Heterogeneous assay for pyrophosphate detection

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FIELD OF THE INVENTION

This invention relates generally to a heterogeneous assay, and in particular, to assay methods using fluorescent nucleotide triphosphates having a fluorophore moiety attached to the .gamma.-phosphate that are especially useful for pyrophosphate detection.

BACKGROUND OF THE INVENTION

The primary sequences of nucleic acids are crucial for understanding the function and control of genes and for applying many of the basic techniques of molecular biology. The ability to do rapid and reliable DNA sequencing is therefore a very important technology. The DNA sequence is an important tool in genomic analysis as well as other applications, such as genetic identification, forensic analysis, genetic counseling, medical diagnostics, etc. With respect to the area of medical diagnostic sequencing, disorders, susceptibilities to disorders, and prognoses of disease conditions, can be correlated with the presence of particular DNA sequences, or the degree of variation (or mutation) in DNA sequences, at one or more genetic loci. Examples of such phenomena include human leukocyte antigen (HLA) typing, cystic fibrosis, tumor progression and heterogeneity, p53 proto-oncogene mutations and ras proto-oncogene mutations (see, Gyllensten et al., PCR Methods and Applications, 1: 91-98 (1991); U.S. Pat. No. 5,578,443, issued to Santamaria et al.; and U.S. Pat. No. 5,776,677, issued to Tsui et al.).

Various approaches to DNA sequencing exist. The dideoxy chain termination method serves as the basis for all currently available automated DNA sequencing machines. (see, Sanger et al., Proc. Natl. Acad. Sci., 74: 5463-5467 (1977); Church et al., Science, 240: 185-188 (1988); and Hunkapiller et al., Science, 254: 59-67 (1991)). Other methods include the chemical degradation method, (see, Maxam et al., Proc. Natl. Acad. Sci., 74: 560-564 (1977), whole-genome approaches (see, Fleischmann et al., Science, 269, 496 (1995)), expressed sequence tag sequencing (see, Velculescu et al, Science, 270, (1995)), array methods based on sequencing by hybridization (see, Koster et al., Nature Biotechnology, 14, 1123 (1996)), and single molecule sequencing (SMS) (see, Jett et al., J. Biomol. Struct. Dyn. 7, 301 (1989) and Schecker et al., Proc. SPIE-Int. Soc. Opt. Eng. 2386, 4 (1995)).

Fluorescent dyes can be used in a variety of these DNA sequencing techniques. A fluorophore moiety or dye is a molecule capable of generating a fluorescence signal. A quencher moiety is a molecule capable of absorbing the fluorescence energy of an excited fluorophore, thereby quenching the fluorescence signal that would otherwise be released from the excited fluorophore. In order for a quencher to quench an excited fluorophore, the quencher moiety must be within a minimum quenching distance of the excited fluorophore moiety at some time prior to the fluorophore releasing the stored fluorescence energy.

Fluorophore-quencher pairs have been incorporated into oligonucleotide probes in order to monitor biological events based on the fluorophore and quencher being separated or brought within a minimum quenching distance of each other. For example, probes have been developed wherein the intensity of the fluorescence increases due to the separation of the fluorophore-quencher pair. Probes have also been developed which lose their fluorescence because the quencher is brought into proximity with the fluorophore. These fluorophore-quencher pairs have been used to monitor hybridization assays and nucleic acid amplification reactions, especially polymerase chain reactions (PCR), by monitoring either the appearance or disappearance of the fluorescence signal generated by the fluorophore molecule.

The decreased fluorescence of a fluorophore moiety by collision or direct interaction with a quencher is due mainly to a transfer of energy from the fluorophore in the excited state to the quencher. The extent of quenching depends on the concentration of quencher and is described by the Stem-Volmer relationship:

F.sub.o /F=1+K.sub.SV [Q]

wherein F.sub.o and F correspond to the fluorescence in the absence and presence of quencher, respectively, and [Q] is the quencher concentration. A plot of F.sub.o /F versus [Q] yields a straight line with a slope corresponding to the Stem-Volmer constant, K.sub.SV. The foregoing equation takes into account the dynamic and collisional quenching which is the dominant component of the quenching reaction. However, deviations from linearity are observed when contributions by static quenching becomes significant, or when the quenching is not efficient (see, A. M. Garcia, Methods in Enzymology, 207, 501-511 (1992)).

In general, fluorophore moieties preferably have a high quantum yield and a large extinction coefficient so that the dye can be used to detect small quantities of the component being detected. Fluorophore moieties preferably have a large Stokes shift (i.e., the difference between the wavelength at which the dye has maximum absorbance and the wavelength at which the dye has maximum emission) so that the fluorescent emission is readily distinguished from the light source used to excite the dye.

One class of fluorescent dyes which has been developed is the energy transfer fluorescent dyes. For instance, U.S. Pat. Nos. 5,800,996, and 5,863,727, issued to Lee et al., disclose donor and acceptor energy fluorescent dyes and linkers useful for DNA sequencing. In energy transfer fluorescent dyes, the acceptor molecule is a fluorophore which is excited at the wavelength of light emitted by the excited donor molecule. When excited, the donor dye transmits its energy to the acceptor dye. Therefore, emission from the donor is not observed. The emission from the donor dye excites the acceptor dye, and causes the acceptor dye to emit at its characteristic wavelength (i.e., a wavelength different from that of the donor dye, therefore observed as a color different from that of the donor). The advantage of this mechanism is twofold; the emission from the acceptor dye is more intense than that from the donor dye alone (see, Li et al., Bioconjugate Chem., 10: 242-245, (1999)) and attachment of acceptor dyes with differing emission spectra allows differentiation among molecules by fluorescence using a single excitation wavelength.

Nucleotide triphosphates having a fluorophore moiety attached to the .gamma.-phosphate are of interest as this modification still allows the modified NTPs to be enzyme substrates. For instance, Felicia et al., describe the synthesis and spectral properties of a "always-on" fluorescent ATP analog, adenosine-5'-triphosphoro-.gamma.-1-(5-sulfonic acid)-naphthyl ethylamindate (.gamma.-1,5-EDANS)ATP. The analog is a good substrate for E. Coli RNA polymerase and can be used to initiate the RNA chain. The ATP analog is incorporated into the RNA synthesized and is a good probe for studies of nucleotide-protein interactions, active site mapping and other ATP-utilizing biological systems (see, Felicia et al., Arch. Biochem Biophys., 246: 564-571 (1986)).

In addition, Sato et al., disclose a homogeneous enzyme assay that uses a fluorophore moiety (bimane) attached to the .gamma.-phosphate group of the nucleotide and a quencher moiety attached to the 5-position of uracil. The quencher moiety is in the form of a halogen, bound to the C-5 position of the pyrimidine. The quenching that is effected by this combination is eliminated by cleavage of the phosphate bond by the phosphodiesterase enzyme. The halogen quencher used in the assay is very inefficient producing only about a two fold decrease in fluorescent efficiency.

A need currently exists for effective nucleotide triphosphate molecules containing a fluorophore and a quencher for use in pyrophosphate detection assays. Accordingly, a need exists for assays using probes which exhibit distinguishable fluorescence characteristics when a fluorophore is attached to the nucleotide through the .gamma.-phosphate and when it is unattached to the nucleotide. A further need exists for assays using probes wherein the fluorophore and a quencher are positioned on the probe such that the quencher moiety can effectively quench the fluorescence of the fluorophore moiety. These and further objectives are provided by the methods and probes of the present invention.

SUMMARY OF THE INVENTION

A need currently exists for effective nucleotide triphosphate molecules containing a fluorophore and a quencher for use in pyrophosphate detection assays. Pyrophosphate detection is useful for monitoring a number of enzymatic reaction mechanisms such as nucleic acid polymerase reactions. As such, in certain aspects, the present invention provides a heterogeneous assay method for detecting pyrophosphate cleavage, the components of the assay comprising a labeled NTP, a target nucleic acid, a primer nucleic acid and a polymerase, the method comprising:

(a) flowing the labeled nucleotide triphosphate (NTP) having a .gamma.-phosphate with a fluorophore moiety attached thereto and a quencher moiety sufficiently proximal to the fluorophore moiety to prevent fluorescence of the fluorophore moiety, past an immobilized component selected from the group consisting of the polymerase and the target nucleic acid;

(b) incorporating the labeled NTP on the primer strand hybridized to the target nucleic acid using the polymerase and releasing the .gamma.-phosphate with the fluorophore moiety attached thereto; and

(c) detecting the fluorescent moiety thereby detecting pyrophosphate cleavage.

Preferably, in the methods of the present invention, the enzyme is immobilized on a solid support and the nucleotide triphosphates comprise dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP, UTP and mixtures thereof. The detection of the fluorescent moieties is preferably accomplished using single molecule detection with for example, a charge couple device (CCD) camera.

In another aspect, the present invention provides a nucleotide triphosphate (NTP) probe, comprising: a NTP having a .gamma.-phosphate with a fluorophore moiety attached thereto; a quencher moiety sufficiently proximal to the fluorophore moiety to prevent fluorescence of the fluorophore moiety; wherein the fluorophore moiety exists quenched with at least about a 5 fold quenching efficiency when the .gamma.-phosphate is attached to the NTP and unquenched when the .gamma.-phosphate is detached from the NTP. In preferred aspects, the quencher moiety is attached to the nucleobase.

In yet another aspect, the present invention provides kits and integrated systems for practicing the assays described herein. In certain aspects, the present invention provides a kit for assaying pyrophosphate cleavage, comprising: (a) a plurality of NTPs each having a .gamma.-phosphate with a distinguishing fluorophore moiety attached thereto and each having a quencher moiety sufficiently proximal to the distinguishing fluorophore moiety to prevent fluorescence of the distinguishing fluorophore moiety; wherein the distinguishing fluorophore moiety exists quenched with at least about a 5 fold quenching efficiency when the .gamma.-phosphate is attached to each of the plurality of dNTP moieties and each is unquenched when the .gamma.-phosphate is detached from each of the plurality of dNTP moieties; and (b) a polymerase. Preferably, the polymerase is immobilized on a solid support.

These and other aspects and advantages will become more apparent when read with the accompanying figures and the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Panel A illustrates pyrophosphate cleavage with a polymerase; Panel B illustrates an embodiment of the present invention.

FIG. 2 Panel A illustrates an optical set of the present invention; Panel B illustrates a single molecule sequencing embodiment of the present invention; Panel C illustrates an embodiment of the present invention.

FIG. 3 illustrates DABCYL and dinitrophenyl derivatives of the present invention.

FIG. 4 illustrates compounds of the present invention.

FIG. 5 illustrates synthesis of a compound of the present invention.

FIG. 6 illustrates synthesis of a compound the present invention.

FIG. 7 illustrates synthesis methods for embodiments of the present invention. General Scheme for the conversion of NHS dyes to thiol reactive groups using lodo or bromo alkane derivatives.

FIG. 8 illustrates synthesis methods for embodiments of the present invention.

FIG. 9 illustrates synthesis of a compound of the present invention.

DEFINITIONS

The term "heterogeneous" assay as used herein refers to an assay method wherein at least one of the reactants in the assay mixture is attached to a solid phase, such as a solid support.

The term "oligonucleotide" as used herein includes linear oligomers of nucleotides or analogs thereof, including deoxyribonucleosides, ribonucleosides, and the like. Usually, oligonucleotides range in size from a few monomeric units, e.g. 3-4, to several hundreds of monomeric units. Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5'-3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted.

The term "nucleoside" as used herein refers to a compound consisting of a purine, deazapurine, or pyrimidine nucleoside base, e.g., adenine, guanine, cytosine, uracil, thymine, deazaadenine, deazaguanosine, and the like, linked to a pentose at the 1' position, including 2'-deoxy and 2'-hydroxyl forms, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).

The term "nucleotide" as used herein refers to a phosphate ester of a nucleoside, e.g., mono, di and triphosphate esters, wherein the most common site of esterification is the hydroxyl group attached to the C-5 position of the pentose. Nucleosides also include, but are not limited to, synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g. described generally by Scheit, Nucleotide Analogs (John Wiley, N.Y., 1980). Suitable NTPs include both naturally occurring and synthetic nucleotide triphosphates, and are not limited to, ATP, dATP, CTP, dCTP, GTP, dGTP, TTP, dTTP, UTP and dUTP. Preferably, the nucleotide triphosphates used in the methods of the present invention are selected from the group of dATP, dCTP, dGTP, dTTP, dUTP and mixtures thereof.

The term "primer" refers to a linear oligonucleotide which specifically anneals to a unique polynucleotide sequence and allows for amplification of that unique polynucleotide sequence.

The phrase "sequence determination" or "determining a nucleotide sequence" in reference to polynucleotides includes determination of partial as well as full sequence information of the polynucleotide. That is, the term includes sequence comparisons, fingerprinting, and like levels of information about a target polynucleotide, or oligonucleotide, as well as the express identification and ordering of nucleosides, usually each nucleoside, in a target polynucleotide. The term also includes the determination of the identification, ordering, and locations of one, two, or three of the four types of nucleotides within a target polynucleotide.

The term "solid-support" refers to a material in the solid-phase that interacts with reagents in the liquid phase by heterogeneous reactions. Solid-supports can be derivatized with proteins such as enzymes, peptides, oligonucleotides and polynucleotides by covalent or non-covalent bonding through one or more attachment sites, thereby "immobilizing" the protein or nucleic acid to the solid-support.

The phrase "target nucleic acid" or "target polynucleotide" refers to a nucleic acid or polynucleotide whose sequence identity or ordering or location of nucleosides is to be determined using methods described herein.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

I. Methods

A. Pyrophosphate Cleavage

In certain embodiments, the present invention provides a heterogeneous assay for the detection of pyrophosphate. The detection of pyrophosphate is advantageous in a number of biological reactions. For example, in a DNA polymerase reaction, wherein the polymerase selects a single DNA molecule from solution and thereafter incorporates the nucleotide at the 3'-end of a primer strand, the natural consequence of such incorporation is the release of pyrophosphate. If the assay solution comprises the four deoxynucleotide triphosphates, each dNTP labeled with a different color of fluorescent dye attached to the .gamma.-phosphate, it is then possible to sequentially record the activity of the polymerase operating on a target DNA. The nucleotide sequence of the target DNA can thereafter be read directly from the order of released dyes attached to the pyrophosphate.

As such, the present invention provides a heterogeneous assay method for detecting pyrophosphate cleavage, the components of the assay comprising a labeled NTP, a target nucleic acid, a primer nucleic acid and a polymerase, the method comprising: (a) flowing the labeled nucleotide triphosphate (NTP) having a .gamma.-phosphate with a fluorophore moiety attached thereto and a quencher moiety sufficiently proximal to the fluorophore moiety to prevent fluorescence of the fluorophore moiety, past an immobilized component selected from the group consisting of the polymerase and the target nucleic acid; (b) incorporating the NTP on a primer strand hybridized to the target nucleic acid using an enzyme and releasing the .gamma.-phosphate with the fluorophore moiety attached thereto; and (c) detecting the fluorescent moiety thereby detecting pyrophosphate cleavage. In the heterogeneous assay of the present invention, either the polymerase or the target nucleic acid is attached to a solid phase, such as a solid support. Preferably, in the methods of the present invention, the polymerase is immobilized on a solid support.

In certain aspects, the polymerase is a DNA polymerase such as DNA polymerase I, II or III. In other aspects, suitable polymerases include, but are not limited to, a DNA dependent RNA polymerase and reverse transcriptase such as an HIV reverse transcriptase. Specific examples include, but are not limited to, T7 DNA polymerase, T5 DNA polymerase, E. Coli DNA polymerase I, T4 DNA polymerase, T7 RNA polymerase and Taq DNA polymerase. Those of skill in the art will know of other enzymes or polymerases suitable for use in the present invention. In certain aspects, the polymerase is bathed in a flowing solution comprising: unlabeled, single-stranded DNA fragments hybridized to an oligonucleotide primer and a mixture of NTPs.

In certain aspects of the present invention, a labeled nucleotide triphosphate (NTP) having a .gamma.-phosphate with a fluorophore moiety attached thereto is incorporated into a polynucleotide chain. As illustrated in FIG. 1A, dNTP incorporation into a growing oligonucleotide by a DNA polymerase results in pyrophosphate cleavage. In this reaction, the phosphate ester bond between the .alpha. and .beta. phosphates of the incorporated nucleotide is cleaved by the DNA polymerase, and the .beta.-.gamma.-diphosphate (pyrophosphate) is released in solution. As used herein, the term pyrophosphate also includes substitution of any of the oxygen atoms of the pyrophosphate group with a nitrogen or a sulfur atom or combinations thereof to generate thiopyrophosphate, dithiopyrophosphate, etc.

As shown in FIG. 1B, in compounds of the present invention wherein a fluorophore is attached to the .gamma.-phosphate, the fluorophore is released from the nucleotide along with the pyrophosphate group. In certain aspects, cleavage of the pyrophosphate switches the fluorophore moiety into a fluorescent state i.e., the fluorophore is dequenched. This event can then be detected using an ultrasensitive fluorescence detector. Using single molecule detection for example, fluorescent signals appear at the locations of the individual molecules being observed. In certain aspects, each type of nucleotide is labeled with a different fluorophore so that the incorporated nucleobases can be sequentially identified by the released fluorophores. Preferably, the nucleotide triphosphate (NTP) of the present methods include, but are not limited to, deoxyadenosine triphosphate, deoxycytosine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate, deoxyuridine triphosphate or mixtures thereof, each with a unique fluorophore attached to the .gamma.-phosphate.

As is described in detail hereinbelow, the nucleotides of the present invention, both purine and pyrimidine varieties, are modified at various sites with a fluorophore moiety and a quencher moiety. In certain aspects, the combination of fluorophore and quencher are attached to the same position of the nucleotide separated by a linker. In others aspects, the moieties are at distinct points on the nucleotide. Once the quenched dNTPs are produced, they can be used to sequence DNA strands by direct single molecule detection. The fluorescence is detected when the labeled dNTPs are incorporated into the strand (the de-quench