Title: 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.
Patent Number: 6,902,900 Issued on 06/07/2005 to Davies, et al.
| Inventors:
|
Davies; Martin (Kent, GB);
Bruce; Ian (East Sussex, GB);
Wolter; Andreas (Esmarchstrasse, DE)
|
| Assignee:
|
Prolico, LLC (Boulder, CO)
|
| Appl. No.:
|
278047 |
| Filed:
|
October 21, 2002 |
| Current U.S. Class: |
435/6; 534/727; 536/23.1; 536/24.3; 536/24.32; 536/25.32; 536/25.33; 536/25.34 |
| Intern'l Class: |
C12Q 001/68; C07H021/00; C07H021/02; C07H021/04; C09B039/00 |
| Field of Search: |
536/231,243.2,253.2,253.3,253.4,243,243.3,256,253
563/243
534/727
435/6,911,912
|
References Cited [Referenced By]
U.S. Patent Documents
| 5210015 | May., 1993 | Gelfand et al.
| |
| 5998135 | Dec., 1999 | Rabbani et al.
| |
| 6251600 | Jun., 2001 | Winger et al.
| |
| 2001/0014452 | Aug., 2001 | Makino et al.
| |
| 2002/0034754 | Mar., 2002 | Reed et al.
| |
| Foreign Patent Documents |
| 1215498 | Jun., 2002 | EP.
| |
| WO 00/4075/1 | Jul., 2000 | WO.
| |
Other References
Sahoo et al. Pyrene Excimer Fluorescence: A spatially sensitive probe to monitor
lipid-induced helical rearrangement of apolipophorin II. Biochemistry. vol. 39.
pp. 6594-6601, 2000.
Cardullo et al. (Dec. 1988) Proc. Natl. Acad. Sci. USA 85:8790-8794.
Espy et al. (Feb. 2000) J. Clin. Microbiol. 38:795-799.
Heid et al. (1996) Genome Research 6:986-994.
Higuchi et al. (Apr. 1992) Biotechnology 10:412-417.
Holland et al. (Aug. 1991) Proc. Natl. Acad. Sci. USA 88:7276-7280.
Marras et al. (1999) Genetic Analysis 14: 151-156.
Shinozuka et al. (1994) J. Chem. Soc., Chem. Comm. 1377-1378.
Tyagi and Kramer (Mar. 1996) Nat. Biotechnol. 14:303-308.
Whitcombe et al. (Aug. 1999) Nat. Biotechnol. 17:804-807.
Wittmer et al. (Jan. 1997) BioTechniques 22:130-138.
Masuko et al. Nucleic Acid Research, (2000), vol. 28, No. 8 pp. e34, i-viii.
|
Primary Examiner: Horlick; Kenneth R.
Assistant Examiner: Wilder; Cynthia
Attorney, Agent or Firm: Swanson & Bratschun, L.L.C.
Parent Case Text
This application claims benefit of U.S. Provisional No. 60/336,432 filed Oct.
19, 2001.
Claims
1. 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 nucleotide of the nucleic probe or at nucleotides of
the nucleic acid probe that are directly adjacent to each other;
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. detecting or quantifying said nucleic acid analyte by measuring the change
in the fluorescence of the nucleic acid probe resulting from the specific interaction
of at least one high affinity dye with the nucleic acid analyte upon the hybridization
of the nucleic acid probe with the nucleic acid analyte.
2. The method of claim 1 wherein the dye that has a high affinity to double stranded
nucleic acids is an intercalator.
3. The method of claim 1 wherein the dye that has a high affinity to double stranded
nucleic acids is a groove binder.
4. The method of claim 1 wherein the nucleic acid probe comprises a fluorescent
intercalator and a non-fluorescent quencher.
5. The method 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 method 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 method of claim 1 wherein the nucleic acid probe comprises a fluorescent
intercalator and two dyes that form an excimer pair.
8. The method of claim 1 wherein the nucleic acid probe comprises a fluorescent
intercalator and two dyes that form an exciplex pair.
9. The method of claim 1 wherein the nucleic acid probe comprises a fluorescent
groove binder and a non-fluorescent quencher.
10. The method 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 fluorescent groove binder and
the second dye.
11. The method 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 fluorescent groove binder and
the second dye.
12. The method of claim 1 wherein the nucleic acid probe comprises a fluorescent
groove binder and two dyes that form an excimer pair.
13. The method of claim 1 wherein the nucleic acid probe comprises a fluorescent
groove binder and two dyes that form an exciplex pair.
14. The method of claim 1 carried out as a homogeneous assay to detect or quantify
a nucleic acid analyte in a sample.
15. The method of claim 14 wherein the homogeneous assay is a polymerase chain
reaction (PCR).
16. The method of claim 15 wherein the nucleic acid probe functions as a primer
in the polymerase chain reaction, providing for a real-time detection or quantification
of the amplification product.
17. The method of claim 15 wherein said nucleic acid probe functions as a hybridization
probe in a polymerase chain reaction, providing for a real-time detection or quantification
of the amplification product.
18. The method of claim 1 conducted in a multiplexed format.
19. The method of claim 18 performed by applying nucleic acid microarrays.
20. The method of claim 19 wherein the nucleic acid probe is provided on a microarray
and the nucleic acid analyte is provided in solution.
21. The method of claim 20 wherein the nucleic acid analyte is hybridized to
a microarray and the nucleic acid probe is brought into contact with the microarray.
22. 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 nucleotide of the nucleic
probe or at nucleotides of the nucleic acid probe that are directly adjacent to
each other;
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.
23. The method of claim 22 wherein the nucleic acid analyte is hybridized to
a microarray and the removal of the unhybridized nucleic acid probe is conducted
by washing the microarray under conditions that leave the hybridized nucleic acid
probe on the array.
24. The method of claim 22 wherein the pH-sensitive dye is selected from a substituted
trityl group and wherein 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.
25. The method of claim 24 wherein the substituted trityl group is a derivative
of the dimethoxytrityl group.
Description
FIELD OF INVENTION
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
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.
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.
Current methods used to detect and measure nucleic acid analytes are briefly
described below.
PCR Methods
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 (λ
max
excitation and λ
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.
FISH Methods
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.
Nucleic Acid Microarrays
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.
Fluorescence Energy-Transfer (FRET) Methods
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.
Molecular Beacon Methods
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.
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.
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.
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.
Hydrolysis Probes
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™
probe technology, Perkin-Elmer Applied Biosystems, Foster City, Calif., USA) and
is amplified 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.
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.
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.
Hairpin Probes
Hairpin probes or molecular beacons are described by Tyagi et at. (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™ 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.
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
m of the loop portion of the hairpin structure with a
complementary nucleic acid sequence and the T
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.
Hybridization Probes
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™ 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™ 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.
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.
Probeless Detection
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.
patent application Ser. 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.
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.
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.
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.
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
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.
In the preferred embodiments of the invention the nucleic acid probes, as defined
herein, are comprised of the following combinations of covalent dyes:
I) a fluorescent intercalator and a non-fluorescent quencher;
II) a fluorescent intercalator and a donor dye of a FRET system;
III) a fluorescent intercalator and an acceptor dye of a FRET system;
IV) an intercalator and two dyes forming an excimer pair;
V) an intercalator and two dyes forming an exciplex pair;
VI) a fluorescent groove binder and a non-fluorescent quencher;
VII) a fluorescent groove binder and a donor dye of a FRET system;
VIII) a fluorescent groove binder and an acceptor dye of a FRET system;
IX) a groove binder and two dyes forming an excimer pair; and
X) a groove binder and two dyes forming an exciplex pair.
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.
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.
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.
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
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).
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).
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).
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.
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.
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).
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).
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).
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.
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 X 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
"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.
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.
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.
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.
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.
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.
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.
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.
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 Bemacchi et al. (2001) Nucleic Acids Res. 29:e62, which is incorporated herein
by reference in its entirety, or by other mechanisms.
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.
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.
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.
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.
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.
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.
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.
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.
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:
I) a fluorescent intercalator and a non-fluorescent quencher;
II) a fluorescent intercalator and a donor dye of a FRET system;
III) a fluorescent intercalator and an acceptor dye of a FRET system;
IV) an intercalator and two dyes forming an excimer pair;
V) an intercalator and two dyes forming an exciplex pair;
VI) a fluorescent groove binder and a non-fluorescent quencher;
VII) a fluorescent groove binder and a donor dye of a FRET system;
VIII) a fluorescent groove binder and an acceptor dye of a FRET system;
IX) a groove binder and two dyes forming an excimer pair; and
X) a groove binder and two dyes forming an exciplex pair.
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.
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.
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.
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 a
