5' modifications such as
capping. The nucleic acid can be derived from a completely chemical
synthesis process, such as a solid phase mediated chemical
synthesis, or from a biological origin, such as through isolation
from almost any species that can provide DNA or RNA, or from
processes that involve the manipulation of nucleic acids by
molecular biology tools, such as DNA replication, PCR
amplification, reverse transcription, or from a combination of
those processes. Virtually any modification of the nucleic acid and
nucleic acids of virtually any origin are contemplated by this
invention.
[0077] "Covalently attached" in the context of this invention
describes an attachment of one molecular moiety to another
molecular moiety through covalent chemical bonds, i.e. chemical
bonds that are established through the pairing of electrons from
the atoms that are bonded together.
[0078] A "dye" in the context of this invention is any organic or
inorganic molecule that absorbs electromagnetic radiation at a
wavelength greater than or equal to 340 nm.
[0079] A "fluorescent dye" as defined herein is any dye that emits
electromagnetic radiation of longer wavelength by a fluorescence
mechanism upon irradiation by a source of electromagnetic
radiation, including but not limited to a lamp, a photodiode or a
laser.
[0080] A "linker" is defined herein as a molecular entity that
covalently connects a dye to a nucleic acid. A linker can be of any
chemical nature known to those skilled in the art. Typically, a
linker contains functional groups that are attachment points that
covalently connect the linker to the nucleic acid and the dye.
Examples of functional groups that provide the attachment points
include, but are not limited to, amino groups, thiol groups,
carboxy groups, diene groups, dienophile groups, ester groups and
phosphodiester groups, as well as, virtually any chemical
functional groups that are known. A linker, aside from containing
functional groups as attachment points for the nucleic acid and
dyes, can consist of any chemical moiety that can carry at a
minimum two functional groups to provide attachment points.
Chemical moieties which are suitable as linker structures include,
but are not limited to, linear, cyclic and branched structures and
any combination thereof.
[0081] A "nucleic acid probe" as defined herein is a nucleic acid
that carries a multitude of covalently attached dyes, with at least
one of the dyes being fluorescent. Preferably, a nucleic acid probe
contains two or three covalently attached dyes. Nucleic acid probes
as defined herein are additionally characterized by a close
molecular proximity of all attached dyes. A "close molecular
proximity" in the context of the present invention means that the
corresponding dyes are attached to the same nucleotide of the
nucleic acid or to two adjacent nucleotides of the nucleic acid.
The attachment of any dye to the nucleic acid consists of a linker
as defined herein that is covalently attached to both the nucleic
acid and the dye. The linker can connect one or more dyes to the
nucleic acid.
[0082] A "detector dye" as defined herein is a fluorescent dye
which is covalently attached to a nucleic acid probe as defined in
this invention and which changes its fluorescent properties upon
the interaction of the nucleic acid probe with an analyte.
[0083] An "intercalator" as defined herein is a dye which is
covalently bound to a nucleic acid probe and which is capable of
interacting with double stranded DNA by intercalation.
[0084] A "groove binder" as defined herein is a dye which is
covalently bound to a nucleic acid probe and which is capable of
interacting with double stranded DNA by binding to the minor groove
or the major groove of the double stranded DNA.
[0085] A "quencher" as defined herein is a dye that reduces the
emission of fluorescence of another dye. The reduction of
fluorescence emission can be caused by a radiationless energy
transfer through space (Fluorescence Resonance Energy Transfer
(FRET)), see Yang et al. (1997) Methods Enzymol. 278:417-44, which
is incorporated herein by reference in its entirety, or by the
formation of ground state heterodimers, see Bernacchi et al. (2001)
Nucleic Acids Res. 29:e62, which is incorporated herein by
reference in its entirety, or by other mechanisms.
[0086] A "donor" as defined herein is a dye that is part of a FRET
system in which the dye transfers energy to another dye by a
radiationless process. Generally, in such a system the fluorescence
of the dye decreases when it is part of a FRET system. FRET is
described in detail in Yang et al. (1997) Methods Enzymol.
278:417-44.
[0087] An "acceptor" as defined herein is a dye that is part of a
FRET system in which the dye accepts energy from another dye by a
radiationless process. Generally, in such a system the fluorescence
of the dye increases when excited at the wavelength of the
corresponding donor of the FRET system when compared to the
fluorescence of the dye when it is not part of a FRET system, see
Yang et al. (1997) Methods Enzymol. 278:417-44.
[0088] An "excimer pair" as defined herein consists of a pair of
identical dyes that form an excimer upon exposure to
electromagnetic radiation. The dyes are covalently bound through a
linker structure that ensures their close molecular proximity. The
excimer formed by the excimer pair is fluorescent. An overview of
excimers is provided in De Schryver et al. (1987) Acc. Chem. Res.
20:159-66 and Birks (1967) Nature 214:1187-90, each of which is
incorporated herein by reference in its entirety.
[0089] An "exciplex pair" as defined herein consists of a pair of
non-identical dyes that form an exciplex upon exposure to
electromagnetic radiation. The dyes are covalently bound through a
linker structure that ensures their close molecular proximity. The
exciplex is fluorescent. Exciplexes are described by Birks (1967)
Nature 214:1187-90.
[0090] A "pH sensitive dye" as defined herein is a dye that is
covalently bound to a nucleic acid probe and which undergoes a
change in its absorption properties upon a change of pH. The change
of the absorption properties of the dye can be due to the
protonation or the deprotonation of the dye and can encompass an
enhancement of the dyes absorption or a decrease of the dyes
absorption at a given wavelength.
[0091] A "homogeneous assay" as defined herein is a process to
detect or quantify a nucleic acid analyte that requires no separate
analyte manipulation or post-assay processing to record the result
of the assay. Homogeneous assays are carried out in closed tubes,
meaning that no further addition of reagents or supplementary
chemicals is necessary to record the result once the assay is
started. Homogeneous assays allow recordation of the result of the
assay in real time, meaning that the result of the assay can be
continuously recorded as the assay progresses in time.
[0092] The present invention includes the use of nucleic acid
probes comprised of a number of covalently attached non-identical
dyes in novel methods for the detection and quantitation of
analytes. The nucleic acid probes are further characterized by a
close molecular proximity of the dyes that are covalently attached
to the nucleic acid.
[0093] In one embodiment, the present invention includes a method
for the detection or quantification of a nucleic acid analyte
comprising the steps of: (a) providing a nucleic acid probe,
wherein said nucleic acid probe is comprised of a nucleic acid that
is derivatized with two or more non-identical covalently attached
dyes, wherein at least one dye is fluorescent, and wherein at least
one dye has a high affinity to double stranded nucleic acids,
wherein the dyes are attached at either the same or at adjacent
nucleotides of the nucleic acid probe; (b) contacting said nucleic
acid probe with a nucleic acid analyte so as to allow for the
hybridization of the nucleic acid probe with the nucleic acid
analyte; and (c) measuring the change in the fluorescence of the
nucleic acid probe that occurs upon the hybridization of the
nucleic acid probe with the nucleic acid analyte.
[0094] In preferred embodiments of the invention the nucleic acid
probes, used in the method of this invention, are comprised of the
following combinations of covalent dyes:
[0095] I) a fluorescent intercalator and a non-fluorescent
quencher;
[0096] II) a fluorescent intercalator and a donor dye of a FRET
system;
[0097] III) a fluorescent intercalator and an acceptor dye of a
FRET system;
[0098] IV) an intercalator and two dyes forming an excimer
pair;
[0099] V) an intercalator and two dyes forming an exciplex
pair;
[0100] VI) a fluorescent groove binder and a non-fluorescent
quencher;
[0101] VII) a fluorescent groove binder and a donor dye of a FRET
system;
[0102] VIII) a fluorescent groove binder and an acceptor dye of a
FRET system;
[0103] IX) a groove binder and two dyes forming an excimer pair;
and
[0104] X) a groove binder and two dyes forming an exciplex
pair.
[0105] In one embodiment of the present invention, the dye that has
a high affinity to double stranded nucleic acids is an
intercalator. In another embodiment, the dye that has a high
affinity to double stranded nucleic acids is a groove binder.
[0106] In another embodiment, the present invention includes a
method for the detection or quantification of a nucleic acid
analyte comprising the steps of: (a) providing a nucleic acid
probe, wherein said nucleic acid probe is comprised of a nucleic
acid that is derivatized with two non-identical covalently attached
dyes, of which at least one dye is fluorescent, and of which one
dye is a pH-sensitive dye, wherein the dyes are attached at either
the same or at adjacent nucleotides of the nucleic acid probe; (b)
contacting said nucleic acid probe with a nucleic acid analyte so
as to allow for the hybridization of the nucleic acid probe with
the nucleic acid analyte; (c) removing the unhybridized nucleic
acid probe from the mixture; and (d) measuring the fluorescence of
the hybridized nucleic acid probe under one or more conditions
where the pH of the medium is defined.
[0107] In a preferred embodiment, the pH-sensitive dye is selected
from the group including, but not limited to a substituted trityl
group, such as a derivative of the dimethoxytrityl group and the
covalent attachment of the substituted trityl group to the nucleic
acid probe is provided through a linkage of one of the aromatic
rings of the substituted trityl group to one of the nucleotides of
the nucleic acid probe. The method of claim 24 wherein the
substituted trityl group is a derivative of the dimethoxytrityl
group.
[0108] Included in the present invention is a method of preparing
nucleic acid probes, said probes comprised of a nucleic acid that
is derivatized with two or more non-identical covalently attached
dyes comprising the steps of: (a) providing an oligonucleotide that
is derivatized with a diene-moiety and an amino group; (b) reacting
the oligonucleotide with a first dye that is derivatized with a
dienophile-moiety; and (c) reacting the oligonucleotide with a
second dye that is derivatized with an amine-reactive moiety. The
present invention also includes the probes prepared according to
the method of this invention, wherein said nucleic acid probes are
comprised of a nucleic acid that is derivatized with two or more
non-identical covalently attached dyes, wherein at least one dye is
fluorescent, and wherein at least one dye has a high affinity to
double stranded nucleic acids, wherein the dyes are attached at
either the same or at adjacent nucleotides of the nucleic acid
probe.
[0109] Also included in the present invention is a method of
preparing an oligonucleotide that is derivatized with a diene
moiety and an amino group, said method comprising the steps of: (a)
synthesizing an oligonucleotide sequence using standard solid phase
phosphoramidite synthesis, using nucleoside phosphoramidites as the
reactants; (b) conducting a last phosphoramidite synthesis cycle
wherein a phosphoramidite is applied in the coupling step of the
synthesis cycle that carries a diene-moiety and a protected amino
group; and (c) deprotecting the oligonucleotide. The present
invention includes the oligonucleotides prepared by this
method.
[0110] Conjugation Chemistry
[0111] The covalent attachment of dyes to nucleic acids can be
achieved by a variety of methods known to those of skill in the
art. The covalent attachment of dyes to nucleic acids is reviewed
in Davies et al. (2000) Chem. Soc. Rev. 29:97-107, which is
incorporated herein by reference in its entirety. Examples include,
but are not limited to: incorporation of the dyes during the
synthesis of nucleic acids, typically solid phase synthesis,
post-synthetic labeling of either synthetic nucleic acids or
nucleic acids derived through enzymatic reactions, e.g. the PCR
reaction, and enzymatic methods of incorporation of dyes into
nucleic acids, e.g. the use of dye conjugated deoxynucleotide
triphosphates in primer elongation reactions such as a PCR
reaction.
[0112] The incorporation of dyes into oligonucleotides using solid
phase synthetic methods entails the conversion of the dyes into
their phosphoramidite derivatives, which are then employed in the
phosphoramidite solid phase synthetic method similar to nucleoside
phosphoramidites. Using this method, the amidite derived from a
particular dye is mixed with an activator such as 1H-tetrazole or
4,5-dicyanoimidazole, in a suitable solvent, and reacted with a
hydroxy group of a support bound oligonucleotide to form a covalent
phosphodiester bond between the oligonucleotide and the dye. The
attachment of dyes using the phosphoramidite method is reviewed by
Beaucage et al. (1993) Tetrahedron 49:1925-63 (1993), which is
incorporated herein by reference in its entirety.
[0113] Post-synthetic labeling of synthetic nucleic acids or
nucleic acids derived from enzymatic reactions involves the
incorporation of functional groups into the nucleic acids, which
serve as anchor points for the attachment of dyes. The dye is then
derivatized with a chemical group or moiety that can be reacted
with the functional group of the nucleic acid to promote the
formation of a covalent bond between the nucleic acid and the dye.
The functional group incorporated into the nucleic acid can be any
group that is capable of reacting selectively with the group or
moiety that is incorporated into the dye. Examples of functional
groups which can be incorporated into nucleic acids and groups or
moieties which can be incorporated into dyes, which can then
selectively react include, but are not limited to, amino
groups/active esters, e.g. hydroxysuccinimide esters, thiol
groups/electrophilic groups and dienes/dienophiles, e.g.
maleimides. Methods known to those skilled in the art to promote a
covalent bond between a nucleic acid and a dye are reviewed by
Grimm et al. (2000) Nucleosides & Nucleotides 19:1943-65, which
is incorporated herein by reference in its entirety.
[0114] The incorporation of functional groups into synthetically
derived nucleic acids can be achieved using a variety of methods. A
standard method known to those skilled in the art is the use of
linker phosphoramidites during solid phase synthesis. Linker
molecules useful in the solid phase phosphoramidite method consist
of an amidite moiety, a spacer and a functional group that is
protected if the functional group interferes with the amidite
synthesis. Examples include, but are not limited to, linkers to
introduce amino-functions, e.g. (1), thiol-functions, e.g. (2), or
diene functions, e.g. (3), below. There are a number of
commercially available phosphoramidite linkers. 1
[0115] The incorporation of functional groups into nucleic acids
derived from enzymatic reactions can be achieved by a variety of
methods. The standard method involves the use of primers that
contain a functional group at the 5'-end in PCR reactions. The use
of such primers results in the generation of PCR reaction products
(amplicons) that contain the functional group at the 5'-end of the
strand of the amplicon that is derived from the primer with the
functional group. An example is the use of 5'-amino derivatized
primers, as described by Kohsaka et al. (1993) Nucleic Acids Res.
21:3469-3472, which is incorporated herein by reference in its
entirety.
[0116] One method of enzymatically incorporating dyes into nucleic
acids is the use of dye-conjugated nucleoside triphosphates in
primer elongation reactions. This method is used extensively in
modifications of the Sanger sequencing method for nucleic acids, as
reviewed by Kashdan et al. in "Recombinant DNA Principles
Methodologies," Greene and Rao (eds), Dekker 1998, NY, which is
incorporated herein by reference. Other methods take advantage of
the enzymes T4 polynucleotide kinase or deoxynucleotityl
transferase, which incorporate dye-conjugated nucleosides through
their triphosphates at the 5'-end or the 3'-end of the nucleic
acid, respectively.
[0117] Covalent attachment of more than one dye to a nucleic acid
can be achieved by a variety of methods, including the use of
standard solid phase synthesis, post-synthetic labeling and
enzymatic methods, as described above, and any combination thereof
of these methods. The dyes can be attached through the use of more
than one monofunctional linkers that allow the covalent attachment
of one dye only, or through multifunctional linkers that allow the
attachment of more than one dye through the same linker.
[0118] The nucleic acid probes disclosed herein are comprised of
two or more non-identical dyes, covalently attached either to the
same nucleotide of a nucleic acid or to adjacent nucleotides of a
nucleic acid to provide a close molecular proximity of the dyes.
The linkage of the dyes to the nucleic acid can be provided using a
variety of methods as described in detail below.
[0119] Attachment with a Minimum of Two Different Linkers
[0120] According to this embodiment of the invention the linkers
can be of the same or of a different chemical nature and can
contain the same or different functional groups that provide the
attachment points for the dyes. The linkers can be attached to
almost any position of the nucleic acid including, but not limited
to, the 3'-terminus of the nucleic acid, the 5'-terminus of the
nucleic acid and/or an internal position on the nucleic acid.
Attachment points of the linkers to the nucleic acid include, but
are not limited to, the base of a nucleotide, the sugar of a
nucleotide and/or the phosphate group of a nucleotide.
[0121] Multiple dye incorporation according to this method may
involve protective group chemistry to address the sequential
incorporation of the dyes and has been described for the synthesis
of molecular beacons, see Tyagi et al. (1996) Nat. Biotechnol.
14:303-308. The synthesis of TAQMAN.TM. probes, and other probes
that require the covalent attachment of two dyes into nucleic acids
are described in Aubert et al. (2000) Nucleic Acids Res. 28:818-25,
which is incorporated herein by reference in its entirety.
[0122] Examples of methods to synthesize nucleic acid probes with
multiple non-identical dyes in close molecular proximity, which are
attached to the probe with different linkers are outlined below and
can easily be adopted by those skilled in the art.
[0123] 1. Attachment of one dye using post synthetic labeling at
the nucleoside base of the 5'-end of the nucleic acid and
attachment of a second dye on the same nucleoside using
phosphoramidite synthesis at the 5'-end. In one embodiment of this
invention, a sequence modifier phosphoramidite, e.g.
5'-dimethoxytrityl-5-[-N-(trifluoracetylaminohexyl)-
-3-acrylimido]-2'-deoxyuridine-3'-phosphoramidite (commercially
available from Glen Research, Sterling, Va., catalog number
10-1039, compound (4)), and a dye phosphoramidite, e.g.
fluorescein-phosphoramidite (commercially available from Proligo
LLC. Boulder, Colo., catalog number M010181, compound (5)), are
consecutively added as the last coupling units in the solid phase
synthesis of an oligonucleotide. Upon deprotection of the
oligonucleotide with aqueous ammonia, the 5'-terminal nucleoside is
covalently attached to two linkers. The first linker provides a
primary amino group at the 5-position of the uridine base and the
second linker consists of a phosphodiester group with a spacer and
a conjugated dye. The primary amino group of the first linker can
be employed in a reaction with an N-hydroxysuccinimide ester
derivative or another amine-reactive derivative of another dye to
provide a nucleic acid probe that contains two dyes attached
through different linkers to the 5'-end nucleoside of the
oligonucleotide. 2
[0124] 2. The attachment of one dye by the post synthetic labeling
of the nucleoside base at the 5'-end of the nucleic acid and
attachment of a second dye at the same nucleoside through a
protected linker phosphoramidite at the 5'-end, which is further
derivatized post-synthetically after deprotection. In another
embodiment of this invention, a sequence modifier phosphoramidite,
e.g. compound (4), and a terminus modifier phosphoramidite, e.g.
compound (1) (commercially available from a number of suppliers),
are added consecutively, as the last coupling units in the solid
phase synthesis of an oligonucleotide. Upon deprotection of the
oligonucleotide with aqueous ammonia the 5'-terminal nucleoside is
covalently attached to two linkers. The first linker provides a
free primary amino group at the 5-position of the uridine base and
the second linker consists of a phosphodiester group with a spacer
and a protected amino group. The primary amino group of the first
linker can be employed in a reaction with an N-hydroxysuccinimide
ester derivative or another amine-reactive derivative of a first
dye. A second dye can then be attached selectively after removal of
the monomethoxytrityl protective group from the amine with 80%
acetic acid, by the reaction of the oligonucleotide with an amine
reactive functional group on the second dye. The resulting nucleic
acid probe contains two dyes attached through different linkers at
the 5'-end nucleoside of the oligonucleotide.
[0125] 3. Attachment of one dye at the 3'-end of an oligonucleotide
through the use of a dye-modified CPG in the synthesis of the
oligonucleotide and the attachment of a second dye through
post-synthetic labeling at the nucleoside located at the 3'-end of
the oligonucleotide. In this embodiment of the invention, a 3'-dye
CPG, e.g. 3'-fluorescein-CPG (commercially available from Glen
Research, Sterling, Va., catalog number 20-2963, compound (6)) is
used to derivatize the oligonucleotide with one of the dyes. A
sequence modifier phosphoramidite, e.g. compound (4), is employed
in the first coupling to the support. Upon deprotection of the
oligonucleotide with aqueous ammonia the 3'-terminal nucleoside is
covalently attached to two linkers. The first linker provides a
free primary amino group at the 5-position of the uridine base and
the second linker consists of a phosphodiester group with a spacer
and a conjugated dye. The primary amino group of the first linker
can be employed in a reaction with an N-hydroxysuccinimide ester
derivative or another amine-reactive derivative of another dye to
provide a nucleic acid probe that contains two dyes attached
through different linkers at the 5'-end nucleoside of the
oligonucleotide. 3
[0126] Methods for the synthesis of nucleic acid probes with
multiple dyes in close molecular proximity that are attached with
different linkers are not limited to the provided examples. There
are numerous other functional groups, which will be known to those
of skill in the art, that can be employed in the post-synthetic
labeling described, various attachment points of the linkers
involved and other sequence modifier phosphoramidites of varying
chemical structure that can be employed in the synthesis of the
probes. Examples of other sequence modifiers are described in
Nelson et al. Nucleic Acids Res. 20:6253-6259; Grzybowski et al.
(1993) Nucleic Acids Res. 21:1705-1712; Behrens et al. (1999)
Nucleosides & Nucleotides 18: 291-305; Behrens et al. (1995)
Bioorganic & Medicinal Chemistry Letters 5:1785-90 and Endo et
al. (1994) Tetrahedron Letters 35:5879-5882, each of which is
specifically incorporated herein by reference in its entirety.
[0127] Attachment with a Branched Linker that Connects Two or More
Dyes to a Single Position
[0128] A branched linker contains a minimum of two functional
groups of the same or different chemical nature, which provide the
attachment points for the dyes and another functional group, which
provides the attachment point to the nucleic acid probe. Branched
linkers have been synthesized and applied to oligonucleotides for
the purpose of generating dendrimeric DNA structures that provide
signal amplification in hybridization assays through networks of
covalently connected labeled probes. For instance, the branching
phosphoramidites (7) and (8) serve as suitable linkers for the
generation of dendrimers (http://www.interactiva-
.de/knowledge/nucleicchem/modifiedoligos.htmL, information
retrieved September 2001). Other examples of branched linkers are
phosphoramidites (9) and (10), as described by Shchepinov et al.
(1999) Nucleic Acids Res. 27:3035-3041 and Shchepinov et al. (1997)
Nucleic Acids Res. 25:4447-4454 (1997), which were used to generate
dendrimeric oligonucleotides of various structures. Each of these
references is specifically incorporated herein by reference in its
entirety. Polushin (2000) Nucleic Acids Res. 28:3125-3133, which is
hereby incorporated by reference in its entirety, has described the
synthesis and the application of the branched linker structures
(11) and (12). In this embodiment of the invention, the branching
linker and a nucleoside are incorporated in one phosphoramidite
structure. Phosphoramidites 7-12 are useful for incorporating
multiple reporter molecules into a synthetic oligonucleotide on a
branched linker. 4
[0129] Included in the present invention is a method for the
synthesis and use of the bifunctional branched linkers (13) and
(14). Phosphoramidites (13) and (14) can be incorporated in the
solid phase phosphoramidite synthesis of oligonucleotides as the
last coupling unit to generate oligonucleotides with a branching
linker at the 5'-terminus. Both linkers are non-symmetrical and
provide a convenient way to covalently attach two non-identical
dyes to the oligonucleotide. 5
[0130] The synthesis of linker (13) is described in Example 1
(Scheme 1). With reference to Scheme 1,4-aminobutyric acid was
converted to either 4-Fmoc-aminobutyric acid
(Fmoc=fluorenemethyloxycarbonyl) or 4-MMT-aminobutyric acid
(MMT=monomethoxytrityl) by reaction with either Fmoc-chloride or
MMT-chloride. Both derivatives were further converted to their
p-nitrophenylesters by reaction with p-nitrophenol and DCC
(DCC=dicyclohexylcarbodiimide). Both esters were then successively
coupled with 1,3-diamino-2-propanol in the presence of a tertiary
base and the resulting product was phosphitylated at the secondary
hydroxyl group to provide linker (13).
[0131] Linker (13) carries a base-labile Fmoc protecting group,
which is removed during the deprotection of the oligonucleotide
with aqueous ammonia. The resulting free primary amino group can be
employed in a reaction with an N-hydroxysuccinimide ester
derivative or another amine-reactive derivative of a dye to
generate an oligonucleotide with one covalently attached dye and
one protected primary amino group. Following the removal of the
monomethoxytrityl group of the second amino group another
amine-reactive dye can be reacted with the oligonucleotide to
provide a nucleic acid probe that contains two non-identical dyes
attached through a branched linker. Examples 3 and 5 describe the
synthesis of an oligonucleotide sequence conjugated to bifunctional
linker (13).
[0132] The synthesis of the linker (14) is described in Example 2
(Scheme 2). With reference to Scheme 2,6-aminocaproic acid was
converted to either its N-(cyclohexa-2,4-dienyl)methoxycarbonyl
derivative or its N-Fmoc derivative by reaction with
cyclohexa-2,4-dienylmethanol and CDI (CDI=carbonyldiimidazol) or
Fmoc-chloride, respectively. Both derivatives were further
converted to their pentaflourophenylesters by reaction with
pentafluorophenol and DCC. Both esters were then coupled with
1,3-diamino-2-propanol in the presence of a tertiary base and the
resulting product was phosphitylated at the secondary hydroxyl
group to provide linker (14).
[0133] Linker (14) carries a base-labile Fmoc protective group,
which is removed during the deprotection of the oligonucleotide
with aqueous ammonia. The resulting free primary amino group can be
employed in a reaction with an N-hydroxysuccinimide ester
derivative or another amine-reactive derivative of a dye to
generate an oligonucleotide with one covalently attached dye and
one diene group. The diene can subsequently be employed in an
aqueous Diels-Alder reaction with a dye that is derivatized with a
dienophile, such as a maleimide, as described by Hill et al. (2001)
J. Org. Chem. 66:5352-58, which is incorporated herein by reference
in its entirety. The product of the Diels-Alder reaction is a
nucleic acid probe that contains two non-identical dyes attached
through a branched linker. Linker (14) provides the additional
advantage that the attachment of both dyes can be performed
simultaneously in a one-pot reaction. A one-pot reaction
dramatically reduces the effort that is applied with the synthesis
and purification in a stepwise sequential manner. Example 4
describes the synthesis of a dT10 oligonucleotide sequence
conjugated to bifunctional linker (14).
[0134] Example 6 describes the coupling of the linker (14) to the
5'-termini of oligonucleotides using solid phase phosphoramidite
synthesis with DCI (4,5-dicyanoimidazole) as the activator of the
amidite. Repeated couplings, i.e. repeated delivery of the amidite
and activator solution, proved to be advantageous with linker (14)
and three successive coupling steps were routinely employed. Also
included in Example 6 is the optional introduction of a
hexaethyleneglycol (HEG) spacer in between the oligonucleotide
sequence and the linker moiety. For this purpose a DMT-protected
HEG amidite is coupled to the oligonucleotide sequence before
linker (14) is introduced. It is well known that the nucleobases of
an oligonucleotide, in particular guanine, may contribute to the
quenching of attached fluorescent reporter dyes, as described by
Seidel et al. (1996) J. Phys. Chem. 100:5541-5553, which is
incorporated herein by reference in its entirety. A spacer moiety
such as the HEG spacer or an oligo-thymidine unit, as also
incorporated in some of the linker-modified oligonucleotides of
this invention, may reduce this kind of interference. Such spacer
moieties may further improve the efficiency of the final doubly
labeled nucleic acid probe as they impart increased range and
higher flexibility to the dyes, thereby facilitating their
interaction with the target sequence upon hybridization.
[0135] Attachment of Dye Combinations that are Chemically
Conjugated to the Nucleic Acid with a Single, Non-Branched
Linker
[0136] In one embodiment of the present invention, the linker is
comprised of a combination of at minimum two dyes, which are
covalently linked together. Examples of such dyes include, but are
not limited to, terminator dyes, which are being used in
sequencing. See for example Lee et al. (1997) Nucleic Acids Res.
25:2816-2822, which is incorporated herein by reference in its
entirety, which describes the use of 4-aminomethylbenzoic acid as a
bifunctional linker to connect substituted fluorescein and
rhodamine dyes. The two dyes are attached to oligonucleotides
through a single linker at the respective fluorescein dye
(BigDye.TM. technology).
[0137] Nucleic Acid Probes
[0138] Intercalators and groove binders are classes of dyes that
have an affinity to double stranded DNA. Upon interaction with the
DNA, an intercalator is characterized by its insertion between
adjacent base pairs of the DNA in an intercalative manner,
stabilized by van der Waals dispersion interactions with the base
pairs surrounding it. Examples of intercalators include, but are
not limited to, proflavine, ethidium bromide, acridine-derivatives,
polyaromatic hydrocarbons, e.g. pyrene-derivatives and
perylene-derivatives. The nucleic acid probes of this invention
include, but are not limited to, the examples of intercalators
provided above, as well as all other dyes that interact with double
stranded DNA in an intercalative manner.
[0139] Groove binders are characterized by their non-covalent
binding to the outer side of a double helical structure. Examples
of groove binders include Hoechst 33258, 2-phenylindole dyes, e.g.
4',6-diamidino-2-phenyli- ndole, berenil and netropsin. The nucleic
acid probes of this invention include, but are not limited to, the
examples of groove binders provided above, as well as all other
dyes that interact with double stranded DNA through non-covalent
binding to the outer side of the double helical structure. Further
examples of intercalators and groove binders, a discussion
regarding their structural features and their mechanism of binding
to DNA, as well as, the resulting implications for the structure of
double stranded DNA are provided in Neidle, "DNA Structure and
Recognition," Oxford University Press, which is incorporated herein
by reference in its entirety.
[0140] The asymmetric cyanine dye thiazole orange (TO) is another
example of a compound that has a strong affinity to double stranded
nucleic acids. Further interesting attributes of TO are a low
intrinsic fluorescence, a strong enhancement of fluorescence upon
binding to nucleic acids, and a high fluorescence quantum yield if
complexed to nucleic acids. The fluorescence enhancement that
occurs upon binding to nucleic acids is probably due to the
restriction of the rotation around the central bond of the dye in
the bound state, as discussed by Nygren et al. (1998) Biopolymers
46:39-51, which is incorporated herein by reference in its
entirety. The mechanism of binding of TO to nucleic acids has not
been fully explored, but it is assumed that intercalation
contributes to the observed strong affinity. However, groove
binding cannot be ruled out as well. Solutions of the dye TO are
well suited for staining nucleic acids in gels, as has been
demonstrated for monomeric or dimeric TO. See e.g. Rye et al.
(1992) Nucleic Acid Res. 20:2803-2812, which is incorporated herein
by reference in its entirety. In addition, sequence-specific
hybridization probes based on the non-natural nucleic acid backbone
PNA (peptide nucleic acids) that are labeled with TO have been
employed in homogenous assays. The PNA-TO conjugates indicate the
presence of a target sequence by an enhanced fluorescence compared
to the unhybridized state. PNA-TO conjugates are described by
Kubista et al., U.S. Pat. No. 6,329,144 and by Svanvik et al.
(2000) Anal. Biochem. 281:26-35, each of which is incorporated
herein by reference in its entirety. Conjugates of TO with DNA
oligonucleotides are less suitable as hybridization probes than the
PNA based probes because of their high intrinsic fluorescence,
which results from the binding of the positively charged dye to the
negatively charged backbone of the natural phosphodiester
oligonucleotide. PNA is employed in the hybridization probes of the
prior art in order to overcome this drawback because PNA provides
an uncharged backbone structure.
[0141] For the purpose of the present invention, a dye has a high
affinity to double stranded nucleic acids if it binds to double
stranded nucleic acids with an association constant of 10.sup.3
M.sup.-1 or greater. In some instances, dyes with a high affinity
to double stranded nucleic acids bind with association constants of
the magnitude of 10.sup.7 to 10.sup.9 M.sup.-1. As discussed for
the dye thiazole orange, it may not be well established whether a
dye with a high affinity to double stranded nucleic acids binds to
the nucleic acids by intercalation or by groove binding. It is,
however, not of prime importance in the context of the present
invention to have a precise knowledge regarding the exact mechanism
of binding of the dye to nucleic acids. For the present invention
such dyes may be classified either as intercalators or groove
binders although the exact mechanism of binding may be determined
later to be different from such a classification.
[0142] The present invention is drawn to nucleic acid probes that
are derivatized with two or more non-identical covalently attached
dyes wherein one of the dyes has a high affinity to double stranded
nucleic acids. Examples of dyes with a high affinity to double
stranded nucleic acids that can be employed in the instant
invention include, but are not limited to ethidium and thiazole
orange, as well as, any other dye with a high affinity to double
stranded nucleic acids.
[0143] The synthesis of ethidium dye derivative (15), which is
suitable in post-synthetic labeling methods based on aqueous
Diels-Alder chemistry, is described in Example 7 (Scheme 3).
Ethidium bromide was converted to ethidium tetraphenylborate in
order to increase the solubility of the ethidium dye in common
organic solvents. The tetraphenylborate salt was reacted with
6-maleimidocaproic acid chloride and the resulting conjugate was
reconverted to the bromide salt using an anion exchange resin. The
ethidium derivative (15) is comprised of a maleimide group, which
is reactive towards diene groups in an aqueous media and can be
employed to conjugate ethidium to oligonucleotides that carry the
diene containing bifunctional linker (14). 6
[0144] The synthesis of thiazole orange dye derivative (16), which
is suitable in post-synthetic labeling methods with
amino-functionalized oligonucleotides, is described in Example 8
(Scheme 4). The synthesis was performed in accordance with
procedures described in U.S. Pat. No. 6,348,596 and PCT publication
number WO 96/22383 A1, each of which is incorporated herein by
reference in its entirety, with several modifications. Quaternary
salts of benzothiazole and lepidin were synthesized as
intermediates with methyl-p-toluenesulfonate and 6-bromohexanoic
acid as alkylating agents. The alkylated heterocycles were
condensed in the presence of triethylamine in dichloromethane at
room temperature to give a hexanoic acid derivative of thiazole
orange, which was converted to its N-hydroxysuccinimide ester (16)
with dicyclohexylcarbodiimide. Thiazole orange derivative (16)
contains an activated ester group that will react with free primary
amines in aqueous solutions. Compound (16) is suitable to attach
the dye thiazole orange to oligonucleotides that carry the
bifunctional linker (14). 7
[0145] A preferred embodiment of the invention for synthesizing
doubly labeled oligonucleotides is described in Example 9.
Oligonucleotides that have been modified with the bifunctional
linker (14) as described in Example 6 are simultaneously reacted
with one dye that is derivatized with an amine-reactive group, such
as an N-hydroxy-succinimide ester and a second dye that is
derivatized with a dienophile, such as a maleimide. Both concurrent
reactions are efficiently conducted at 37.degree. C. over a period
of 3 hours at neutral pH. A final purification by gel
electrophoresis yields the doubly labeled oligonucleotide probes
that are ready to be used for assaying nucleic acid analytes. The
general formula representing the doubly labeled probes as
synthesized according to Example 9 is depicted in formula (17) and
a list of examples of probes with two covalently attached dyes that
were prepared by this method is provided in Table 1.
1TABLE 1 8 9 Doubly labeled probes synthesized according to Example
9 Probe 5'-Modification No. Sequence Dye I.sup.1) Dye II.sup.2) as
introduced by SEQ ID NO: 17.1 5'-d(TTT-TTT-TTT-TTT-TTT-TTT-
-TTT-)3' QSY 7 Ethidium (14) 1 17.2
5'-d(TTT-TTT-TTT-TTT-TTT-TTT-TT- T)-3' 5(6)-FAM Ethidium (14) 1
17.3 5'-d(CGC-TCA-TCT-TCA-AGT-CCA-CC- C-T)-3' QSY-7 Ethidium (14) 2
17.4 5'-d(CGC-TCA-TCT-TCA-AGT-CCA-CCC- -T)-3' 5(6)-FAM Ethidium
(14) 2 17.5 5'-d(CGC-TCA-TCT-TCA-AGT-CCA-C- CC-G)-3' 5(6)-TAMRA
Ethidium (14) 3 17.6 5'-d(CGC-TCA-TCT-TCA-AGT-C- CA-CCC-T)-3'
5(6)-FAM Ethidium (14) 2 17.7 5'-d(CGC-TCA-TCT-TCA-AGT-
-CCA-CCC-T)-3' QSY-7 Ethidium (14) 2 17.8
5'-d(CGC-TCA-TCT-TCA-AGT-- CCA-CCC-G)-3' 5(6)-TAMRA Ethidium (14) 3
17.9 5'-d(TTT-TT- 5(6)-FAM Ethidium (14) 4
CGC-TCA-TCT-TCA-AGT-CCA-CCC-T)-3' 17.10 5'-d(TTT-TT- 5(6)-FAM
Ethidium (14) 5 CGC-TCA-TCT-TCA-AGT-CCA-CCC- -G)-3' 17.11
5'-d(TTT-TT- TO QSY-7 (14) 4 CGC-TCA-TCT-TCA-AGT-CCA-CCC-T)-3'
17.12 5'-d(TTT-TT- TO 5-TAMRA (14) 4
CGC-TCA-TCT-TCA-AGT-CCA-CCC-T)-3' 17.13 5'-d(TTT-TT- TO 5-TAMRA
(14) 5 CGC-TCA-TCT-TCA-AGT-CCA-CCC-G)-3' 17.14 5'-d(TTT-TT- TO
Texas Red (14) 4 CGC-TCA-TCT-TCA-AGT-CCA-CCC-T)-3' 17.15
5'-d(TTT-TT- TO MDCC (14) 4 CGC-TCA-TCT-TCA-AGT-CCA-CCC-T)-3' 17.16
5'-d(TTT-TT- TO MDCC (14) 5 CGC-TCA-TCT-TCA-AGT-CCA-CCC-G)-3' 17.17
5'-d(CGC-TCA-TCT-TCA-AGT-CCA-CCC-T)-3' TO 5-FAM (14) 2 17.18
5'-d(TTT-TT- TO 5-FAM (14) 4 CGC-TCA-TCT-TCA-AGT-CCA-CCC-T)-3'
17.19 5'-d(CGC-TCA-TCT-TCA-AGT-CCA-CCC-T)-3' TO 5-FAM 1.HEG-amidite
2 2. (14) 17.20 5'-d(CGC-TCA-TCT-TCA-AGT-CCA-CCC-T)-3' TO Texas Red
1.HEG-amidite 2 2. (14) 17.21
5'-d(CGC-TCA-TCT-TCA-AGT-CCA-CCC-T)-3' TO MDCC 1.HEG-amidite 2 2.
(14) 17.22 5'-d(TTT-TT- 5(6)-FAM Ethidium (14) 4
CGC-TCA-TCT-TCA-AGT-CCA-CCC-T)-3' 17.23 5'-d(TTT-TT- TO 5-FAM (14)
4 CGC-TCA-TCT-TCA-AGT-CCA-CCC-T)-3' 17.24 5'-d(TTT-TT- TO MDCC (14)
6 CAC-TGG-GAG-CAT-TGA-GGC-TC)-3' 17.25 5'-d(TTT-TT- TO 5-FAM (14) 6
CAC-TGG-GAG-CAT-TGA-GGC-TC)-3' 17.26 5'-d(TTT-TT- TO 5-TAMRA (14) 6
CAC-TGG-GAG-CAT-TGA-GGC-TC)- -3' 17.27 5'-d(TTT-TT- TO none
1.HEG-amidite 4 CGC-TCA-TCT-TCA-AGT-CCA-CCC-T)-3' 2. (14) 17.28
5'-d(ATT-CTC-AAC-TCT-GAC-TGT-GAG- TO MDCC (14) 7 CAA-CA)-3' 17.29
5'-d(ATT-CTC-AAC-TCT-GAC-TGT-GAG- TO 5-FAM (14) 7 CAA-CA)-3' 17.30
5'-d(ATT-CTC-AAC-TCT-GAC-TGT-GAG- TO 5-TAMRA (14) 7 CAA-CA)-3'
17.31 5'-d(AAC-TCC-TCT-TCA-GTA-AAG-CCC- TO MDCC 1.HEG-amidite 8
ATG-TCC-CGT)-3' 2. (14) 17.32 5'-d(AAA-CTC-CTC-TTC-AGT-AAA-GCC- TO
MDCC 1.HEG-amidite 9 CAT-GTC-CCG-T)-3' 2. (14) 17.33
5'-d(TTC-AGT-AAA-GCC-CAT-GTC-CCG- - TO MDCC 1.HEG-amidite 10 TT)-3'
2. (14) .sup.1)attached via corresponding NHS-ester.
.sup.2)attached via corresponding maleimide.
[0146] In one embodiment of the present invention, the nucleic acid
probe is designed to carry both a covalently attached intercalator
and at minimum one other dye. At least one of the attached dyes is
fluorescent and its fluorescence properties depend on whether or
not the intercalator is incorporated into double stranded DNA.
Nucleic acid probes that are designed according to this embodiment
are useful to detect and quantify nucleic acids. The design of the
nucleic acid probes of this embodiment can be applied in the
following variations.
[0147] The nucleic acid probe is comprised of a covalently attached
intercalator and one other covalently attached dye (1.-3.). In this
embodiment, either of the two dyes or both of the dyes can be
fluorescent.
[0148] 1. The intercalator is fluorescent and serves as the
detector dye and the second dye is a non-fluorescent quencher. As a
single stranded nucleic acid the nucleic acid probe displays either
none or only minimal fluorescence that is caused by incomplete
quenching of the fluorescence from the intercalator. Upon
hybridization of the nucleic acid probe to a nucleic acid analyte
of complementary sequence increased fluorescence is observed due to
the interaction of the intercalator with the formed double stranded
nucleic acid. The incorporation of the intercalator into the double
stranded nucleic acid increases the distance between the
intercalator and the quencher and therefore reduces the efficiency
of the quenching which in turn enhances the fluorescence of the
intercalator. It is expected that the observed fluorescence will be
markedly different from the fluorescence of the unattached
intercalator, it is well known that the fluorescence of a
non-conjugated intercalator differs from the fluorescence of the
same intercalator after incorporation into double stranded DNA. See
Haugland in Handbook of Fluorescent Probes and Research Chemicals,
Molecular Probes, Inc., Eugene, Oreg., which is incorporated herein
by reference in its entirety. The differences in fluorescence
between the non-conjugated intercalator and the intercalator after
incorporation into double stranded DNA could encompass the emission
wavelength, the fluorescence intensity and the fluorescence
lifetime. The underlying principle of this method is illustrated in
FIG. 1. Probes that have a structure according to the principle
outlined in FIG. 1 are denoted as type 1 nucleic acid probes in the
context of this invention.
[0149] The present invention includes a universal quencher moiety
to attach a fluorescent intercalator to oligonucleotides, as well
as, a non-fluorescent quencher moiety to attach a non-fluorescent
intercalator to oligonucleotides with a single, non-branched
linker. The universal quencher structure consists of a dabcyl
moiety (dabcyl=p-dimethylamino-az- obenzene) that can be covalently
linked to a variety of intercalators via a spacer of varying
length, as illustrated by structure (18). Notably, the linker that
is attached at the 3-position of the dabcyl group, i.e. meta to the
carboxyl group, can also be attached at the 2-position, i.e. ortho
to the carboxyl group. The universal quencher can be covalently
linked to oligonucleotides at the carboxyl group of the dabcyl
substructure through a variety of methods. Examples include, but
are not limited to, the attachment via a phosphoramidite
derivative, as described e.g. with (19), or the attachment through
post-synthetic derivatization techniques, e.g. through the
N-hydroxysuccinimide ester derivative of (18), or through another
amine-reactive derivative of (18), that is reacted with an amino
functionalized oligonucleotide, and other post-synthetic
conjugation methods. 10
[0150] 2. The intercalator is fluorescent and serves as the
detector dye and the second dye is another fluorescent dye that
serves as the donor dye of a FRET system composed of the
intercalator and the second dye. In this embodiment, both dyes
modify the fluorescent properties of each other through the FRET
process. As a single stranded nucleic acid, the nucleic acid probe
displays fluorescence upon excitation of either the donor or the
intercalator, but excitation of the donor leads to a greatly
decreased fluorescence of the donor compared to the fluorescence of
the non-conjugated donor. Simultaneously, an increased fluorescence
of the intercalator compared to the excitation of the
non-conjugated intercalator is observed due to the energy transfer.
After hybridization of the nucleic acid probe to a nucleic acid
analyte of complementary sequence increased fluorescence is
observed upon excitation of the donor due to the interaction of the
intercalator with the formed double stranded nucleic acid. The
incorporation of the intercalator into the double stranded nucleic
acid increases the distance between the intercalator and the donor
and therefore reduces the efficiency of the FRET process, which in
turn enhances the fluorescence of the donor. In this system the
fluorescence of the intercalator is simultaneously decreased when
the donor is excited.
[0151] The underlying principle of this method is illustrated in
FIG. 2. Probes that have a structure according to the principle
outlined in FIG. 2 are referred to herein as type II nucleic acid
probes. In this method of detection, the universal quencher
structure (18) can simultaneously serve as quencher and
intercalator, with another fluorochrome donor dye attached to the
nucleic acid probe in close molecular proximity. The universal
quencher, exemplified by the dabcyl moiety of compound (18), is
advantageous in that it can efficiently quench the fluorescence of
a wide range of fluorochromes, including those most commonly used
in nucleic acid sequencing and detection applications. Upon
hybridization the donor displays enhanced fluorescence due to the
less effective quenching caused by the interaction of the
intercalator with the double stranded DNA.
[0152] In this embodiment the intercalator may or may not change
its fluorescent properties upon hybridization of the nucleic acid
probe to a complementary target sequence. Changes in the
fluorescent properties of the intercalator upon hybridization may
include a shift in the maxima of the fluorescence excitation and/or
emission spectra or a decreased or increased fluorescence
intensity. For example, the dye thiazole orange displays a
remarkable increase in its fluorescence intensity upon its
interaction with double stranded nucleic acids which is accompanied
by a small shift in the maxima of the fluorescence excitation and
emission spectra, as discussed by Rye et al. (1992) Nucleic Acid
Res. 20:2803-2812. In this case the hybridization of a type II
nucleic acid probe of the invention could potentially not only
result in a higher fluorescence intensity of the donor dye upon
excitation of the donor, but, in addition, also to a higher
fluorescence intensity of the intercalator upon excitation of the
donor, because the intercalator in the hybridized form of the probe
can be more efficiently excited through a FRET mechanism upon
excitation of the donor due to its stronger overall
fluorescence.
[0153] 3. The intercalator is fluorescent and serves as the
detector dye and the second dye is another fluorescent dye that
serves as the acceptor dye of a FRET system comprised of the
intercalator and the second dye. In this embodiment, both dyes
modify the fluorescent properties of each other through the FRET
process. As a single stranded nucleic acid the nucleic acid probe
displays fluorescence upon excitation of either the intercalator or
the acceptor, but excitation of the intercalator leads to decreased
fluorescence compared to the excitation of the non-conjugated
intercalator and increased fluorescence of the acceptor compared to
the excitation of the non-conjugated acceptor. After hybridization
of the nucleic acid probe to a nucleic acid analyte of
complementary sequence increased fluorescence is observed upon
excitation of the intercalator due to the interaction of the
intercalator with the formed double stranded nucleic acid. The
incorporation of the intercalator into the double stranded nucleic
acid increases the distance between the intercalator and the
acceptor and therefore reduces the efficiency of the FRET process,
which in turn enhances the fluorescence of the intercalator. For
many known intercalators fluorescence is further enhanced as a
result of the intercalation process. (Haugland in "Handbook of
Fluorescent Probes and Research Chemicals," Molecular Probes, Inc.,
Eugene, Oreg.). In this system, the fluorescence of the acceptor is
simultaneously decreased when the intercalator is exited. The
underlying principle of this method of detection is illustrated in
FIG. 3. Probes that have a structure according to the principle
outlined in FIG. 3 are referred to herein as type III nucleic acid
probes.
[0154] In this embodiment the intercalator may or may not change
its fluorescent properties upon hybridization of the nucleic acid
probe to a complementary target sequence, including shifts in the
maxima of the fluorescence excitation and emission spectra and a
decrease or increase of the fluorescence intensity, as discussed
for type II nucleic acid probes. In the case of an increased
fluorescence intensity of the intercalator upon hybridization the
hybridization of a type III nucleic acid probe of the invention
could potentially not only result in a higher fluorescence
intensity of the intercalator upon excitation of the intercalator,
but, in addition, also to a higher fluorescence intensity of the
acceptor dye upon excitation of the intercalator, because the
acceptor dye in the hybridized form of the probe can be more
efficiently excited through a FRET mechanism upon excitation of the
intercalator due to the stronger overall fluorescence of the
intercalator.
[0155] The nucleic acid probe is comprised of a covalently attached
intercalator and two other covalently attached dyes (4.-5.). The
dyes that are not intercalators are covalently linked through a
spacer and form a dye pair that is fluorescent.
[0156] 4. In this embodiment of the instant invention, the dye pair
is an excimer pair that forms an excimer upon exposure to
electromagnetic radiation. The excimer pair is used as the detector
dye. As a single stranded nucleic acid the nucleic acid probe
displays fluorescence that is caused by the excimer. Upon
hybridization of the nucleic acid probe to a nucleic acid analyte
of complementary sequence decreased fluorescence of the excimer is
observed due to the interaction of the intercalator with the formed
double stranded nucleic acid. The incorporation of the intercalator
into the double stranded nucleic acid increases the distance
between the dyes of the excimer pair and therefore reduces the
efficiency of the formation of the excimer, which in turn reduces
the fluorescence caused by the excimer pair. The fluorescence
caused by the excimer pair can be advantageously monitored because
excimer pairs are known to have comparatively large Stokes shifts,
which leads to increased signal to noise ratios. Furthermore, the
efficiency of the formation of an excimer is very sensitive to the
molecular distance of the excimer partners, see De Schryver et al.
(1987) Acc. Chem. Res. 20:159-66. The underlying principle of this
embodiment is illustrated in FIG. 4. Probes that have a structure
according to the principle outlined in FIG. 4 are denoted herein as
type IV nucleic acid probes.
[0157] 5. In this embodiment to the instant invention, the dye pair
is an exciplex pair that forms an exciplex upon exposure to
electromagnetic radiation. The exciplex pair is used as the
detector dye. As a single stranded nucleic acid, the nucleic acid
probe displays fluorescence that is caused by the exciplex. Upon
hybridization of the nucleic acid probe to a nucleic acid analyte
of complementary sequence decreased fluorescence is observed due to
the interaction of the intercalator with the formed double stranded
nucleic acid. The incorporation of the intercalator into the double
stranded nucleic acid increases the distance between the dyes of
the exciplex pair and therefore reduces the efficiency of the
formation of the exciplex, which in turn reduces the fluorescence
caused by the exciplex pair. The fluorescence caused by the
exciplex pair can be advantageously monitored because exciplex
pairs are known to have comparatively large stokes shifts, which
leads to increased signal to noise ratios. Furthermore, the
efficiency of the formation of an exciplex is very sensitive to the
molecular distance of the exciplex partners, similar to the
efficiency of the formation of an excimer, as discussed above. This
embodiment of the instant invention is illustrated in FIG. 5.
Probes that have a structure according to the principle outlined in
FIG. 5 are denoted herein as type V nucleic acid probes.
[0158] In another embodiment, the nucleic acid probe is designed to
carry both a covalently attached groove binder and at minimum one
other dye. At least one of the attached dyes is fluorescent and its
fluorescence properties depend on the interaction of the groove
binder with double stranded DNA. Nucleic acid probes that are
designed according to this embodiment are useful to detect and
quantify nucleic acids. The design of the nucleic acid probes of
this embodiment can be applied in the following variations.
[0159] The nucleic acid probe is comprised of a covalently attached
groove binder and one other covalently attached dye (6.-8.). In
this embodiment, either of the two dyes or both dyes can be
fluorescent dyes.
[0160] 6. In this embodiment, the groove binder is fluorescent and
serves as the detector dye and the second dye is a non-fluorescent
quencher. As a single stranded nucleic acid the nucleic acid probe
displays either none or only minimal fluorescence that is caused by
incomplete quenching of the fluorescence from the groove binder.
Upon hybridization of the nucleic acid probe to a nucleic acid
analyte of complementary sequence increased fluorescence is
observed due to the interaction of the groove binder with the
formed double stranded nucleic acid. The incorporation of the
groove binder into the double stranded nucleic acid increases the
distance between the groove binder and the quencher and therefore
reduces the efficiency of the quenching, which in turn enhances the
fluorescence of the groove binder. It is expected that the observed
fluorescence will markedly different from the fluorescence of the
unattached groove binder, because in many literature examples the
fluorescence of a non-conjugated groove binder differs from the
fluorescence of the same groove binder after incorporation into
double stranded DNA. The differences in fluorescence between the
non-conjugated groove binder and the groove binder after
incorporation into double stranded DNA could encompass the emission
wavelength, the fluorescence intensity and the fluorescence
lifetime. In many examples enhanced fluorescence is observed upon
the interaction of a groove binder with double stranded DNA.
Haugland in "Handbook of Fluorescent Probes and Research
Chemicals," Molecular Probes, Inc., Eugene, Oreg. The underlying
principle of this embodiment of the invention is illustrated in
FIG. 6. Probes that have a structure according to the principle
outlined in FIG. 6 are referred to herein as type VI nucleic acid
probes.
[0161] 7. In this embodiment, the groove binder is fluorescent and
serves as the detector dye and the second dye is also fluorescent
and serves as the donor dye of a FRET system composed of the groove
binder and the second dye. Both dyes modify the fluorescent
properties of each other through the FRET process. As a single
stranded nucleic acid the nucleic acid probe displays fluorescence
upon excitation of either the donor or the groove binder, but
excitation of the donor leads to a greatly decreased fluorescence
of the donor compared to the fluorescence of the non-conjugated
donor. Simultaneously, an increased fluorescence of the groove
binder compared to the excitation of the non-conjugated groove
binder is observed due to the energy transfer. After hybridization
of the nucleic acid probe to a nucleic acid analyte of
complementary sequence increased fluorescence is observed upon
excitation of the donor due to the interaction of the groove binder
with the formed double stranded nucleic acid. The incorporation of
the groove binder into the double stranded nucleic acid increases
the distance between the groove binder and the donor and therefore
reduces the efficiency of the FRET process, which in turn enhances
the fluorescence of the donor. In this system the fluorescence of
the groove binder is simultaneously decreased when the donor is
exited. The underlying principle of this embodiment of the
invention is illustrated in FIG. 7. Probes that have a structure
according to the principle outlined in FIG. 7 are denoted herein as
type VII nucleic acid probes.
[0162] In this embodiment the groove binder may or may not change
its fluorescent properties upon hybridization of the nucleic acid
probe to a complementary target sequence, including shifts in the
maxima of the fluorescence excitation and emission spectra and a
decrease or increase of the fluorescence intensity. In the case of
an increased fluorescence intensity of the groove binder upon
hybridization the hybridization of a type VII nucleic acid probe of
the invention could potentially not only result in a higher
fluorescence intensity of the donor dye upon excitation of the
donor dye, but, in addition, also to a higher fluorescence
intensity of the groove binder upon excitation of the donor dye,
because the groove binder in the hybridized form of the probe can
be more efficiently excited through a FRET mechanism upon
excitation of the donor dye due to the stronger overall
fluorescence of the groove binder.
[0163] 8. According to this embodiment, the groove binder is
fluorescent and serves as the detector dye and the second dye is
another fluorescent dye that serves as the acceptor dye of a FRET
system composed of the groove binder and the second dye. Both dyes
modify the fluorescent properties of each other through the FRET
process. As a single stranded nucleic acid the nucleic acid probe
displays fluorescence upon excitation of either the groove binder
or the acceptor, but excitation of the groove binder leads to
decreased fluorescence compared to the excitation of the
non-conjugated groove binder and increased fluorescence of the
acceptor compared to the excitation of the non-conjugated acceptor.
After hybridization of the nucleic acid probe to a nucleic acid
analyte of complementary sequence increased fluorescence is
observed upon excitation of the groove binder due to the
interaction of the groove binder with the formed double stranded
nucleic acid. The incorporation of the groove binder into the
double stranded nucleic acid increases the distance between the
groove binder and the acceptor and therefore reduces the efficiency
of the FRET process, which in turn enhances the fluorescence of the
groove binder. For many known groove binders the fluorescence is
further enhanced due to the intercalation process. An enhancement
of the fluorescence due to intercalation in double stranded DNA was
observed for many groove binders, see Haugland in "Handbook of
Fluorescent Probes and Research Chemicals," Molecular Probes, Inc.,
Eugene, Oreg., for examples. In this system the fluorescence of the
acceptor is simultaneously decreased when the groove binder is
excited. The underlying principle of this embodiment is illustrated
in FIG. 8. Probes that have a structure according to the principle
outlined in FIG. 8 are denoted herein as type VIII nucleic acid
probes.
[0164] In this embodiment the groove binder may or may not change
its fluorescent properties upon hybridization of the nucleic acid
probe to a complementary target sequence, including shifts in the
maxima of the fluorescence excitation and emission spectra and a
decrease or increase of the fluorescence intensity. In the case of
an increased fluorescence intensity of the groove binder upon
hybridization the hybridization of a type VIII nucleic acid probe
of the invention could potentially not only result in a higher
fluorescence intensity of the groove binder upon excitation of the
groove binder, but, in addition, also to a higher fluorescence
intensity of the acceptor dye upon excitation of the groove binder,
because the acceptor dye in the hybridized form of the probe can be
more efficiently excited through a FRET mechanism upon excitation
of the groove binder due to the stronger overall fluorescence of
the groove binder.
[0165] The nucleic acid probe carries a covalently attached groove
binder and two additional covalently attached dyes (9.-10.). The
dyes that are not groove binders are covalently linked through a
spacer and form a dye pair that is fluorescent.
[0166] 9. In this embodiment, the dye pair forms an excimer upon
exposure to electromagnetic radiation. As a single stranded nucleic
acid the nucleic acid probe displays fluorescence that is caused by
the excimer. Upon hybridization of the nucleic acid probe to a
nucleic acid analyte of complementary sequence decreased
fluorescence of the excimer is observed due to the interaction of
the groove binder with the formed double stranded nucleic acid. The
incorporation of the groove binder into the double stranded nucleic
acid increases the distance between the dyes of the excimer pair
and therefore reduces the efficiency of the formation of the
excimer, which in turn reduces the fluorescence caused by the
excimer pair. The underlying principle of this embodiment is
illustrated in FIG. 9. Probes that have a structure according to
the principle outlined in FIG. 9 are denoted as type IX nucleic
acid probes.
[0167] 10. In this embodiment, the dye pair forms an exciplex upon
exposure to electromagnetic radiation. As a single stranded nucleic
acid the nucleic acid probe displays fluorescence that is caused by
the exciplex. Upon hybridization of the nucleic acid probe to a
nucleic acid analyte of complementary sequence decreased
fluorescence is observed due to the interaction of the groove
binder with the formed double stranded nucleic acid. The
incorporation of the groove binder into the double stranded nucleic
acid increases the distance between the dyes of the exciplex pair
and therefore reduces the efficiency of the formation of the
exciplex, which in turn reduces the fluorescence caused by the
exciplex pair. The underlying principle of this embodiment is
illustrated in FIG. 10. Probes that have a structure according to
the principle outlined in FIG. 10 are referred to herein as type X
nucleic acid probes.
[0168] The design of nucleic acid probes that carry both a first
covalently attached dye that has a high affinity to double stranded
nucleic acids and at minimum one other dye wherein at least one of
the dyes is fluorescent and its fluorescence properties depend on
the interaction of the first dye with a double stranded nucleic
acid is illustrated in Examples 10 and 11, below.
[0169] In Example 10, the fluorescence of the nucleic acid probe
(17.2) which carries the covalently attached dyes fluorescein and
ethidium was measured as a function of temperature in the range
40.degree. C. to 90.degree. C. The experiments were performed at an
excitation wavelength of 470 nm and the emission was recorded at
530 nm and 645 nm, both in the presence and absence of the
complementary oligonucleotide d(A.sub.29G) under otherwise
identical conditions of probe and buffer concentration. The
observed fluorescence intensities are displayed in FIG. 14. The
fluorescence intensity of the probe (17.2) in the absence of a
complementary sequence is represented by curve A at the emission
wavelength 530 nm and by curve A' at the emission wavelength 645
nm. The fluorescence intensity of probe (17.2) in the presence of
the complementary oligonucleotide d(A.sub.29G) is represented by
curve B at the emission wavelength 530 nm and by curve B' at the
emission wavelength 645 nm. The fluorescence of the unhybridized
probe decreases slightly with rising temperature in a semi-linear
manner at both recorded wavelengths. The fluorescence of the
hybridized probe differs considerably from the fluorescence of the
unhybridized probe. In the temperature range of 40.degree. C. to
approximately 55.degree. C., the fluorescence intensity of the
hybridized probe at the emission wavelength 530 nm is roughly 33%
higher than the fluorescence intensity of the unhybridized probe.
At the emission wavelength 645 nm the fluorescence intensity of the
hybridized probe is roughly double as high as the fluorescence
intensity of the unhybridized probe. In the temperature range of
approximately 55.degree. C. to 65.degree. C., the fluorescence
intensity of the hybridized probe shows a strong dependence of the
temperature and decreases to the intensity level of the
unhybridized probe at both recorded emission wavelengths. The rapid
drop in fluorescence intensity is presumably due to the
dissociation of the probe from the complement in this temperature
range and exhibits the typical characteristics of a melting curve
of a double stranded nucleic acid, which is a clear indication of a
specific interaction. The T.sub.m value of the probe/complement
hybrid, as reflected by the inflection point, is approximately
57.degree. C.
[0170] Probe (17.2) is an example of a type II nucleic acid probe
of the invention comprising a FRET system established by two
fluorescent dyes, the fluorescent intercalator ethidium, which
serves as the detector dye and the fluorescent dye fluorescein,
which serves as the donor dye of a FRET system. After hybridization
of the nucleic acid probe an increased fluorescence of the donor
dye fluorescein is observed at 530 nm upon its excitation at 470 nm
due to the interaction of the intercalator with the formed double
stranded nucleic acid. In addition, probe (17.2) also displays an
increased fluorescence of the intercalator ethidium at 645 nm upon
excitation of the donor fluorescein at 470 nm, which is potentially
related to the increased overall fluorescence of ethidium upon
intercalation. The latter feature of probe (17.2) is particularly
useful as the excitation and emission wavelengths are separated by
175 nm. Such a big difference between the excitation and emission
wavelengths provides the basis for a greatly reduced background in
fluorescence measurements, because all other background
fluorescence that is excited at 470 nm is highly unlikely to
display fluorescence emission at 645 nm. A very large Stokes shift
would be required for a background fluorescence dye to emit light
at 645 nm upon excitation at 470 nm and therefore any fluorescence
that is introduced by spurious contamination with fluorescent
impurities is expected to be practically eliminated in measurements
with probe (17.2) and similar probes.
[0171] In Example 11, the fluorescence of the nucleic acid probes
(17.11), (17.20) and (17.21) were measured as a function of
temperature in the range of 30.degree. C. to 90.degree. C. The
experiments were performed with probe specific excitation and
emission wavelengths in the presence as well as in the absence of
the complementary oligonucleotide d(AGGGTGGACTTGAAGATGAGCGAAAAA)
(SEQ ID NO:11) under otherwise identical conditions of probe and
buffer concentration. The recorded fluorescence intensities in the
presence and in the absence of the complementary sequence
(I.sub.F.sup.hyb and I.sub.F.sup.free) and their ratios
(I.sub.F.sup.hyb/I.sub.F.sup.free) are displayed in FIGS. 15 to 18
as a function of temperature.
[0172] Probe (17.11) carries the covalently attached dyes QSY-7 and
thiazole orange. The fluorescence intensities of the probe in the
absence and in the presence of the complementary sequence at an
excitation wavelength of 510 nm and an emission wavelength of 528
nm, and the ratio of both intensities, is displayed in FIG. 15 by
the curves A, B and C, respectively. The excitation and emission
wavelength match the maxima in the excitation and emission spectra
of thiazole orange in this experiment. Both the fluorescence of the
unhybridized probe and the fluorescence of the hybridized probe
decrease continuously with rising temperature. A distinct decrease
of the fluorescence intensity of probes that were singly labeled
with the dye thiazol orange with increasing temperature was already
observed by Svanvik et al. (2000) Anal. Biochem. 281:26-35, which
is incorporated herein by reference in its entirety, and may be
attributed to the properties of the dye thiazole orange, as
discussed by Nygren et al. (1998) Biopolymers 46: 39-51. The
temperature-dependent fluorescence did not mirror a melting curve
for probe (17.11) in the presence of the complementary sequence,
but since the fluorescence intensity of the hybridized probe became
identical to the fluorescence intensity of the unhybridized probe
at about 80.degree. C., complete dissociation at 80.degree. C. can
be assumed. The fluorescence of the hybridized probe is
considerably higher than the fluorescence of the unhybridized probe
in the temperature range 30.degree. C. to approximately 75.degree.
C. At 30.degree. C. the hybridized probe is approximately 4-fold
more fluorescent than the unhybridized probe and a maximum of
fluorescence enhancement of approximately 7-fold enhancement upon
hybridization is observed in the temperature range of 55.degree. C.
to 65.degree. C.
[0173] Probe (17.11) is an example of a type I nucleic acid probe
of the invention comprising a fluorescent intercalator and a
non-fluorescent quencher. The fluorescent intercalator thiazole
orange serves as the detector dye and QSY7 serves as the quencher.
Upon hybridization of the nucleic acid probe to a complementary
sequence increased fluorescence is observed due to the interaction
of the intercalator with the formed double stranded nucleic acid.
In the unhybridized state the fluorescence of the intercalator is
effectively quenched through the presence of the non-fluorescent
quencher. In the hybridized state the fluorescence of the
intercalator thiazole orange is less effectively quenched.
[0174] Probe (17.20) carries the covalently attached dyes thiazole
orange and Texas Red. The fluorescence intensities of the probe in
the absence and in the presence of the complementary sequence at an
excitation wavelength of 510 nm and an emission wavelength of 625
nm, and the ratio of both intensities, is displayed in FIG. 16 by
the curves A, B and C, respectively. The excitation wavelength of
510 nm reflects the maximum in the excitation spectrum of thiazole
orange and the emission wavelength of 625 nm reflects the maximum
in the emission spectrum of Texas Red in this experiment. The
fluorescence intensity of the hybridized probe is higher than the
fluorescence intensity of the unhybridized probe at all
temperatures and both fluorescence intensities decrease
continuously with rising temperature. The fluorescence enhancement
that is obtained upon hybridization is approximately 1.5-fold at
30.degree. C. and the maximum enhancement is approximately 2-fold
in the temperature range 58.degree. C. to 68.degree. C.
[0175] Probe (17.20) is an example of a type III nucleic acid probe
of the invention comprising a FRET system established by two
fluorescent dyes, the fluorescent intercalator thiazole orange that
serves as the detector dye and the second dye being the fluorescent
dye Texas Red that serves as the acceptor dye of a FRET system.
After hybridization of the nucleic acid probe an increased
fluorescence of the acceptor dye Texas Red is observed at 625 nm
upon the excitation of the intercalator thiazole orange at 510 nm
due to the interaction of the intercalator with the formed double
stranded nucleic acid. Most likely the increase in the fluorescence
intensity of Texas Red is due to the stronger fluorescence of
thiazole orange in the hybridized state for this probe. Probe
(17.20) is assumed to be a very useful probe due to its very high
fluorescence intensity and the large separation of the excitation
and the emission wavelength that differ by 115 nm, which provides
the basis for a greatly reduced background in fluorescence
measurements.
[0176] Probe (17.21) carries the covalently attached dyes thiazole
orange and MDCC
(7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin-
). The fluorescence intensities of the probe in the absence and in
the presence of the complementary sequence and the ratio of both
intensities, is displayed in FIG. 17 and FIG. 18 by the curves A, B
and C, respectively. In FIG. 17, an excitation wavelength of 510 nm
and an emission wavelength of 525 nm were employed, reflecting the
maxima in the excitation and emission spectra of thiazole orange in
this particular experiment. In FIG. 18, an excitation wavelength of
420 nm and an emission wavelength of 525 nm were employed, the
excitation wavelength reflecting the maximum in the excitation
spectrum of MDCC and the emission wavelength reflecting the maximum
in the emission spectrum of thiazole orange this particular
experiment. With both excitation wavelengths the fluorescence
intensity of the hybridized probe is higher than the fluorescence
intensity of the unhybridized probe over the whole temperature
range of 30.degree. C. to approximately 75.degree. C. and in both
cases the fluorescence intensities of the hybridized probe and the
unhybridized probe decrease continuously with rising temperature.
With an excitation at 420 nm the fluorescence enhancement that is
obtained upon hybridization is approximately 1.2-fold at 30.degree.
C. and the maximum enhancement is approximately 2-fold in the
temperature range 55.degree. C. to 67.degree. C. With an excitation
at 510 nm the fluorescence enhancement that is obtained upon
hybridization is approximately 2-fold at 30.degree. C. and the
maximum enhancement is approximately 4-fold.
[0177] Probe (17.21) is an example of a type II nucleic acid probe
of the invention comprising a FRET system established by two
fluorescent dyes, the fluorescent intercalator thiazole orange
serves as the detector dye and the fluorescent dye MDCC serves as
the donor dye of a FRET system. After hybridization of the nucleic
acid probe an increased fluorescence is observed at 528 nm upon
excitation of the probe at 420 nm or 510 nm due to the interaction
of the intercalator with the formed double stranded nucleic acid.
Most likely the increase of the fluorescence of thiazole orange
upon intercalation allows for a higher energy transfer in this
probe.
[0178] In yet another embodiment of the present invention, the
nucleic acid probe is designed to carry both a covalently attached
pH sensitive dye and at a minimum one other covalently attached
dye. At least one of the dyes is fluorescent and the fluorescence
intensity is modulated by the pH of the medium employed in the
assay. The pH sensitive dye and the fluorescent dye form a FRET
system. Under certain conditions of pH the probe is fluorescent,
whereas upon a change of the pH, the pH sensitive dye becomes an
effective quencher and the fluorescence of the probe is no longer
measurable or drops down to a low intrinsic background level.
Probes that carry both a covalently attached pH sensitive dye and
at minimum one other covalently attached dye are denoted as type XI
nucleic acid probes.
[0179] pH-sensitive dyes are characterized by a change in their
absorption characteristics upon a change of the pH of the medium.
Typical examples of pH-sensitive dyes are the common pH-indicators.
Examples of such pH-indicators include, but are not limited to,
methylorange which changes color from red to yellow-orange upon a
change of the pH from <3 to >4.5, bromothymolblue which
changes color from yellow to blue upon a change of the pH from
<6 to >7.5, thymolblue which changes color from yellow to
blue upon a change of the pH from <8 to >9.5, and
phenolphthalein which changes color from colorless to violet upon a
change of the pH from <8 to >10. All of these dyes and other
pH-sensitive dyes can potentially be applied in the probes of the
present invention if they are covalently attached to the probe
together with a second fluorescent dye.
[0180] pH-sensitive nucleic acid probes can be obtained by
incorporation of a modified trityl group as one of the dyes of the
probe. For instance, a DMT (dimethoxytrityl) group can be employed
together with a fluorescent dye, as exemplified in structures (20)
and (21) below. In an acidic medium the DMT group is converted to
the cationic species (20), which has a considerable absorption at
approximately 500 nm. Under these conditions, the nucleic acid
probe displays minimal or no fluorescence because the DMT group
acts as an efficient quencher of the fluorescent dye. Under neutral
or basic conditions the DMT group is reconverted to its OH-form
(structure (21)), which has essentially no absorption at 500 nm. In
this form the nucleic acid probe is highly fluorescent, because the
fluorescence of the second dye is no longer quenched. Modified
trityl groups were described as pH sensitive fluorescence labels by
Shchepinov et al. (2000) Tetrahedron Lett. 41:4943-4948, which is
incorporated herein by reference in its entirety. This reference
discloses modified trityl groups as pH-sensitive fluorescent dyes,
but does not incorporate them into oligonucleotides, although
applications with oligonucleotides are proposed. The probes
disclosed herein, however, are more versatile, in that almost any
pH-sensitive chromophore or dye can be employed as the pH-sensitive
element of the probe, regardless of whether the chromophore or dye
has an intrinsic fluorescence or not. This allows the introduction
of desirable properties such as photostability, a very high
brightness and a high quantum yield into the probe by the selection
of the fluorescent dye, which is not limited by the requirement of
being fluorescent. Probes that carry both a covalently attached
trityl group and a fluorescent dye are illustrated in FIG. 11.
11
[0181] Applications in Homogeneous Assays
[0182] In one embodiment of the present invention, a nucleic acid
probe as described above, is used in homogeneous assays to detect
or quantify nucleic acid targets. In such assays, a fluorescent
signal is generated upon the presence of a complementary nucleic
acid sequence in the analyte. The fluorescent signal is monitored
and quantified with fluorescence detectors, such as fluorescence
spectrophotometers, commercial systems that allow the monitoring of
fluorescence in PCR reactions, e.g. instruments produced by
Perkin-Elmer Applied Biosystems, Foster City, Calif., or
LIGHTCYCLER.TM. instruments of Roche Diagnostics, Indianapolis,
Ind., or, in some instances, by the human eye. With type I, II,
III, VI, VII or VIII nucleic acid probes a fluorescent signal is
generated or increased in the presence of a complementary sequence
in the target nucleic acid analyte. With type IV, V, IX or X
nucleic acid probes a fluorescent signal is decreased in the
presence of a complementary sequence in the target nucleic acid
analyte, see FIGS. 1-10 for an illustration.
[0183] In one embodiment, a homogeneous assay is conducted without
the addition of reagents, such as buffers and other non-reactive
ingredients. Other non-reactive ingredients including, but are not
limited to, EDTA, magnesium salts, sodium chloride, potassium
chloride, inorganic phosphates, BSA (bovine serum albumin),
gelatin, DMF, DMSO, urea, chaotropic salts or other non-reactive
ingredients known to those skilled in the art which are commonly
employed in nucleic acid based diagnostic assays can also be used
with this method. In this embodiment of the invention, the nucleic
acid probe hybridizes with a complementary nucleic acid sequence,
if present in the target, to form a stretch of double stranded DNA.
In turn one of the dyes, which is covalently attached to the
nucleic probe, interacts with the stretch of double stranded DNA,
resulting in the generation of a fluorescent signal. With
appropriate target standards and concentration versus signal
standard curves the method can easily be used to quantitate the
target. In addition to single stranded target nucleic acids, double
stranded target nucleic acids can also be detected by the nucleic
acid probe following denaturation. Targets that can be specifically
detected and/or quantified with this method include, but are not
limited to, plasmid DNA, cloning inserts in plasmid DNA, RNA
transcripts, ribosomal RNA, PCR amplicons, restriction fragments,
synthetic oligonucleotides, as well as any other nucleic acids and
oligonucleotides.
[0184] In another particular embodiment of the invention a
homogeneous assay is conducted simultaneously with a PCR reaction.
With this type of assay all components that are necessary to
conduct a PCR reaction on the target nucleic acid analyte are added
simultaneously with the nucleic acid probe and the target. The
components of the PCR reaction include primers, a thermostable DNA
polymerase, an aqueous buffer, magnesium chloride and
deoxynucleotide triphosphates, and may also include other
non-reactive ingredients, including, but not limited to, salts,
BSA, gelatin, DMSO, chaotropic salts, as discussed above. As the
PCR reaction progresses increasing amounts of double stranded PCR
amplicons are formed which are denatured within the course of a PCR
cycle. In these assays, the specific nucleic acid probe contains a
stretch of nucleic acid sequence that is complementary to a stretch
of nucleic acid sequence on the formed amplicon. Upon hybridization
of the nucleic acid probe to its complementary stretch of nucleic
acid sequence on the single stranded amplicon a fluorescent signal
is generated in the annealing step of the PCR cycle that is
proportional to the amount of amplicon formed. In the course of the
elongation of the primer on the single stranded amplicon the
nucleic acid probe is displaced from the amplicon, either through
the 5'-3'-exonucleolytic activity of the polymerase, which removes
the nucleic acid stepwise one nucleotide after the other from the
5'-end of the probe, or through thermal denaturation in the
elongation step of the PCR reaction.
[0185] The principle of homogeneous assays conducted simultaneously
with a PCR reaction is illustrated with type I nucleic acid probes
in FIG. 12. With reference to FIG. 12, after combining the nucleic
acid probe with two standard sequence specific DNA primers and the
double stranded DNA to be amplified in the appropriate reaction
medium, denaturation and annealing, a fluorescent signal is
generated because the nucleic acid probe anneals to the denatured
DNA, resulting in increased fluorescence. After the primer
elongation step the DNA is in doubled stranded form and
fluorescence is not observed or minimized (to the residual low
fluorescence of the nucleic probe). In the next cycle of the PCR
reaction the fluorescent signal is increased at the annealing step,
because the number of single stranded amplicons has doubled in the
reaction. This scheme continues with increasing cycle number
displaying increased fluorescence that is proportional to the
amount of amplicon formed.
[0186] In another variation of this embodiment of the invention,
the nucleic acid probe acts as one of the primers of the PCR
reaction. In this variation the specific nucleic acid probe is
incorporated in the PCR amplicon upon the progression of the
reaction. Upon annealing of the nucleic acid probe that acts as the
primer to the single stranded amplicon a fluorescent signal is
generated, which can be recorded in the annealing step of the PCR
reaction and which is proportional to the amount of amplicon
formed. This method is more versatile than the method described
above because only one additional primer is required. An additional
advantage of this variation of the method is that the amplicon
product of the assay carries the covalently attached dyes that were
part of the nucleic acid probe in one strand and can be further
employed as nucleic probes themselves, e.g. in microarray
applications, as described below.
[0187] This variation of the method of this invention is
illustrated in FIG. 13. With reference to FIG. 13, after combining
the nucleic acid probe with one standard sequence specific DNA
primer and the double stranded DNA to be amplified in the
appropriate reaction medium, denaturation and annealing, a
fluorescent signal is generated when the nucleic acid probe acts as
one of the primers of the PCR reaction because it anneals to the
denatured DNA, which results in increased fluorescence. After the
primer elongation step the DNA is in doubled stranded form, but
contains the nucleic acid probe incorporated in the sequence and a
fluorescent signal is still observable. In the next cycle of the
PCR reaction the fluorescent signal is increased at the annealing
step, because the number of single stranded amplicons has doubled
in the reaction. This scheme continues with increasing cycle number
displaying increased fluorescence that is proportional to the
amount of amplicon formed.
[0188] The described method allows the monitoring of a PCR reaction
in real time, which has several benefits compared to a conventional
end point analysis, e.g. the analysis of PCR reactions through gel
electrophoresis. Firstly, the method allows the direct observation
of the reaction, which provides a quality control of the PCR. Since
the success of any PCR reaction, especially on complex targets like
the human genome, is dependent on the exact nature of the target
structure and not automatically guaranteed, a quality control early
in the process is highly desirable. PCR reactions that fail to
produce an amplicon could be discarded without further analysis,
which provides great savings regarding the amount of post-PCR
handling and materials usage to the investigator. Secondly, many
PCR reactions not only provide specific amplicons, but also
non-specific products from incorrect annealing of the primers to
the target. Typically, the non-specific products require a higher
cycle number than the specific product to become abundant in the
reaction. Monitoring the reaction in real time allows the
investigator to stop the reaction at a cycle number that provides
enough specific amplicon and does not provide impeding quantities
of non-specific amplicons which eliminates the need to perform a
careful optimization of a PCR to obtain specific amplicons. This is
particularly valuable in high-throughput labs as the amount of
necessary optimization work can easily exceed the amount of work
involved with the final generation of the amplicon product.
[0189] The doubly labeled probes prepared with the diene/amino
linker (14) are suitable for PCR applications. They are perfectly
stable under the conditions of PCR temperature cycling and can be
employed as primers of the PCR. Example 12 demonstrates the
stability of the nucleic acid probe (17.13) which carries the
covalently attached dyes TAMRA and thiazole orange. The probe is
dissolved in a PCR buffer and subjected to 40 temperature cycles in
a PCR machine. The probe is analyzed by HPLC and compared to a
reference sample. Virtually no difference in the overall purity and
in the impurity profile was found between the probe that was
subjected to the temperature cycling conditions and the reference
sample. This result demonstrates that the covalent linkage between
the nucleic acid probe and the attached dyes as introduced with
linker (14) is stable under the conditions of PCR. It also
demonstrates the stability of the attached dyes TAMRA and thiazole
orange under such conditions.
[0190] Example 13 describes several PCR reactions employing the
nucleic acid probes of the invention as primers in the PCR
reaction. Specific PCR products of the expected length were
obtained with a variety of probes that were employed as one of the
primers of the PCR. Different combinations of dyes were included
and PCR products that correspond to 3 different human genes were
obtained. The synthesis of the PCR products was verified by agarose
gel electrophoresis. Example 13 demonstrates that the nucleic acid
probes of the invention can be employed as primers in a PCR
reaction and that the presence of such probes in a PCR does not
inhibit the preparation of specific amplification products.
[0191] Example 14 describes the application of the probes of the
present invention in several PCR reactions that were monitored in
real time through the measurement of the fluorescence of the
probes. Pre-amplified DNA was used as a target nucleic acid in this
example. In one of the reactions the nucleic acid probe (17.21),
which carries the covalently attached dyes MDCC and thiazole
orange, was employed in the detection of a nucleic acid sequence
that corresponds to a part of the human adenine deaminase (ADA)
gene. The probe was excited at 490 nm and the monitoring of the PCR
reaction was performed at 530 nm. The observed fluorescence is
displayed as a function of the PCR cycle number in FIG. 19. After
some cycles in which a low overall fluorescence was monitored, a
sharp rise of the fluorescence within 4-5 cycles was observed
indicating the formation of the PCR product. In another example,
the nucleic acid probe (17.24) was employed in the detection of a
nucleic acid sequence that corresponds to a part of the human
prothrombin gene. The probe was excited at 490 nm and the
monitoring of the PCR reaction was performed at 530 nm. A similar
result as obtained with nucleic acid probe (17.21) was obtained and
the observed fluorescence is displayed as a function of the PCR
cycle number in FIG. 21. In yet another example, nucleic acid probe
(17.14), which carries the covalently attached dyes thiazole orange
and Texas Red was employed in the detection of the same ADA nucleic
acid sequence that was targeted with probe (17.21). The probe was
excited at 490 nm and the monitoring of the PCR reaction was
performed at 625 nm in this case. A similar result as obtained with
nucleic acid probes (17.21) and (17.24) was obtained, indicating
the formation of the PCR product by a rise of the observed
fluorescence after some initial cycles with low observable
fluorescence. The formation of specific PCR products of the
expected size was verified by agarose gel electrophoresis for each
of the described examples. Example 14 demonstrates the capacity of
the nucleic acid probes of the invention for the detection of
nucleic acid targets in homogeneous assays and in the detection of
PCR products in real-time.
[0192] In all homogeneous assays that are conducted simultaneously
with a PCR reaction, the fluorescent signal can be used to
determine the amount of target nucleic acid analyte present prior
to the start of the PCR reaction. The method is based on the
monitoring of the fluorescence signal in all annealing steps of the
subsequent cycles of the PCR reaction. In the initial cycles of the
PCR a very low fluorescence intensity is observed because the
quantity of the amplicon formed does not support a measurable
fluorescence output from the assay. After the initial cycles, as
the amount of formed amplicon increases, the fluorescence intensity
increases to a measurable level and reaches a plateau in later
cycles when the PCR enters into a non-logarithmic phase. Through a
plot of the fluorescence intensity versus the cycle number, the
specific cycle at which a measurable fluorescence signal is
obtained from the PCR reaction can be deduced and used to
back-calculate the quantity of the target before the start of the
PCR. The number of the specific cycles that is determined by this
method is named CT (cycle threshold). The method is described in
detail in Heid et al. (1996) Genome Methods 6:986-94, with
reference to hydrolysis probes. It can be used effectively to
quantify target nucleic acid analytes with a very large dynamic
range, at least a >100,000-fold range.
[0193] Example 15 describes the application of the nucleic acid
probes of the present invention in the quantification of a target
sequence. Probe (17.21) was employed in a series of PCR reactions
that were conducted in the presence of different amounts of the
target nucleic acid sequence. A serial dilution of a pre-amplified
target sequence was used for this purpose. The target sequence
relates to a part of the human adenine deaminase (ADA) gene. The
probe was excited at 490 nm and the monitoring of the PCR reaction
was performed at 530 nm. The observed fluorescence is displayed as
a function of the PCR cycle number in FIG. 20. Curves A and B in
the figure relate to the amplification of approximately 0.1% of a
pre-formed PCR product. After some cycles in which a low overall
fluorescence the observed fluorescence rises sharply indicating the
formation of the PCR product. The application of a threshold for
the fluorescence in the first cycles leads to a cycle number where
the observed fluorescence rises above the threshold CT. CT-values
of 12.3 and 12.1 were observed for curves A and B. Curve C in the
figure relates to the amplification of approximately 0.01% of a
pre-formed PCR product. A CT-value of 15.5 was observed for curve
C. Curves D and E in FIG. 20 relate to the amplification of
approximately 0.001% of a pre-formed PCR product. CT-values of 19.7
and 19.9 were observed for curves D and E. The curves F and G in
FIG. 20 relate to the amplification of approximately 0.0001% of a
pre-formed PCR product. CT-values of 22.9 and 25.2 were observed
for curves F and G. The observed cycle threshold values CT
correlate well with the geometrical progression of the PCR
reaction. Assuming that the same amount of hybridized probe is
necessary to generate a fluorescence signal that matches the
threshold in each of the experiments a 10-fold amplification is
required to compensate for a 10-fold reduced number of target
copies in the reaction. A 10-fold amplification corresponds to 3.3
cycles of the PCR at 100% amplification efficiency, which matches
the observed average difference between the CT-values for 10-fold
diluted targets, i.e. an average difference of 3.2 between the
CT-values of curves A/B and C, an average difference of 4.3 between
the CT-values of curves C and D/E, and an average difference of
4.25 between the CT-values of curves D/E and F/G. The slightly
higher values that were observed can be explained by a PCR
efficiency that is slightly below 100%. The formation of specific
PCR products of the expected size was verified by agarose gel
electrophoresis for each of the described experiments. Example 15
demonstrates the utility of the nucleic acid probes of the
invention for the quantification of nucleic acid targets in
homogeneous assays and in real-time PCR based assays.
[0194] The methods described above can be applied to conventional
PCR reactions with a DNA target nucleic acid analyte as well as to
many variations thereof, including, but not limited to, RT-PCR
(reverse transcription PCR) with RNA nucleic acid analytes, site
specific mutagenesis using PCR, inverse PCR reactions and other
variations. The methods are useful to detect and quantify genetic
variations in the target nucleic acid analyte using established PCR
techniques, see McPherson et al. eds., in "PCR A Practical
Approach," Oxford University Press, and Innis et al. eds., in "PCR
Applications, Protocols for Functional Genomics," each of which is
incorporated herein by reference in its entirety. In particular, an
allele specific PCR reaction can be performed to detect single
nucleotide polymorphisms as described in Newton et al. (1989)
Nucleic Acids Res. 17:2503-2516, and Sommer et al. (1989) Mayo
Clin. Proc. 64:1361-1372, each of which is incorporated herein by
reference.
[0195] In another particular embodiment of the invention, a
homogeneous assay is conducted simultaneously with an in vitro
transcription assay. In this type of assay, a plasmid or another
source of DNA sequence that carries a promoter sequence provides
the formation of single stranded RNA transcripts. The assay
typically contains the nucleic acid probe and the source of DNA, a
RNA polymerase, e.g. T7 RNA polymerase, an aqueous buffer,
ribonucleotide triphosphates and other non-reactive ingredients.
The other non-reactive ingredients include, but are not limited to,
dithiothreitol, dithioerythrol, magnesium chloride, salts, RNAse
inhibitors and other ingredients. In the course of the assay
multiple RNA transcripts are formed from a single DNA source
template. The transcript contains a stretch of nucleic acid
sequence, which is complementary to a stretch of nucleic acid
sequence on the probe. Upon hybridization of the nucleic acid probe
to the transcript a fluorescent signal is generated, which is
proportional to the amount of transcript formed. This embodiment of
the invention is illustrated schematically in FIG. 13. A
homogeneous transcription assay with an oligonucleotide probe that
contains the covalently attached intercalator oxazol yellow has
been described by Ishiguro et al. (1996) Nucleic Acids Res.
24:4992-97 (1996), which is incorporated herein by reference.
However, in the approach described by Ishiguro et al., the
described fluorescence signal was of rather low intensity and
needed to be measured against a fluorescence background of a
comparable intensity.
[0196] Homogeneous assays in accordance with this invention can be
multiplexed, i.e. more than one target nucleic acid analyte can be
detected in one assay. In a multiplex assay, more than one specific
nucleic acid probe, which differ in the nature of their covalently
attached dyes, is added to the mixture to be assayed. The dyes are
chosen to produce distinguishable fluorescent signals from each
specific nucleic acid probe. The signals of the different dye
combinations of the nucleic acid probes are recorded simultaneously
to detect and/or quantify the corresponding target nucleic acids at
the same time. Multiplexing greatly reduces the cost of analysis
and can tremendously increase throughput in high volume
settings.
[0197] Applications in Nucleic Acid Microarrays
[0198] In another embodiment of the present invention, a nucleic
acid probe as described herein is employed in assays that are
conducted on nucleic acid microarrays to detect or quantify nucleic
acid targets. In such assays, a fluorescent signal is generated on
a nucleic acid microarray upon the presence of a complementary
nucleic acid sequence in the analyte. Nucleic acid microarrays,
also called nucleic acid chips, consist of ordered arrays of
nucleic acids that are covalently attached to a solid surface, see
Schena, ed., in "DNA Microarrays A Practical Approach," Oxford
University Press, and Marshall et al. (1998) Nat. Biotechnol.
16:27-31, each of which is incorporated herein by reference, for a
comprehensive description of nucleic acid microarrays. The
fluorescent signal that is generated in the assay can be monitored
and quantified with fluorescence detectors, including fluorescence
imagers, e.g. commercial instruments supplied by Hitachi Corp., San
Bruno, Calif. or confocal laser microscopes (confocal fluorescence
scanners), e.g. commercial instruments from General Scanning, Inc.,
Watertown, Mass. With type I, II, III, VI, VII or VIII nucleic acid
probes a fluorescent signal is generated or increased upon the
presence of a complementary sequence in the target nucleic acid
analyte. With type IV, V, IX or X nucleic acid probes a fluorescent
signal is decreased upon the presence of a complementary sequence
in the target nucleic acid analyte.
[0199] In assays that are conducted on nucleic acid microarrays the
target nucleic acid analyte may be a mixture of nucleic acid
sequences, consisting of up to hundreds of nucleic acid sequences,
and in some instances of up to tens of thousands of nucleic acid
sequences. This instance particularly applies to expression
analysis, where many or all mRNA sequences that are present in a
biological system, e.g. a certain cell type from a cell culture,
are analyzed, see Hunt et al., eds., in "Functional Genomics A
Practical Approach," Oxford University Press, for a comprehensive
description of expression analysis, which is incorporated herein by
reference. Typically, the mRNA sequences are amplified by reverse
transcription PCR with universal primers prior to their use as
analytes in the assay. In this setting all nucleic acid sequences
present in the analyte are simultaneously applied to the microarray
for analysis, thus allowing the interaction of all nucleic acid
sequences of the analyte with all nucleic acids that are present on
the array. In other instances, the target nucleic acid analyte
contains a limited number of up to a hundred nucleic acid sequences
and in some instances only one nucleic acid sequence. In this case
the limited number of sequences typically contain more than one
stretch of specific nucleotide sequence to be analyzed, e.g. more
than one single nucleotide polymorphism. The nucleic acid sequences
of this setting may be amplified by PCR with the aid of specific
primers prior to their analysis on the microarray.
[0200] Generally, in analysis on microarrays the fluorescent
signals generated are converted to sequence specific results
through the known relation of the location of a spot on the array
and the nucleotide sequence attached to it.
[0201] In a particular embodiment of the invention mRNA molecules
from a biological source are detected and/or quantified. Typically,
the mRNA molecules are converted to cDNA molecules and/or further
amplified by PCR to provide the target nucleic acid analyte to be
applied in the assay on the microarray. In many instances, such
assays are conducted with mRNA samples obtained from a biological
system under different environmental conditions, such as exposures
to varying concentration of a drug candidate or mixtures of drug
candidates, which can provide data on the efficacy, the safety
profile, the mechanism of action and other properties of the drug
candidates that are required in drug development. In a particular
variation such samples are differentially labeled during the
reverse transcription and/or the PCR process to allow their
simultaneous application in the microarray based assay and
therefore a direct comparison of the quantity of specific mRNA
obtained under the different environmental conditions applied to
the biological system. This technique is known as "differential
display," see Hunt et al., eds., in "Functional Genomics A
Practical Approach," for a comprehensive description of this
method. Differential display is a technique useful to compare the
state of a biological system before and after exposure to
artificial conditions, especially exposure to drugs and drug
candidates to evaluate pharmacogenomic properties, or the exposure
to pathogens and their toxins. This technique can be extended to a
large variety of other assays that compare biological systems,
including, but not limited to the comparison of cells from
different tissues of an organism, e.g. different tissues from a
human body and the comparison of similar organisms, e.g. bacterial
strains, and other assays.
[0202] In the context of the present invention, the labeling of the
cDNA molecules of the analyte is performed with a nucleic acid
probe that acts as one of the unspecific primers in a reversed
transcription PCR reaction prior to the analysis. The product of
such a PCR reaction is a mixture of cDNA amplicons, each of which
contains the dyes of the probe and therefore represents a nucleic
acid probe by itself. The resulting mixture of amplicons is then
exposed under hybridization conditions to a microarray with an
ordered pattern of known oligonucleotide sequences that contain
stretches of complimentary nucleotide sequence to some or all of
the probes. Upon hybridization on the array a fluorescent signal is
generated at those spots of the array that contain stretches of
nucleotide sequence which are complementary to nucleotide sequences
in the probes indicating the presence of such sequences in the mRNA
that was used to generate the probes. In the differential display
technique two sets of labeled amplicons, generated as described
above, with differentially labeled nucleic acid probes as primers
of the reverse transcription PCR reaction are used simultaneously
on the microarray. Each set of amplicons is labeled with a set of
dyes unique to the corresponding nucleic acid probe in this
setting. The hybridization of the mixture of analytes results in
specific fluorescence signals on the array that allow the
comparison of the mRNA populations of the original samples.
[0203] In another embodiment of the invention, the nucleic acid
probes to detect or quantify nucleic acid targets in assays that
are conducted on nucleic acid microarrays are derived from genomic
DNA in order to analyze for the presence or absence of
polymorphisms in the genomic DNA. The polymorphisms can be
deletions, insertions, or base substitutions or other polymorphisms
of the genomic DNA. Typically the polymorphisms are single
nucleotide polymorphisms (SNPs), i.e. single base substitutions in
the genomic DNA.
[0204] In one case, the genomic DNA is amplified in one or more PCR
reactions with nucleic acid probes, as defined herein, that act as
specific primers of the PCR. The resulting amplicons of such PCR
reactions contain the primers in their sequence and thus the dyes,
which were incorporated into the nucleic acid probe. The amplicons
themselves are therefore nucleic acid probes, as defined in this
invention. The amplicons contain one or more polymorphic sites of
interest. The microarray that is used in the assay contains ordered
nucleic acid sequences that are complementary and/or partly
complementary to the polymorphic sites in such a manner that the
polymorphic sites can be identified in the assay. Typically, the
microarray contains sequences, which differ in one nucleotide
corresponding to the polymorphic site and allow the discrimination
of the possible variations at the polymorphic site upon
hybridization of the amplicons. A sequence that is fully
complementary will generate a fluorescent signal, whereas a
sequence with a corresponding possible variation of the
polymorphism, in many cases a single nucleotide variation, will not
hybridize as efficiently as the fully complementary sequence under
the conditions of the assay, and therefore will generate a weaker
fluorescent signal or no fluorescent signal at all. The employed
array typically contains more than one or all possible variations
of nucleotide sequence corresponding to the polymorphic site of
interest, e.g. both variations of a SNP, and therefore allows the
detection and/or quantitation of more than one or all variations of
the polymorphic site, e.g. both variations of a SNP. Therefore, in
SNP detection typically both homozygote and heterozygote variations
of the SNP can be detected.
[0205] This method can be employed with specific primers, which are
nucleic acid probes that carry the same set of covalently attached
dyes, or with specific primers, that are nucleic acid probes which
carry differentially labeled sets of covalently attached dyes. With
respect to the latter variation polymorphisms from different genes
may be individually labeled. The PCR reactions that employ the
nucleic acid probes as primers may be performed individually, or as
multiplexed PCR reactions.
[0206] In yet another variation, the genomic DNA is amplified in
one or more PCR reactions with unmodified specific primers, which
are not labeled. The resulting amplicons contain the polymorphisms
of interest and are employed in an assay with a microarray that
contains ordered spatially arranged nucleic acid probes in
accordance with this invention. The nucleic acid probes contain
stretches of nucleotide sequences that are complementary and/or
partly complementary to the polymorphic sites in such a manner that
the polymorphic sites can be identified in the assay. In this
variation the assay is conducted similarly to the assay described
above for unlabeled nucleotide sequences on the array and labeled
PCR amplicons and all the variations thereof as described
above.
[0207] The use of the nucleic acid probes of this invention in
assays that are conducted on nucleic acid microarrays to detect or
quantify nucleic acid targets offers several advantages compared to
other methods that are based on microarrays. The advantages are
based on the very low fluorescent background of the nucleic acid
probes defined herein. In particular, type I, II, III, VI, VII and
VIII nucleic acid probes have a very low intrinsic fluorescence and
are in some instances essentially non-fluorescent.
[0208] In a conventional assay that is based on microarrays with
fluorescent methods, the array needs to be stringently washed in
order to remove non-hybridized fluorescent nucleotide sequences
that interfere with the detection of the fluorescent signal and
increase the signal to noise ratio in the assay. In these assays a
delicate balance must be achieved with respect to the stringency of
the washing. A stringency that is too high removes correctly
hybridized sequences, which decreases positive signals in the
assay, and a stringency that is too low increases false positive
signals leading to unreliable and erroneous results. Often,
unspecific absorption of fluorescent nucleic acids on the surface
of microarrays is a major problem in these assays as the background
fluorescence generated from such absorption processes can't be
removed effectively. This is particularly true for surfaces
modified with chemical agents that provide aminated surfaces. An
aminated surface provides a positively charged matrix that attracts
all negatively charged nucleic acid species in a sample in a
non-specific manner.
[0209] The nucleic acid probes described herein require less
stringent washes because the probes generate positive signals if
and only if they are correctly hybridized on the array. Under
optimized conditions the nucleic acid probes can be applied even
without washes, because the probes themselves are non-fluorescent
and do not produce a background. This feature greatly diminishes
the cost of microarray based assays, increases the signal to noise
ratio of the assay and therefore its reliability and allows the
analysis of smaller and/or more dilute nucleic acid target
samples.
[0210] Applications of pH Sensitive Probes
[0211] In another embodiment of the instant invention, type XI
nucleic acid probes derivatized with a pH sensitive dye are used to
provide a switchable fluorescence signal in an assay. Upon
hybridization with a complementary nucleic acid analyte a
fluorescence signal is generated that is switchable, meaning it can
be turned on and off by an adjustment of the pH of the mixture.
This is particularly useful on microarrays, especially in
competitive assays, e.g. in differential display techniques. In
this variation, two 2 differentially labeled nucleic acid probes
with pH sensitive quenchers can be employed as primers in reverse
transcription assays to generate differentially labeled populations
of cDNAs from e.g. two different biological samples obtained under
different environmental conditions from the same organism or cell
culture. One of the probes can carry a quencher that becomes
effective under higher pH conditions, e.g. pH 7-10 (basic
quencher), and the other probe can carry a pH sensitive quencher
that becomes effective under lower pH conditions, e.g. at pH 4-7
(acidic quencher). Upon hybridization on a microarray in a
differential display technique, as described above, the fluorescent
signals on the microarray can be switched in response to the pH of
the hybridization buffer. At acidic pH, e.g. at pH 5, the
fluorescence from the probe that carries the acidic quencher can be
measured and the fluorescence from the probe that carries the basic
quencher is non-detectable. At basic pH, e.g. pH 9, this situation
is reversed, as the fluorescence from the probe that carries the
acidic quencher is non-detectable and the fluorescence from the
probe that carries the basic quencher is measurable. At neutral pH,
both probes provide signals. The switching of the signals allows a
clear distinction of the signals that result from both types of
probes on the array and avoids all problems that are associated
with crosstalk from two different dyes or dye combinations that are
commonly employed in differential display techniques.
[0212] In another embodiment type XI nucleic acid probes with pH
sensitive dyes are used as pH sensitive fluorescence sensors to
detect variations in pH within a sample. Samples include, but are
not limited to, environmental samples, cellular or other bodily
fluids, biological membranes and other samples. In this embodiment
the nucleic acid part of the nucleic acid probe provides water
solubility to the dye combination that is covalently attached with
the dyes. This allows the use of dye combinations that can't be
applied as regular pH indicators due to solubility problems in
water. The method can also be used with nucleic acid conjugates,
e.g. protein, peptide or lipid conjugates. The conjugation
transfers additional specificity to the probes, e.g. a specificity
for membranes from lipophilic conjugation, or a specificity for
antigens from the conjugation of an antibody.
[0213] The following examples are provided for illustrative
purposes only and are not intended to limit the scope of the
invention.
EXAMPLES
Example 1
Synthesis of bifunctional linker (13):
1-(N-4-(9-fluorenylmethoxycarbonyl)
aminobutyryl)-amino-3-(N-4-(4-methoxytrityl)-aminobutyryl)-aminopropan-2--
ol cyanoethoxy-diisopropylaminophosphoramidite
[0214] The synthesis of bifunctional linker 13 is outlined in
Scheme 1. 12
[0215] N4-(9-fluorenylmethoxycarbonyl)aminobutyric acid.
4-Aminobutyric acid (12.4 g, 120 mmol) was dissolved in a solution
of Na.sub.2CO.sub.3 (50.9 g, 480 mmol) in water (450 mL). To this
mixture a solution of 9-fluorenylmethyl chloroformate
(Fmoc-chloride, 25.8 g, 100 mmol) in 1,4-dioxane (60 mL) was added
under vigorous stirring at ambient temperature. The initially
formed precipitate disappeared during the reaction (approx. 3
hours). After the Fmoc-chloride was completely consumed
(TLC:CH.sub.2Cl.sub.2/EtOH, 90:10; R.sub.f (product) 0.67), the
mixture was carefully adjusted to pH 2 by adding concentrated HCl.
The slurry obtained was overlaid with ethyl acetate (.about.2.5 L),
mixed and the two phases were allowed to separate. The organic
phase was washed with water (3.times.500 mL), dried over
Na.sub.2SO.sub.4 and evaporated to dryness.
[0216] The residue obtained was dissolved in boiling ethanol (150
mL) and allowed to crystallize at room temperature. The precipitate
was filtrated and washed with ethanol (2.times.20 mL). Again the
precipitate was dissolved in boiling ethanol (100 mL), allowed to
crystallize at room temperature, filtrated and washed with ethanol
(2.times.10 mL). The residue was dried in vacuo yielding 20.4 g
(62.5%) of the product.
[0217] N4-(9-fluorenylmethoxycarbonyl)-aminobutyryl-p-nitrophenyl
ester. N4-(9-fluorenylmethoxycarbonyl)aminobutyric acid (18.6 g, 57
mmol), 4-nitrophenol (8.7 g, 63 mmol) and 4-dimethylaminopyridine
(0.7 g, 6 mmol) were dissolved in 1,4-dioxane (550 mL) and cooled
to 10.degree. C. To this solution dicyclohexylcarbodiimide (DCC,
11.9 g, 58 mmol) taken up in 1,4-dioxane (50 mL) was added in one
portion. After stirring over night at room temperature the reaction
mixture was quenched (TLC:EtOAc/hexane, 50:50; R.sub.f (product)
0.63) by adding acetic acid (2 mL). After 1 hour the solution was
filtered to remove precipitated urea. The obtained filter cake was
washed with ethyl acetate (2.times.250 mL) and the combined organic
phases were evaporated to dryness and taken up in ethyl acetate
(500 mL). The organic phase was washed with aqueous NaHCO.sub.3
(5%, 3.times.200 mL), saturated NaCl (3.times.200 mL), dried over
Na.sub.2SO.sub.4 and evaporated to dryness. A stirred solution of
the obtained residue in ethyl acetate (200 mL) was subjected to a
slow addition of hexanes (800 mL). The resulting suspension was
allowed to crystallize overnight, filtered, washed with hexanes
(2.times.50 mL) and dried in vacuo.
[0218] To remove polar contaminants the residue was dissolved in
boiling ethanol (200 mL) and allowed to crystallize overnight at
room temperature. The crystals were filtered, washed with cold
ethanol (2.times.20 mL) and dried in vacuo. The crude product (2.2
g, 48%) was subjected to prep. HPLC (CH.sub.2Cl.sub.2/ethyl
acetate, 100:0 to 97:3), yielding 10.3 g (41.0%) of product that
was 99.5% pure by HPLC. .sup.1H NMR (200 MHz, CDCl.sub.3): .delta.
8.29-7.24 (m, 12H), 4.94 (bs, 1H), 4.45 (d, 2H), 4.24 (t, 1H),
3.50-3.28 (m, 2H), 2.67 (t, 2H), 2.11-1.90 (m, 2H).
[0219] N4-(4-methoxytrityl)aminobutyric acid. 4-Aminobutyric acid
(10.3 g, 100 mmol) was suspended in a DMSO/pyridine mixture (100
mL, 80:20 v/v). 4-Methoxytrityl chloride (34.0 g, 110 mmol) was
added in portions (8.0 g, 8.0 g, 7.0 g, 7.0 g and 4.0 g) to the
stirred suspension in 15 minute intervals at ambient temperature
and the reaction mixture was stirred overnight at room temperature.
After all of the 4-aminobutyric acid was consumed
(TLC:CH.sub.2Cl.sub.2/EtOH, 90:10; R.sub.f (product) 0.52), the
mixture was diluted with CH.sub.2Cl.sub.2 (500 mL). The organic
phase was washed with saturated NaCl (1.times.500 mL, 1.times.300
mL), dried over Na.sub.2SO.sub.4 and evaporated to dryness. The
crude material (40.3 g) was purified by prep. HPLC
(CH.sub.2Cl.sub.2/ethanol+0.2% triethylamine, 100:0 to 97:3) to
yield 25.2 g (67%) of 97.3% pure product (HPLC). .sup.1H NMR (200
MHz, CDCl.sub.3): .delta. 7.51-6.75 (m, 14H), 3.77 (s, 3H), 2.38
(t, 2H), 2.24 (t, 1H), 1.73-1.94 (m, 2H).
[0220] N4-(4-methoxytrityl)-aminobutyryl-p-nitrophenyl ester. To a
solution of N4-(4-methoxytrityl)aminobutyric acid (25.2 g, 67 mmol)
in ethyl acetate (250 mL) was added a mixture of 4-nitrophenol
(10.4 g, 75 mmol) and DCC (13.9 g, 67 mmol) in ethyl acetate (200
mL). After the addition of 4-dimethylamino-pyridine (820 mg, 7
mmol), the reaction mixture was stirred overnight at room
temperature. Upon consumption of all of the
N4-(4-methoxytrityl)aminobutyric acid (TLC:EtOAc/hexane, 33:66;
R.sub.f (product) 0.65), the reaction was quenched by adding water
(1 mL). After 1 hour the mixture was filtered to remove the
precipitated urea. The obtained filter cake was washed with ethyl
acetate (300 mL) and the combined organic phases were extracted
with aqueous K.sub.2CO.sub.3 (5%, 2.times.200 mL), aq. NaHCO.sub.3
(5%, 2.times.200 mL), water (200 mL), dried over Na.sub.2SO.sub.4
and evaporated to dryness. The crude material was purified by prep.
HPLC (CH.sub.2CL.sub.2) to yield 20.7 g (62%) of 92.6% pure (HPLC)
product.
[0221]
1-Amino-N-3-(N-4-(4-methoxytrityl)-aminobutyryl)-aminopropan-2-ol.
A solution of N4-(4-methoxytrityl)-aminobutyryl-p-nitrophenyl ester
(20.4 g, 41 mmol) in CH.sub.2Cl.sub.2 (150 mL) was added to a
solution of 1,3-diaminopropan-2-ol (7.4 g, 82 mmol) and
triethylamine (5.7 mL, 41 mmol) in CH.sub.2Cl.sub.2 (200 mL) over a
period of 2 hours at ambient temperature. The reaction mixture was
stirred overnight at room temperature. Upon complete consumption of
the nitrophenyl ester (TLC:CH.sub.2Cl.sub.2/EtOH, 80:20; R.sub.f
(product) 0.06), the suspension was washed with aqueous
K.sub.2CO.sub.3 (5%, 2.times.250 mL), aq. NaHCO.sub.3 (5%,
2.times.250 mL), water (250 mL), dried over Na.sub.2SO.sub.4 and
evaporated to dryness. The crude material (15.6 g) was purified by
prep. HPLC (1. CH.sub.2Cl.sub.2/ethanol, 100:0 to 80:20; 2.
methanol), which yielded 9.0 g (49%) of 97.3% pure (HPLC) product.
.sup.1H NMR (200 MHz, CDCl.sub.3): .delta. 7.60-6.75 (m, 14H), 6.15
(t, 1H), 3.75 (s, 3H), 3.64-3.38 (m, 2H), 3.29-3.05 (m, 1H), 2.80
(dd, 1H), 2.56 (dd, 1H), 2.37-2.11 (m, 4H), 1.97-1.72 (m, 3H).
.sup.13C NMR (50 MHz, CDCl.sub.3): .delta. 173.8, 157.8, 146.3,
138.2, 129.7, 128.4, 127.7, 126.1, 113.0, 70.9, 70.3, 55.2, 44.6,
43.1, 34.6, 26.7.
[0222]
1-(N-4-(9-fluorenylmethoxycarbonyl)aminobutyryl)-amino-3-(N4-(4-met-
hoxytrityl)-aminobutyryl)-aminopropan-2-ol. A solution of
1-amino-N-3-(N-4-(4-methoxytrityl)-aminobutyryl)-aminopropan-2-ol
(9.0 g, 20.1 mmol) and ethyldiisopropylamine (3.5 mL, 20.1 mmol) in
CH.sub.2Cl.sub.2 (100 mL) was added to a solution of
N4-(9-fluorenylmethoxycarbonyl)-aminobutyryl-p-nitrophenyl ester
(9.0 g, 20.1 mmol) in CH.sub.2Cl.sub.2 (100 mL) over a period of 20
minutes at ambient temperature. The reaction mixture was stirred
overnight at room temperature (TLC:CH.sub.2Cl.sub.2/EtOH, 90:10;
R.sub.f (product) 0.54). The solution was extracted with aqueous
K.sub.2CO.sub.3 (5%, 2.times.200 mL), aq. NaHCO.sub.3 (5%,
2.times.200 mL), water (200 mL), dried over Na.sub.2SO.sub.4 and
evaporated to dryness. Upon adding ethyl acetate (100 mL) to the
residue, the mixture was heated to reflux until a clear solution
was obtained. Hexane (50 mL) was added over a period of 1 minute to
the stirred mixture. The suspension was allowed to crystallize for
4 hours at room temperature, filtered, washed with hexane/ethyl
acetate (100 mL, 33:66) and dried in vacuo. The crystals obtained
were purified via prep. HPLC to yield 9.3 g (61%) of the desired
product, which was 99.0% pure by HPLC. .sup.13C NMR (50 MHz,
CDCl.sub.3): .delta. 174.6, 174.1, 157.7, 156.9, 146.2, 143.8,
141.3, 138.1, 129.7, 128.4, 127.7, 127.0, 126.1, 125.0, 120.0,
113.0, 70.2, 70.1, 66.5, 55.2, 47.2, 43.0, 40.0, 34.5, 33.2, 26.6,
26.0.
[0223]
1-(N-4-(9-fluorenylmethoxycarbonyl)aminobutyryl)-amino-3-(N-4-(4-me-
thoxytrityl)-aminobutyryl)-aminopropan-2-ol
cyanoethoxydiisopropylaminopho- sphoramidite.
1-(N-4-(9-fluorenylmethoxycarbonyl)aminobutyryl)-amino-3-(N--
4-(4-methoxytrityl)-aminobutyryl)-aminopropan-2-ol (8.0 g, 10.6
mmol) was co-evaporated twice with dry THF (100 mL, each) and dried
in vacuo. The residue was dissolved in THF (100 mL), cooled to
5.degree. C. on an ice bath and diisopropylethylamine (2.8 mL, 15.9
mmol) was added. Chloro-2-cyanoethoxydiisopropylaminophosphane
(2.89 g, 12.2 mmol) was added to the solution in one portion. The
ice bath was removed and the reaction mixture was stirred for 1.25
hours at ambient temperature (TLC:THF/EtOAc, 25:75; R.sub.f
(product) 0.58). The solution was diluted with ethyl acetate (400
mL), washed with aqueous NaHCO.sub.3 (5%, 3.times.250 mL), dried
over Na.sub.2SO.sub.4 and evaporated to dryness. The crude material
was purified by prep. HPLC (ethyl acetate/hexane 70:30 to 80:20)
yielding 3.9 g (38%) of a white foam, which was subjected to
additional precipitation steps due to an insufficient purity as
measured by .sup.31P-NMR (.about.75%). The stirred solution of the
residue in ethyl acetate (15 mL) was triturated with hexanes (100
mL) and the resulting suspension was allowed to separate for 30
minutes and the upper phase was decanted. This process was repeated
and the resulting oil was dried in vacuo to give 3.4 g (32%) of a
white foam, the purity of which was 97.5% by HPLC, and 96.8% by
.sup.31P-NMR. .sup.1H NMR (200 MHz, CDCl.sub.3): .delta. 7.98-6.78
(m, 22H), 6.77-6.45 (m, 1H), 5.42-5.21 (m, 1H), 4.55-4.33 (m, 2H),
4.07-3.49 (m, 9H), 3.40-3.05 (m, 3H), 3.02-2.80 (m, 1H), 2.75-2.53
(m, 2H), 2.42-2.12 (m, 6H), 2.06-1.63 (m, 5H), 1.55-1.08 (m, 12H).
.sup.31P NMR (81 MHz, CDCl.sub.3): .delta. 148.64, 148.71.
Example 2
Synthesis of the bifunctional linker (14):
1-(N-6-(9-fluorenylmethoxycarbo-
nyl)aminocaproyl)-amino-3-(N-6-(cyclohexa-2,4-dienylmethoxycarbonyl)aminoc-
aproyl)-aminopropan-2-ol
cyanoethoxydiisopropylaminophosphoramidite
[0224] The synthesis of bifunctional linker 14 is outlined in
Scheme 2. 13
[0225] Ethyl-.epsilon.-aminocaproate hydrotosylate. A mixture of
.epsilon.-aminocaproic acid (70.0 g, 533.8 mmol), toluenesulfonic
acid (111.7 g, 1.1 eq.) and ethanol (1.6 L) was heated to reflux
for 20 hours. Thereafter, TLC (CH.sub.2Cl.sub.2/EtOH/TEA, 85:10:5)
revealed complete conversion. The reaction mixture was concentrated
until solids started to precipitate. The precipitation was driven
to completion by adding diethyl ether (1.5 L). The white solid was
filtered, washed with diethyl ether, and dried in vacuo affording
174.0 g (98.3%) of a white powder. .sup.1H NMR: (200 MHz,
CDCl.sub.3): .delta. 7.47 (d, 2H, J=8.2 Hz), 7.10 (d, 2H, J=8.2
Hz), 4.02 (q, J=7.0 Hz), 2.79-2.65 (m, 2H), 2.28 (s, 3H), 2.25 (t,
2H, J=7.0 Hz), 1.16 (t, 3H, J=7.0 Hz), 1.49 (dt, 4H, J=7.6, 7.0
Hz), 1.29-1.22 (m, 2H). .sup.13C NMR: 173.4, 145.6, 138.8, 128.9,
126.1, 60.4, 39.4, 33.9, 27.3, 25.9, 24.6, 21.5, 14.8.
[0226]
Ethyl-N-6-(cyclohexa-2,4-dienylmethoxycarbonyl)-aminocaproate
[0227] Hydroxymethylcyclohexa-2,4-diene (49.0 g, 444.8 mmol),
dissolved in THF (100 mL), was added under inert conditions to a
stirred solution of carbonyldiimidazole (CDI, 72.1 g, 444.8 mmol)
in THF (500 mL) at a rate of 20 mL/min. The mixture was allowed to
stir for 2 hours. An aliquot was removed, dried and dissolved in
CDCl.sub.3 to confirm the completion of the reaction by .sup.1H NMR
analysis. The solvent was removed by evaporation and the residue
was combined with ethyl-.epsilon.-aminocaproa- te hydrotosylate
(162.2 g, 489.3 mmol) in DMF (400 mL). After flushing with argon,
triethylamine (75.0 mL, 1.2 eq.) was added and the mixture was
allowed to stir overnight at ambient temperature. The reaction was
quenched with water (500 mL) and the aqueous phase was extracted
with ethyl acetate (3.times.250 mL). The organic fractions were
combined and washed with saturated bicarbonate and brine. The
organic phase was dried with MgSO.sub.4, filtered and the solvent
removed in vacuo. The resulting oil was purified via flash
chromatography (EtOAc/hexanes 2:8) to yield 53.3 g (35.6%) of a
colorless wax.
[0228] .sup.1H NMR (200 MHz, CDCl.sub.3): .delta. 5.92-5.82 (m,
2H), 5.75-5.70 (m, 1H), 5.64-5.59 (m, 1H), 4.09 (q, 2H, J=7.3 Hz),
4.02-3.90 (m, 2H), 3.19-3.11 (m, 2H), 2.27 (t, 2H, J=7.6 Hz),
2.20-2.00 (m, 2H), 1.66-1.55 (m, 2H), 1.55-1.43 (m, 2H), 1.40-1.29
(m, 2H), 1.22 (t, 3H, J=7.0 Hz). .sup.13C NMR (CDCl.sub.3): .delta.
173.7, 156.8, 126.7, 125.6, 124.1, 66.4, 60.4, 41.0, 34.4, 33.2,
29.9, 26.4, 25.4, 24.8, 14.4.
[0229] N6-(Cyclohexa-2,4-dienylmethoxycarbonyl)-aminocaproic acid.
A mixture of
ethyl-N-6-(cyclohexa-2,4-dienylmethoxycarbonyl)-aminocaproate (60.0
g, 203.1 mmol), 1 N NaOH (600 mL, 3 eq.) and dioxane (600 mL) was
heated to reflux and allowed to stir for 60 minutes. The mixture
was then concentrated to half the original volume and 4 N HCl (200
mL) was added with vigorous stirring. The solids were filtered off,
washed with water and co-evaporated with acetonitrile (3.times.500
mL) to afford 51.2 g (191.4 mmol, 94.2%) of a white powder. .sup.1H
NMR (200 MHz, CDCl.sub.3): .delta. 10.50 (bs, 1H), 5.94-5.83 (m,
2H), 5.76-5.70 (m, 2H), 5.65-5.60 (m, 1H), 4.82 (s, 1H), 4.05-3.97
(m, 2H), 3.15 (q, 2H, J=6.5 Hz), 2.33 (t, 2H, J=7.3 Hz), 2.25-1.99
(m, 2H), 1.68-1.58 (m, 2H), 1.55-1.45 (m, 2H), 1.41-1.32 (m, 2H).
.sup.13C NMR (CDCl.sub.3): .delta. 178.9, 158.5, 127.2, 126.5,
125.5, 124.1, 67.2, 41.0, 34.1, 33.0, 26.3, 25.3, 24.5.
[0230]
Pentafluorophenyl-N-6-(cyclohexa-2,4-dienylmethoxycarbonyl)-aminoca-
proate. Dicyclohexylcarbodiimide (DCC, 44.6 g, 1.15 eq.) was added
to a stirred solution of
N6-(cyclohexa-2,4-dienylmethoxycarbonyl)-aminocaproic acid (50.1 g,
187.3 mmol) in CH.sub.2Cl.sub.2 (400 mL). Pentafluorophenol (45.4
g, 1.32 eq.), dissolved in CH.sub.2Cl.sub.2 (100 mL) was added
under inert conditions. After stirring overnight TLC
(EtOAc/Hex/AcOH, 35:60:5) showed complete conversion. The reaction
mixture was filtered and the filtrate was used crude in the next
reaction. NMR of a purified sample: .sup.1H NMR (200 MHz,
CDCl.sub.3): .delta. 5.96-5.82 (m, 2H), 5.80-5.70 (m, 1H),
5.65-5.60 (m, 1H), 4.71 (s, 1H), 4.01 (d, 2H), 3.20 (q, 2H, J=6.5
Hz), 2.65 (t, 2H, J=7.0 Hz), 2.37-2.01 (m, 2H), 1.75 (q, 2H, J=7.3
Hz), 1.64-1.61 (m, 4H).
[0231]
1-Amino-N-3-(N-6-(cyclohexa-2,4-dienylmethoxycarbonyl)-aminocaproyl-
)-aminopropan-2-ol. A mixture of 1,3-diamino-2-hydroxypropane (67.6
g, 750 mmol) and CH.sub.2Cl.sub.2 (1 L) was stirred until the
diamine was dissolved completely (2 hours). A solution of crude
pentafluorophenyl-N-6-(cyclohexa-2,4-dienylmethoxycarbonyl)-aminocaproate
(187.3 mmol) was added at a rate of approximately 10 mL/min. The
reaction mixture was stirred overnight at ambient temperature,
filtered and the filtrate was concentrated to give an oil. The oil
was purified via flash chromatography (10 L of
EtOH/CH.sub.2Cl.sub.2, 1:4, followed by 10 L of EtOH/AcOH, 19:1) to
afford 44.6 g (60% over 2 steps) of a tan powder (isolated as the
acetate salt). .sup.1H NMR (200 MHz, CDCl.sub.3): .delta. 7.87 (s,
1H), 7.14 (s, 1H), 5.96-5.84 (m, 2H), 5.80-5.70 (m, 1H), 5.63 (dd,
1H, J=9.4, 3.5 Hz), 4.19 (bs, 3H), 3.85 (d, 2H, J=5.9 Hz), 3.46 (s,
1H), 3.03 (dt, 2H, J=4.1, 5.9 Hz), 2.92 (q, 2H, J=6.5 Hz),
2.60-2.37 (m, 6H), 2.04 (t, 2H, J=7.3 Hz), 1.48-1.30 (m, 4H), 1.77
(s, 3H), 1.19 (q, 2H, J=6.5 Hz). .sup.13C NMR (d.sub.6-DMSO):
.delta. 174.9, 173.3, 156.8, 127.5, 126.1, 125.7, 124.4, 67.8,
65.6, 43.6, 43.2, 35.9, 33.2, 29.9, 26.6, 25.7, 25.3, 23.9.
[0232]
Pentafluorophenyl-N-6-(9-fluorenylmethoxycarbonyl)-aminocaproate.
Pentafluorophenol (32.6 g, 1.25 eq.), dissolved in THF (150 mL),
was added to a stirred solution of N6-Fmoc-aminocaproic acid (50.0
g, 141.5 mmol) and DCC (36.5 g, 1.25 eq.) in THF (400 mL) under
inert conditions. The reaction was allowed to stir overnight at
ambient temperature. The solids were filtered off and washed with
THF. The filtrate was concentrated to approximately 300 mL and used
crude in the next reaction.
[0233]
1-(N-6-(9-Fluorenylmethoxycarbonyl)aminocaproyl)-amino-3-(N-6-(cycl-
ohexa-2,4-dienylmethoxycarbonyl)aminocaproyl)-aminopropan-2-ol.
1-Amino-N-3-(N-6-(cyclohexa-2,4-dienylmethoxycarbonyl)-aminocaproyl)-amin-
opropan-2-ol (43.6 g, 109.2 mmol) was mixed with THF (200 mL) and
DMF (100 mL) and the flask was flushed with argon. Triethylamine
(30.4 mL, 2.0 eq.) and crude
pentafluorophenyl-N-6-(9-fluorenylmethoxycarbonyl)-aminoca- proate
(141.5 mmol) were added to the stirred mixture. After 3 hours TLC
(10% EtOH in CH.sub.2Cl.sub.2) established the formation of the
desired product (R.sub.f 0.4) and the absence of starting material
(TLC with 5% AcOH/EtOH). The reaction was quenched by pouring the
mixture into 0.2 M NaH.sub.2PO.sub.4 (1 L) followed by storage at
4.degree. C. overnight. The mixture was extracted with EtOAc
(3.times.500 mL) and the combined organic phases were washed with
saturated NaHCO.sub.3 (2.times.500 mL) and brine (500 mL). The
organic phase was dried with MgSO.sub.4, filtered and concentrated.
Flash chromatography (EtOH/CH.sub.2Cl.sub.2, 20:80) of the crude
material gave 34.10 g (46.3%) of a white solid. .sup.1H NMR (200
MHz, CDCl.sub.3): .delta. 7.72 (d, 2H, J=7.6 Hz), 7.56 (d, 2H,
J=7.6 Hz), 7.37 (t, 2H, J=7.6 Hz), 7.27 (t, 2H, J=7.6 Hz),
7.10-6.89 (m, 2H), 5.97-5.81 (m, 2H), 5.77-5.67 (m, 1H), 5.61 (dd,
1H), 5.27-5.21 (m, 1H), 5.07-5.00 (m, 1H), 4.35 (d, 2H, J=7.0 Hz),
4.19 (t, 1H), 3.96 (d, 2H, J=6.5 Hz), 3.69-3.75 (m, 2H), 3.40-3.28
(m, 2H), 3.25-3.05 (m, 6H), 2.65-2.50 (m, 1H), 2.23-2.03 (m, 6H),
1.60 (q, 2H, J=7.3 Hz), 1.47 (q, 2H, J=7.3 Hz), 1.35-1.25 (m, 2H).
.sup.13C NMR (CDCl.sub.3): .delta. 175.0, 156.8, 144.2, 141.5,
127.9, 127.2, 126.6, 125.7, 125.6, 125.3, 124.2, 120.2, 70.0, 66.7,
66.4, 58.5, 47.5, 42.6, 41.0, 36.4, 33.1, 29.8, 26.4, 25.4,
18.6.
[0234]
1-(N-6-(9-Fluorenylmethoxycarbonyl)aminocaproyl)-amino-3-(N-6-(cycl-
ohexa-2,4-dienylmethoxycarbonyl)aminocaproyl)-aminopropan-2-ol
cyanoethoxydiisopropylaminophosphoramidite. A solution of
dicyanoimidazole (DCI, 75 mg, 0.06 eq.) in CH.sub.3CN (10 mL) was
added to a mixture of
1-(N-6-(9-fluorenylmethoxycarbonyl)aminocaproyl)-amino-3--
(N-6-(cyclohexa-2,4-dienylmethoxycarbonyl)aminocaproyl)-aminopropan-2-ol
(7.35 g, 10.89 mmol) and
bis(diisopropylamino)-2-cyanoethoxyphosphan ("bis-amidite", 3.44 g,
1.05 eq.) in CH.sub.2Cl.sub.2 (200 mL) under inert conditions. The
reaction mixture was stirred overnight at ambient temperature,
diluted to 500 mL with CH.sub.2Cl.sub.2 and washed with saturated
bicarbonate and brine. The aqueous fractions were combined and
back-extracted with CH.sub.2Cl.sub.2. The organic phases were
combined, dried with MgSO.sub.4, filtered and concentrated to give
an oil. The crude product was purified via flash chromatography
(THF/hexane, 60:40) to give 5.65 g (60%) of a highly viscous clear
oil. .sup.1H NMR (d.sub.6-DMSO): .delta. 7.86 (d, 2H, J=7.6 Hz),
7.74 (t, 1H, J=5.9 Hz), 7.66 (d, 3H, J=7.0 Hz), 7.35 (m, 5H), 7.11
(t, 1H, J=5.3 Hz), 5.90 (m, 2H), 5.76 (m, 1H), 5.64 (dd, 1H, J=3.5
Hz, 9.4 Hz), 4.27 (d, 2H, J=6.4 Hz), 4.20 (d, 1H, J=6.4 Hz), 3.86
(d, 2H, J=5.8 Hz), 3.80 (m, 3H), 3.78 (m, 3H), 3.33 (d, 1H, J=7.0
Hz), 3.19 (bs, 3H), 2.92 (t, 4H, J=6.4 Hz), 2.75 (t, 2H, J=5.9 Hz),
2.24-2.15 (m, 1H), 2.07-2.03 (m, 5H), 1.78-1.70 (m, 1H), 1.48-1.35
(m, 7H), 1.22-1.06 (m, 15H). .sup.31P NMR (d.sub.6-DMSO): .delta.
148.5 (s). MS (FAB+) calcd. for C.sub.47H.sub.67N.sub.6O.sub.8P
(MH+) 876.05, found 875.
Example 3
Synthesis of a dT10 Oligonucleotide Sequence Conjugated to
Bifunctional Linker (13)
[0235] A dT10 oligonucleotide sequence was assembled on an Applied
Biosystems model 391 synthesizer on a 1 .mu.mol scale on a CPG 500
dT-support. The synthesis protocol supplied by the instrument
manufacturer was followed with the exception that a 0.25 M solution
of 4,5-dicyanoimidazole (DCI) was used as the activator solution.
The amidite (13) was used in an additional synthesis cycle on the
support as a 0.1 M solution without further modifications of the
cycle. The resulting oligonucleotide was obtained in the trityl-on
mode and subjected to treatment with concentrated aqueous ammonia
for 24 hours at room temperature, which cleaved the oligonucleotide
from the support and removed the N-Fmoc protective group of the
linker in one step. The crude product was 91.9% pure as analyzed by
anion-exchange chromatography and found to contain 1.2% dT10
indicating a coupling efficiency greater than 98%. The product was
positively identified in the crude mixture by a co-injection
technique using a dT10-standard in the HPLC analysis. The
oligonucleotide was treated with 80% aqueous acetic acid for 1 hour
at 55.degree. C. to remove the N-MMT protective group of the linker
resulting in a crude product which was 93.0% pure by anion exchange
HPLC and contained 1.1% dT10.
Example 4
Synthesis of a dT10 Oligonucleotide Sequence Conjugated to
Bifunctional Linker (14)
[0236] A dT10 oligonucleotide sequence was assembled on an Applied
Biosystems model 391 synthesizer on a 1 .mu.mol scale on a CPG 500
dT-support. The synthesis protocol supplied by the instrument
manufacturer was followed with the exception that a 0.25 M solution
of 4,5-dicyanoimidazole (DCI) was used as the activator solution.
The amidite (14) was used in an additional synthesis cycle on the
support as a 0.1 M solution without further modifications of the
cycle. The resulting oligonucleotide was subjected to treatment
with concentrated aqueous ammonia at room temperature, which
cleaved the oligonucleotide from the support and removed the N-Fmoc
protecting group of the linker in one step. The crude material from
this synthesis contained 47.9% product, 16.5% dT10 and 17.7% of
product that was still protected with the N-Fmoc group after 3
hours of deprotection time, as analyzed by anion-exchange
chromatography. The product was positively identified in the crude
mixture by a co-injection technique using a dT10-standard in the
HPLC analysis.
Example 5
Synthesis of the Oligonucleotide Sequence
d(CCACTATCCTTCGCAAGACCCTTCC) (SEQ ID NO:[1] 12) Conjugated to the
Bifunctional Linker (13)
[0237] The oligonucleotide sequence d(CCACTATCCTTCGCAAGACCCTTCC)
(SEQ ID NO: 12) was assembled on an Applied Biosystems model 391
synthesizer on a 1 .mu.mol scale. The synthesis protocol supplied
by the instrument manufacturer was followed. The amidite (13) was
used in an additional synthesis cycle on the support as a 0.1 M
solution without further modifications of the cycle. The resulting
oligonucleotide was obtained in the trityl-off mode and manually
subjected to further treatment with detritylating solution and
acetonitrile in order to ensure the complete removal of the N-MMT
protecting group. The material was subjected to treatment with
concentrated aqueous ammonia solution for 24 hours at room
temperature. The resulting solution was evaporated, re-dissolved in
100 .mu.L of water and precipitated with 200 .mu.L of cold
(-20.degree. C.) ethanol and 20 .mu.L of 4 M ammonium chloride
solution. The mixture was chilled at -20.degree. C. for 15 minutes
and the pellet was recovered by centrifugation at 15,000 rpm for 15
minutes. The supernatant was removed and the pellet was washed with
70% aqueous ethanol. The presence of the target molecule in the
pellet was confirmed by mass spectroscopy (m/z calculated 7779.7,
found 7780.3).
Example 6
Procedure for the Synthesis of an Oligonucleotide Sequence
Conjugated to the Bifunctional Linker (14), Potentially Via a HEG
Spacer
[0238] The desired oligonucleotide sequence was assembled on an
EXPEDITE.TM. 8909 (Applied Biosystems Inc.) synthesizer at 1
.mu.mol scale on a CPG 500-support. Standard protected
deoxynucleoside phosphoramidites were employed (amidites derived
from DMT-dT, DMT-dA(bz), DMT-dG(ib) and DMT-dC(bz)). The synthesis
protocol supplied by the instrument manufacturer was followed with
the exception that a 0.25 M solution of 4,5-dicyanoimidazole (DCI)
was used as the activator solution. In the case in which the
conjugation of the bifunctional linker via the HEG spacer was
intended, a 0.063 M solution of the DMT-protected HEG
phosphoramidite (Cruachem Ltd., Glaskow, UK) was applied in an
additional unmodified synthesis cycle on the support. The amidite
(14) was then coupled to the HEG modified oligonucleotide sequence
on the support in the last synthesis cycle by applying a 0.1 M
solution of (14) and a 0.25 M solution of 4,5-dicyanoimidazole
(DCI). The coupling routine of the synthesis cycle was repeated
three times in this coupling step. Despite the modification of the
coupling routine, a standard synthesis cycle was employed as
recommended by the manufacturer of the instrument. The CPG support
with the resulting linker conjugated oligonucleotide was treated
with concentrated aqueous ammonia at room temperature for 60
minutes to cleave the oligonucleotide from the support. The CPG was
filtered off and the filtrate was incubated at 55.degree. C. over
night to deprotect the nucleobases and to remove the N-Fmoc
protecting group of the linker in one step. The solution of the
oligonucleotide was concentrated to dryness in a vacuum centrifuge.
The residue was dissolved in a mixture of 200 .mu.L water and 44
.mu.L of a 3 M solution of sodium acetate, pH 5.5. 2 mL of
isopropanol was added and the mixture was chilled at -20.degree. C.
for 1 hour. The resulting suspension was centrifuged for 10 minutes
and the supernatant of the pellet was discarded. The pellet was
washed once with 70% ethanol and dried under vacuum.
Example 7
Synthesis of the ethidium derivative (15):
8-(6-maleimidocaproyl)-ethidium bromide
[0239] The synthesis of the maleimide-ethidium conjugate (15) is
outlined in Scheme 3. 14
[0240] Ethidium tetraphenylborate. Ethidium bromide (1.05 g, 2.66
mmol) was dissolved in 400 mL of water. A solution of sodium
tetraphenylborate (2.74 g, 8 mmol) in 200 mL water was added with
stirring at room temperature, producing a bright red solid. The
solid was filtered off, washed with water and dissolved in
CH.sub.2Cl.sub.2. The resulting solution was filtered and
evaporated to dryness. The residue was coevaporated with
acetonitrile to yield 1.49 g (88%) of a purple solid.
[0241] 8-(6-maleimidocaproyl)-ethidium tetraphenylborate.
6-Maleimidocaproic acid (211 mg, 1.0 mmol) was dissolved in 10 mL
CH.sub.2Cl.sub.2. Oxalyl chloride (0.50 mL, 5 equiv.) was added
under inert gas followed by one drop of DMF. The reaction mixture
was stirred for one hour at room temperature. The solvent was
evaporated under vacuum and the residue was co-evaporated once with
10 mL CH.sub.2Cl.sub.2 under vacuum. The resulting
6-maleimidocaproic acid chloride was dissolved in 2 mL
CH.sub.2Cl.sub.2.
[0242] Ethidium tetraphenylborate (660 mL, 1.0 mmol) was dissolved
in 10 mL CH.sub.2Cl.sub.2. Triethylamine (2 equiv.) and the
previously prepared solution of 6-maleimidocaproic acid chloride
were added under inert gas. The reaction mixture was protected from
light and stirred overnight at room temperature. The reaction
mixture was diluted with 50 mL CH.sub.2Cl.sub.2 and extracted with
aqueous NaH.sub.2PO.sub.4 (pH 5.5, 0.2 M, 3.times.50 mL). The
organic phase was dried with MgSO.sub.4, filtered, and evaporated
to dryness to give a red-orange solid.
[0243] 8-(6-maleimidocaproyl)-ethidium bromide.
8-(6-maleimidocaproyl)-eth- idium tetraphenylborate from the
previous step was dissolved in dioxane/N,N-dimethyl-formamide (DMF)
5:1, v/v (10 mL) and then added to 10 mL water.
N,N-dimethylformamide was added in 1 mL portions until a
homogeneous solution was obtained. Polymer-supported bromide
(bromide on Amberlyst A-26, 3 mmol bromide/g, 5 g) was added and
the resulting suspension was shaken at room temperature overnight.
The solids were filtered off and the filtrate was evaporated to
dryness under vacuum. The residue was coevaporated with
acetonitrile to give an orange-red solid. The crude material was
purified by prep. HPLC with 10% ethanol in CH.sub.2Cl.sub.2. A
bis-derivatized ethidium compound eluted first followed by the
desired product, an orange solid, yield 149 mg
[0244] (26% over 2 steps). .sup.1H NMR (300 MHz, d.sub.6-DMSO):
.delta. 10.27 (s, 1H), 8.79 (t, 2H), 8.25 (d, 1H), 7.68-7.79 (m,
5H), 7.38-7.43 (m, 2H), 6.98 (s, 2H), 6.61 (s, 1H), 4.48 (d, 2H),
3.30-3.48 (m, H), 2.25 (t, 2H), 1.38-1.58 (m, 6H), 1.20 (q, 2H). MS
(FAB+) m/z=507.
Example 8
Preparation of NHS-ester functionalized thiazole orange derivative
(16):
6-(4-[3-methyl-2,3-dihydro(benzo-1,3-thiazole)-2-methyliden]-1-quinoliniu-
m)-hexanoic acid N-hydroxysuccinimide ester
[0245] The synthesis of the thiazole orange derivative (16) is
outlined in Scheme 4. 15
[0246] 3-Methyl-2-(methylthio)benzothiazolium tosylate. A mixture
of 2-methylthio-benzothiazole (18.4 g, 0.1 mol) and
methyl-p-toluenesulfonat- e (76 mL, 0.5 mol) was heated to a
temperature between 120.degree. C. to 145.degree. C. for 1 hour.
After cooling to room temperature diethyl ether (200 mL) was added.
The resulting solid was triturated with diethyl ether and isolated
by filtration to give the crude product (42.7 g, quantitative).
.sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 8.12 (d, 1H), 7.92 (d,
1H), 7.62 (d, 3H), 7.3 (t, 1H), 7.02 (d, 2H), 4.05 (s, 3H); 3.0 (s,
3H), 2.23 (s, 3H).
[0247] 6-(4-Methyl-1-quinolinium)-hexanoic acid. Lepidine (14.3 mL,
15.5 g, 0.108 mol) was mixed with 6-bromohexanoic acid (31 g, 0.159
mmol, 1.5 eqiv.) and heated at 130.degree. C. for 6 hours. The
mixture was allowed to cool to room temperature and acetone (100
mL) was added. The resulting solid was triturated with acetone to
provide the crude product (30.9 g, quantitative). .sup.1H-NMR (300
MHz, d.sub.6-DMSO): .delta. 9.44 (d, 1H), 8.60 (d, 1H), 8.55 (dd,
1H), 8.28 (d, 1H), 8.0 (m, 2H), 5.0 (t, 2H), 3.0 (s, 3H), 2.2 (t,
2H), 1.95 (m, 2H), 1.55 (m, 2H), 1.4 (m, 2H).).
[0248]
6-(4-[3-Methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-1-qu-
inolinium)-hexanoic acid. 6-(4-Methyl-1-quinolinium)-hexanoic acid
(10.5 g, 40.8 mmol) and 3-methyl-2-(methylthio)benzothiazolium
tosylate (15 g, 40.8 mmol) were mixed together in dichloromethane
(100 mL). Triethylamine (15 mL, 102 mmol, 2.5 eqiv.) was added and
the slightly turbid solution was stirred at room temperature over
night. The reaction was quenched by the addition of potassium
iodide (23 mL of an aqueous solution (30%, v/v)). The product was
precipitated with hydrochloric acid (80 mL of an aqueous solution
(3%, v/v)). After stirring for 2 hours the red solid was isolated
by filtration and washed with tetrahydrofuran to yield a crude
product (11.8 g, 71.6%), which was not further purified.
.sup.1H-NMR (300 MHz, CD.sub.3OD): .delta. 8.68 (d, 1H), 8.43 (d,
1H), 8.08 (dd, 1H), 7.98 (m, 1H), 7.9 (dd, 1H), 7.76 (m, 1H), 7.68
(d, 1H), 7.62 (m, 1H), 7.5 (d, 1H), 7.42 (m, 1H), 6.94 (s, 1H), 4.6
(t, 2H), 4.0 (s, 3H), 2.32 (t, 2H), 2.0 (m, 2H), 1.7 (m, 2H), 1.5
(m, 2H).
[0249]
6-(4-[3-Methyl-2,3-dihydro(benzo-1,3-thiazole)-2-methyliden]-quinol-
inium)-hexanoic acid N-hydroxysuccinimide ester (16). To a solution
of
6-(4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-1-quinolin-
ium)-hexanoic acid (2 g, 5 mmol) in N,N-dimethyl formamide (150 mL)
was added 1,3-dicyclohexylcarbodiimide (3.2 g, 15.4 mmol) and
N-hydroxysuccinimide (1.76 g, 15.4 mmol). The red turbid solution
was stirred for 2 days at room temperature. After evaporation of
the solvent the remaining residue was re-dissolved in 100 mL
CH.sub.2Cl.sub.2 and filtered. The filtrate was concentrated to
approximately 15 mL and precipitated in diethyl ether (500 mL). The
89,1% pure (HPLC) product was isolated as a red solid (2,9 g).
.sup.1H-NMR (300 MHz, d.sub.6-DMSO): .delta. 8.8 (d, 1H), 8.65 (d,
1H), 8.15 (d, 1H), 8.05 (dd, 2H), 7.8 (dd, 2H), 7.6 (t, 1H), 7.4
(dd, 2H), 6.95 (s, 1H), 4.6 (t, 2H), 4.0 (s, 3H), 2.8 (s, 4H), 2.7
(t, 2H), 1.9 (m, 2H), 1.7 (m, 2H); 1.5 (m, 2H).
Example 9
Procedure for the Preparation of the Doubly Labeled Probes of
Formula (17)
[0250] A linker-conjugated oligonucleotide (10 nmol) prepared
according to the method described in Example 6 was dissolved in
10-20 .mu.L of water. Sodium phosphate buffer (25 .mu.L, 100 mM, pH
7) and solutions of an NHS ester functionalized dye in
N,N-dimethylformamide (2.5 .mu.L, 40 mM, tenfold molar excess) and
a maleimide functionalized dye in N,N-dimethylformamide (2.5 .mu.L,
40 mM, tenfold excess) were added. The volume of the reaction
mixture was adjusted to 50 .mu.L with water and the solution was
incubated for 3 h at 37.degree. C. The resulting product solution
was stored at -20.degree. C.
[0251] Alternatively the labeling reaction was performed using the
following two separate steps:
[0252] 1. A linker-conjugated oligonucleotide (10 nmol) prepared
according to Example 6 was dissolved in 10-20 .mu.L water. Sodium
borate buffer (25 .mu.L, 50 mM, pH 8.5) and a solution of an NHS
ester functionalized dye in N,N-dimethylformamide (2,5 .mu.L, 40
mM, tenfold molar excess) were added. The volume was adjusted to 50
.mu.L with water and the solution was incubated for 3 h at
37.degree. C. The mixture was concentrated to dryness in a vacuum
centrifuge.
[0253] 2. The singly labeled oligonucleotide from step 1 was
dissolved in 10-20 .mu.L water. Sodium phosphate buffer (25 .mu.L,
100 mM, pH 5.5) and a solution of a maleimide functionalized dye in
N,N-dimethylformamide (2,5 .mu.L, 40 mM, tenfold excess) were
added. The volume was adjusted to 50 .mu.L with water and the
solution was incubated for 3 h at 37.degree. C. The resulting
product solution was stored at -20.degree. C.
[0254] The doubly labeled oligonucleotides were purified by gel
electrophoresis (16% acrylamide, 7 M urea; 1.5 mm.times.40
cm.times.21 cm; constant power of 55 Watt). After completion of the
electrophoresis the product bands were visualized by UV-shadowing
and gel slices that contained the major product bands were cut out.
The doubly labeled oligonucleotides were extracted from the gel
slices with 0.5 mL water at 60.degree. C. three times. The
solutions containing the labeled oligonucleotides were concentrated
in vacuo and subjected to ultracentrifugation with a 4 KDa cut-off.
Followed by dilution in water and OD measurement the concentration
of the oligonucleotide product solution was adjusted to 10
.mu.M.
[0255] Examples of doubly labeled oligonucleotide probes that were
prepared according to this procedure are listed in Table 1,
compounds (17.1)-(17.33). The ethidium derivative (15) was used in
all reactions where ethidium is attached to the oligonucleotide and
the thiazole orange (TO) derivative (16) was used in all reactions
where TO is attached to the oligonucleotide. The derivatives of
ethidium and thiazole orange were prepared as described in Example
7 and Example 8. All other dye derivatives were purchased either
from Molecular Probes Inc. (Eugene, Oreg., USA) or from
Sigma-Aldrich Corp. (St. Louis, Mo., USA) and the abbreviations for
the dyes are used in accordance with the catalogue of the supplier.
One letter abbreviations of nucleotides that are printed in bold
stand for LNA (locked nucleic acid) nucleotides.
[0256] The employed fluorescent dyes that were functionalized as an
NHS ester or as a maleimide were the following: Purchased from
Molecular Probes, Inc. (Eugene, Oreg., USA):
[0257] 5(6)-TAMRA-NHS (5-(and 6-)carboxytetramethylrhodamine
succinimidyl ester)
[0258] 5(6)-FAM-NHS (5-(and 6-)carboxyfluorescein succinimidyl
ester)
[0259] 7-QSY-NHS (carboxy-QSY-7-succinimidyl ester)
[0260] 5-TAMRA-M (tetramethylrhodamine-5-maleimide)
[0261] 7-QSY-M (QSY-7-maleimide)
[0262] Texas Red-M (Texas Red C.sub.2 maleimide)
[0263] MDCC-M
(7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coum-
arin)
[0264] Purchased from Sigma-Aldrich Corp. (St. Louis, Mo.,
USA):
[0265] 5-FAM-M (fluorescein-5-maleimide)
[0266] Doubly labeled oligonucleotide probes (17.1) to (17.33) were
characterized by MALDI-TOF mass spectroscopy with a Biflex.TM. III
(Bruker Daltonics Inc., Billerica, Mass., USA) instrument. The
calculated and observed m/z ratios are listed in Table 2.
2TABLE 2 Mass spectroscopic analysis of doubly labeled probes
Doubly labeled m/z m/z probe calculated observed (17.1) 7687.9
7674.0 (17.2) 7404.5 7403.0 (17.3) 8230.3 8224.5 (17.4) 7947.8
7938.9 (17.5) 8026.9 8020.1 (17.6) 7975.8 7976.2 (17.7) 8258.3
8255.9 (17.8) 8054.6 8057.2 (17.9) 9470.8 9469.4 (17.10) 9495.8
9494.0 (17.11) 9812.4 9797.3 (17.13) 9498.5 9497.1 (17.14) 9718.2
9695.7 (17.16) 9400.4 9397.9 (17.17) 7895.8 7890.6 (17.19) 8240.1
8242.8 (17.20) 8541.6 8527.3 (17.21) 8196.1 8192.1 (17.22) 9551.83
9539.3 (17.24) 8967.1 8966.9 (17.25) 9011.1 9011.9 (17.26) 9065.2
9061 (17.27) 9336.3 9341.6 (17.28) 9202.9 9204 (17.29) 9246.9
9245.5 (17.30) 9301 9294.7 (17.31) 10694.8 10698.9 (17.32) 11008
11011.1 (17.33) 8607 8611.3
Example 10
Measurement of the Fluorescence of the Nucleic Acid Probe (17.2) in
the Presence and Absence of a Complementary Sequence as a Function
of Temperature
[0267] The experiments were performed on a LIGHTCYCLER.TM.
instrument (Roche Diagnostics, Indianapolis, Ind., USA). Either
probe (17.2) (d(T).sub.20 with the covalently attached dyes
fluorescein and ethidium, see Table 1, 0.5 .mu.M) or a mixture of
probe (17.2) (0.5 .mu.M) with the complementary oligonucleotide
d(A.sub.29G) were employed. 20 .mu.L of a solution containing the
corresponding oligonucleotides in a PCR buffer (50 mM Tris/HCL, 10
mM KCl, 5 mM (NH.sub.4).sub.2SO.sub.4, 4 mM MgCl.sub.2, pH 8.3 at
25.degree. C.) were subjected to the following temperature
program:
[0268] 1. 95.degree. C. for 5 min
[0269] 2. cool down to 40.degree. C. at 20.degree. C. per
second
[0270] 3. heat up to 90.degree. C. at 0.1.degree. C. per second
[0271] 4. cool down to 40.degree. C. at 0.1.degree. C. per
second
[0272] 5. repeat steps 3 and 4 two times
[0273] During steps 3 and 4, the relative fluorescence intensity at
530 nm and 640 nm were recorded with irradiation of the solution at
the excitation wavelength 470 nm. The averaged data are depicted in
FIG. 14, curves A, A', B and B'.
Example 11
Measurement of the Fluorescence of the Nucleic Acid Probes (17.11),
(17.20, and (17.21) in the Presence and Absence of a Complementary
Sequence as a Function of Temperature
[0274] The experiments were performed on a SPEX FLUOROMAX.TM.-3
fluorescence spectrometer (Horiba Instruments, Inc., CA, USA). The
slit widths of both the emission and the excitation monochromator
were adjusted to 2 nm. For all probes the oligonucleotide
5'-d(AGGGTGGACTTGAAGATGAGCGAAAAA)-3' (SEQ ID NO:11) was employed as
the complementary sequence. 200 .mu.L of a solution containing
either the nucleic acid probe (0.5 .mu.M) or the nucleic acid probe
with its complementary sequence (probe 0.5 .mu.M, complementary
sequence 5 .mu.M) in a PCR buffer (50 mM Tris/HCL, 10 mM KCl, 5 mM
(NH.sub.4).sub.2SO.sub.4- , 4 mM MgCl.sub.2, pH 8.3 at 25.degree.
C.) were subjected to the following temperature program:
[0275] The fluorescence emission intensities were determined by
averaging over the data recorded successively four times at each
5.degree. C. step of temperature increase. The respective
excitation and emission wavelengths were 510/528 nm for probe
(17.11), 510/625 nm for probe (17.20) and both 420/528 nm and
510/528 nm for probe (17.21) For each of the probes the
fluorescence emission intensities observed with and without the
presence of the complementary sequence (I.sub.F.sup.hyb and
I.sub.F.sup.free) and additionally the ratio
I.sub.F.sup.hyb/I.sub.F.sup.- freeb of the fluorescence intensities
with and without the presence of the complementary sequence are
displayed as a function of temperature in FIG. 15 (probe (17.11)),
FIG. 16 (probe (17.20)), FIG. 17 (probe (17.21) with
excitation/emission at 510/528 nm) and FIG. 18 (probe (17.21) with
excitation/emission at 420/528 nm).
Example 12
Stability of the Nucleic Acid Probe (17.13) Under PCR
Conditions
[0276] The nucleic acid probe (17.13)
(5'-d(TTTTTCGCTCATCTTCAAGTCCACCCG)-3- ' (SEQ ID NO:5) with the
covalently attached dyes TAMRA and thiazole orange), see Table 1, 4
nmol) was dissolved in 430 .mu.L PCR buffer (Tris/HCl (50 mM, pH
8.3), KCl (10 mM), (NH.sub.4).sub.2SO.sub.4 (5 mM), MgCl.sub.2 (2
mM)) in the presence of 17.5 units of FastStartTaq DNA-Polymerase
(Roche Diagnostics, Indianapolis, Ind., USA). 50% of the solution
of the oligonucleotide was used as a reference sample. The
reference sample was concentrated to dryness in a vacuum centrifuge
and the residue was reconstituted in 20 .mu.L 0.1 M TEAA buffer pH
7.0 with 5% acetonitrile. The resulting solution was injected in an
analytical HPLC system and the oligonucleotide was analyzed at a
flow rate of 1.0 mL/minute with a gradient elution of 5% to 100%
acetonitrile in 0.1M TEAA buffer pH 7.0 for 15 minutes, followed by
elution with 100% acetonitrile for 10 minutes on a C18 reversed
phase column. The oligonucleotide eluted with a retention time of
21.5 and was approximately 80% pure by integration at 260 nm. The
remaining solution of the oligonucleotide in PCR buffer was
subjected to 40 temperature cycles in a PCR machine (1 minute
94.degree. C., 1 minute 66.degree. C., 1 minute 72.degree. C.),
concentrated to dryness and analyzed as described for the reference
sample. The oligonucleotide was approximately 80% pure by
integration at 260 nm and had an impurity profile that was
virtually indistinguishable from the impurity profile of the
reference sample.
Example 13
PCR with Doubly Labeled Nucleic Acid Probes and Human Genomic
DNA
[0277] The experiments summarized in Table 3, below were performed
on a MINICYCLER.TM. TTC-150 thermal cycler (MJ Research, Inc.,
Reno, Nev., USA). The PCR reactions were set up in a total volume
of 25 .mu.L with each tube containing Tris/HCl buffer (50 mM, pH
8.3), KCl (10 mM), (NH.sub.4).sub.2SO.sub.4 (5 mM), MgCl.sub.2 (2
mM), the deoxynucleotide triphosphates dATP, dGTP, dCTP and dTTP
(200 .mu.M each), primers 1 and 2 according to table 3 (250 nM),
human genomic DNA (10 ng), FastStartTaq DNA-Polymerase (1 unit,
Roche Diagnostics, Indianapolis, Ind., USA) and BSA (0.5 mg/mL).
The reactions were initiated at 94.degree. C. for 10 minutes,
followed by 30 cycles of denaturation at 94.degree. C. for 1
minute, annealing at 66.degree. C. for 1 minute, and elongation at
72.degree. C. for 1 minute. Finally the reaction was kept at
72.degree. C. for 7 minutes. The PCR products were analyzed by
agarose gel electrophoresis and the corresponding PCR products were
detected by staining with SYBR Gold (Molecular probes, Eugene,
Oreg., USA). Specific PCR products of the expected length were
obtained in all cases.
3TABLE 3 PCR with doubly labeled probes and human genomic DNA
Primer 1.sup.1) Dye 1 Dye 2 5'-Modification Primer 2.sup.2)
Gene.sup.3) Amplicon (17.4) 5(6)-FAM Ethidium (14) (22) ADA 254 bp
(17.15) TO MDCC (14) (22) ADA 259 bp (17.16) TO MDCC (14) (22) ADA
259 bp (17.21) TO MDCC HEG/(14) (22) ADA 254 bp (17.24) TO MDCC
(14) (23) Prothrombin 218 bp (17.28) TO MDCC (14) (24) TCR.beta.
372 bp .sup.1)Nucleic acid probes (17.15), (17.16) and (17.24)
contain a 5'-leading sequence d(T).sub.5 that is not part of the
corresponding gene. .sup.2)Primer (22) =
5'-d(GCT-CAA-CAC-AAA-GAT-GTC-TTC-TCT-GTG)-3' (SEQ ID NO:13) Primer
(23) = 5'-d(GGG-TGA-AGG-CTG-TGA-CCG)-3' (SEQ ID NO:14) Primer (24)
= 5'-d(GGG-TGA-AGG-CTG-TGA-CCG)-3' (SEQ ID NO:14) .sup.3)Female
human genomic DNA from Promega GmbH (Mannheim, Germany, catalogue
no. G1521) was employed in all reactions except for the reaction
with the nucleic acid probe (17.16) that represents an
allele-specific primer for a single nucleotide polymorphism in the
adenine deaminase (ADA) gene with a T to G substitution at position
1006, human genomic DNA from the Coriell Institute (Camden, NJ,
USA) that was heterozygous with respect to the polymorphism was
employed with probe (17.16)
Example 14
Real-Time PCR with the Nucleic Acid Probes (17.14), (17.21) and
(17.24)
[0278] The experiments were performed with an "ICYCLER.TM. iQ" PCR
machine (Bio-Rad Laboratories, Inc., USA). The PCR reactions were
set up in a total volume of 25 .mu.L containing Tris/HCl buffer (50
mM, pH 8.3), KCl (10 mM), (NH.sub.4).sub.2SO.sub.4 (5 mM),
MgCl.sub.2 (2 mM), the deoxynucleotide triphosphates dATP, dGTP,
dCTP and dTTP (200 .mu.M each), one of the nucleic acid probes
(17.14), (17.21) or (17.24) as the first primer (800 nM), a second
primer (primer (22)=5'-d(GCT-CAA-CAC-AAA-GAT-GT- C-TTC-TCT-GTG)-3'
(SEQ ID NO:13) with probes (17.14) and (17.21), and primer
(23)=5'-d(GGG-TGA-AGG-CTG-TGA-CCG)-3' (SEQ ID NO:14) with probe
(17.24), 800 nM), FastStartTaq DNA-Polymerase (1 unit, Roche
Diagnostics, Indianapolis, Ind., USA) and BSA (0.5 mg/mL). An
aliquot of a PCR product that was prepared according to Example 13,
was employed as the template in each reaction. The reactions with
the probes (17.14) and (17.21) contained approximately 0.1% of the
254 bp or 259 bp PCR product that corresponds to a part of the
human adenine deaminase (ADA) gene and the reaction with the probe
(17.24) contained approximately 0.1% of the 218 bp PCR product that
corresponds to a part of the human prothrombin gene. A filter set
corresponding to an excitation wavelength of 490 nm and an emission
wavelength of 530 nm was employed with the probes (17.21) and
(17.24) and a filter set corresponding to an excitation wavelength
of 490 nm and an emission wavelength of 625 nm was employed with
the probe (17.14).
[0279] The reactions were initiated with 10 minutes at 94.degree.
C., followed by 30 cycles of denaturation at 94.degree. C. for 1
minute, annealing at 66.degree. C. for 1 minute, and elongation at
72.degree. C. for 1 minute. Finally the reactions were kept at
72.degree. C. for 7 minutes. The fluorescence emission intensities
as a function of the cycle number that were observed with the
nucleic acid probes (17.21) and (17.24) are depicted in FIG. 19 and
FIG. 21. A nearly identical result was obtained with the nucleic
acid probe (17.14). The PCR products were analyzed by agarose gel
electrophoresis and the corresponding products with a length of 254
bp, 259 bp or 218 bp were detected by staining with SYBR Gold
(Molecular probes, Eugene, Oreg., USA).
Example 15
Quantification of a Target Nucleic Acid with the Nucleic Acid Probe
(17.21)
[0280] The experiments were performed with an "ICYCLER.TM. iQ" PCR
machine (Bio-Rad Laboratories, Inc., USA). The PCR reactions were
set up in a total volume of 25 .mu.L containing Tris/HCl buffer (50
mM, pH 8.3), KCl (10 mM), (NH.sub.4).sub.2SO.sub.4 (5 mM),
MgCl.sub.2 (2 mM), the deoxynucleotide triphosphates dATP, dGTP,
dCTP and dTTP (200 .mu.M each), the primer (22)
(5'-d(GCT-CAA-CAC-AAA-GAT-GTC-TTC-TCT-GTG)-3' (SEQ ID NO:13) 800
nM), the nucleic acid probe (17.21) as the second primer (800 nM),
FastStartTaq DNA-Polymerase (1 unit, Roche Diagnostics,
Indianapolis, Ind., USA) and BSA (0.5 mg/mL). Aliquots of the 254
bp PCR product corresponding to the human adenine deaminase (ADA)
gene that was prepared according to Example 13 were employed as the
template in the reactions. Approximately 0.1%, 0.01%, 0.001% and
0.0001% of the PCR product was used. The reactions were performed
in duplicate. A filter set corresponding to an excitation
wavelength of 490 nm and an emission wavelength of 530 nm was
employed.
[0281] The reactions were initiated with 10 minutes at 94.degree.
C., followed by 30 cycles of denaturation at 94.degree. C. for 1
minute, annealing at 66.degree. C. for 1 minute, and elongation at
72.degree. C. for 1 minute. Finally the reactions were kept at
72.degree. C. for 7 minutes. The observed fluorescence emission
intensities as a function of the cycle number are depicted in FIG.
20. Curves A and B depict the observed fluorescence intensity where
0.1% of the total amount of a PCR product was employed as the
template, curve C relates to a reaction where 0.01% of a PCR
product was employed, curves D and E relate to reactions where
0.001% of a PCR product was employed, and curves F and G relate to
reactions where 0.001% of a PCR product was employed. The PCR
products were analyzed by agarose gel electrophoresis and the
corresponding products with a length of 254 bp were detected by
staining with SYBR Gold (Molecular probes, Eugene, Oreg., USA).
Sequence CWU 1
1
14 1 20 DNA Artificial Synthetic Nucleic Acid Probe 1 tttttttttt
tttttttttt 20 2 22 DNA Artificial Synthetic Nucleic Acid Probe 2
cgctcatctt caagtccacc ct 22 3 22 DNA Artificial Synthetic Nucleic
Acid Probe 3 cgctcatctt caagtccacc cg 22 4 27 DNA Artificial
Synthetic Nucleic Acid Probe 4 tttttcgctc atcttcaagt ccaccct 27 5
27 DNA Artificial Synthetic Nucleic Acid Probe 5 tttttcgctc
atcttcaagt ccacccg 27 6 25 DNA Artificial Synthetic Nucleic Acid
Probe 6 tttttcactg ggagcattga ggctc 25 7 26 DNA Artificial
Synthetic Nucleic Acid Probe 7 attctcaact ctgactgtga gcaaca 26 8 30
DNA Artificial Synthetic Nucleic Acid Probe 8 aactcctctt cagtaaagcc
catgtcccgt 30 9 31 DNA Artificial Synthetic Nucleic Acid Probe 9
aaactcctct tcagtaaagc ccatgtcccg t 31 10 23 DNA Artificial
Synthetic Nucleic Acid Probe 10 ttcagtaaag cccatgtccc gtt 23 11 27
DNA Artificial Synthetic Nucleic Acid Probe 11 agggtggact
tgtgaaagag cgaaaaa 27 12 25 DNA Artificial Synthetic Nucleic Acid
Probe 12 ccactatcct tcgcaagacc cttcc 25 13 27 DNA Artificial
Synthetic Nucleic Acid Probe 13 gctcaacaca aagatgtctt ctctgtg 27 14
18 DNA Artificial Synthetic Nucleic Acid Probe 14 gggtgaaggc
tgtgaccg 18
* * * * *
References