Probe-antiprobe compositions and methods for DNA or RNA detection

• US 20090209434A1
• Filed: 08/15/2007
• Published: 08/20/2009
• Est. Priority Date: 08/15/2006
• Status: Active Grant

• Current Assignee: Genetag Technology, Inc.
• Original Assignee: Genetag Technology, Inc.
• Inventors: Shafer, David A.
• Granted Patent: US 8,076,067 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2537/162 Helper probe
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded

Abstract

The invention provides novel compositions and methods for detecting unlabeled nucleic acid targets using labeled polynucleotide probes and partially complementary antiprobes. The interaction of probes, antiprobes and targets result in signaling changes that indicate target frequency. This novel detection mechanism is called a DNA detection switch, and it enable end-point detection, microarray detection and real-time PCR detection of a variety of nucleic acid targets including microbial species and subspecies, drug resistant mutants, and pathogenic strains.

Claims

1. A probe-antiprobe composition to detect a target sequence, comprising:

(i) a polynucleotide probe having a sequence complementary to the target sequence and a first labeling component of a pair of interacting labeling components; and

(ii) a polynucleotide antiprobe comprising a second labeling component of the pair and a sequence that binds to a partial segment of the probe substantially within or outside of the target complementary sequence of the probe;

wherein the composition exhibits;

(a) a first signaling state when the pair of labeling components are in proximity due to the binding of probe and antiprobe; and

(b) a second signaling state when the labeling components are dissociated due to the binding of probe and target, such that when probe, antiprobe and target are hybridized together, a signaling potential of the composition switches from the first to the second signaling state relative to the presence and frequency of the target.

2. The probe-antiprobe composition of claim 1, wherein the labeling components comprise a FRET donor-acceptor pair that is a fluorescent donor compound paired with a fluorescent acceptor moiety, wherein the fluorescent acceptor moiety comprises either a longer wavelength fluorescent compound, a quencher compound, or a guanine-rich sequence segment having about 2 to about 8 guanines;

wherein the signaling state of the probe-antiprobe composition is modulated by the proximity of the donor and acceptor labeling components.

3. The probe-antiprobe composition of claim 1, wherein the labeling components are FAM, TET, HEX, JOE, VIC, ROX, NED, Texas Red, Yakima Yellow, BHQ1, BHQ2, BHQ3, Iowa Black FQ, Iowa Black RQ, TAMRA, DABCYL, ElleQuencher, Eclipse Dark Quencher, Methyl Red, DisperseBlue3, Bodipy 493/503, the Cy dyes Cy2, Cy3, Cy3.5, Cy5, Cy5.5, and Cy7, the AlexaFluor dyes 488, 532, 546, 555, 568, 594, 610, 647, and 680, the PromoFluor dyes 488, 555, 590, 633, 647, and 680, a fluorescent compound, a quencher compound, a color dye compound, a quantum dot, a silver or nanogold compound, or a guanine-rich sequence segment.

4. The probe-antiprobe composition of claim 1, wherein the probe comprises a target-specific sequence of about 20 to about 40 bases and the antiprobe comprises a shorter probe-complementary sequence of about 7 to about 20 bases, said composition suitable for end-point detection, microarray detection, or real-time PCR detection.

5. The probe-antiprobe composition of claim 1, wherein the polynucleotide probe is a two-segment probe comprising sequentially:

(i) the first labeling component, (ii) the first sequence segment complementary to the target sequence, and (iii) a generic second sequence segment that does not match or complement any target sequence in the sample; and

wherein the antiprobe is a generic antiprobe comprising;

(i) the second labeling component and (ii) a sequence complementary to the generic second sequence segment of the probe.

6. The probe-antiprobe composition of claim 5, wherein the labeling components comprise a FRET donor-acceptor pair;

wherein the labeling configuration of the probe and antiprobe is a 5′

donor-labeled probe and a 3′

or 5′

acceptor-labeled antiprobe, a 3′

donor-labeled probe and a 3′

or 5′

acceptor-labeled antiprobe, a 3′

acceptor-labeled probe and a 5′

or 3′

donor-labeled antiprobe, or a 5′

acceptor-labeled probe and a 5′

or 3′

donor-labeled antiprobe.

7. The probe-antiprobe composition of claim 1, wherein the probe and antiprobe components comprise a single polynucleotide labeled with a FRET donor-acceptor pair, comprising a donor-label at one end and an acceptor-label at the other end;

wherein the antiprobe sequence at one end is partially complementary to the probe sequence at the other end;

wherein the length and sequence of the antiprobe and probe segments are selected so that the Tm between the probe segment and target is about two or more degrees higher than the Tm between the antiprobe segment and the probe segment;

wherein the binding of the shorter antiprobe segment to a portion of the probe sequence comprises a first signaling state and the binding of the longer probe segment to the longer target sequence comprises a second signaling state;

wherein the signaling potential of the composition switches from the first to the second signaling state relative to the presence and frequency of the targets;

the composition providing highly specific detection of a matching target sequence and avoiding the detection of a mismatched target sequence, including a single base mismatch.

8. The probe-antiprobe composition of claim 1, wherein the length and position of the antiprobe sequence is selected to be partially complementary to the probe sequence so that the Tm between probe and target is about two or more degrees higher than the Tm between antiprobe and probe;

wherein the labeling components comprise a FRET donor-acceptor pair;

wherein the labeling configuration of the probe and antiprobe is selected from the group consisting of;

a 5′

donor-labeled probe and a 3′

acceptor-labeled antiprobe;

a 3′

donor-labeled probe and a 5′

acceptor-labeled antiprobe;

a 3′

acceptor-labeled probe and a 5′

donor-labeled antiprobe;

a 5′

acceptor-labeled probe and a 3′

donor-labeled antiprobe;

a 5′ and

3′

donor-labeled probe and a 5′ and

3′

acceptor-labeled antiprobe;

a 5′ and

3′

acceptor-labeled probe and a 5′ and

3′

donor-labeled antiprobe;

a probe with 5′

donor-label and 3′

acceptor-label and an antiprobe with 3′

acceptor-label and 5′

donor-label;

a probe with 5′

acceptor-label and 3′

donor-label and an antiprobe with 3′

donor-label and 5′

acceptor-label;

a probe with a central donor-label and an antiprobe with a central acceptor-label; and

a probe with a central acceptor-label and an antiprobe with central donor-label.

9. The probe-antiprobe composition of claim 1, wherein the probe is a primer/probe further comprising a primer sequence, wherein the signaling potential of the composition switches from a first signaling state to a second signaling state when the primer/probe is incorporated into the amplified target, said composition suitable for target amplification and detection.

10. The probe-antiprobe composition of claim 9, wherein the primer/probe comprises a sequence of about 20 to about 40 bases and the antiprobe comprises a sequence of about 16 to about 22 bases;

said composition suitable for real-time PCR detection.

11. The probe-antiprobe composition of claim 10, wherein the primer/probe and antiprobe further comprise artificial sequences inserted between the labeling component and the target-specific sequence of the probe and between the labeling component and the target-specific sequence of the antiprobe;

wherein the Tm between antiprobe and probe is increased to improve probe-antiprobe binding and/or to improve specificity.

12. The probe-antiprobe composition of claim 9, wherein the composition does not comprise an antiprobe and the primer/probe comprises a self-quenching primer/probe having sequentially:

(i) the labeling component comprising a fluorescent compound;

(ii) an inserted artificial cytidine-rich sequence comprising about 2 to about 8 cytidines; and

(iii) the 3′

primer sequence;

wherein the signaling potential of the primer/probe switches from a high signaling state to a low signaling state when it is incorporated into amplified targets.

13. The probe-antiprobe composition of claim 12, wherein a natural self-quenching primer/probe is created by selecting a primer sequence that includes one or more cytidines and no guanines near the label end of the probe.

14. The probe-antiprobe composition of claim 9, wherein the primer/probe comprises two sequence segments, a generic or universal sequence not complementary to the target sequence and a 3′

target-specific primer sequence, wherein the antiprobe comprises a sequence complementary to the generic or universal sequence of the primer/probe.

15. The probe-antiprobe composition of claim 1, wherein the probe comprises a polynucleotide sequence complementary to the target region and one or more labeling components;

wherein the antiprobe comprises an unlabeled polynucleotide that is complementary to a partial sequence of the probe;

wherein the probe and antiprobe sequences are selected to locate the variant base or bases of interest between the ends of the antiprobe sequence and preferably central to the antiprobe sequence; and

wherein the length and position of the probe and antiprobe sequences relative to the target sequence are selected to exhibit three Tm levels that differ by about two or more degrees from each other when these components are hybridized together;

said Tm levels comprising a high Tm level between the probe and the correct matching target;

an intermediate Tm level between the probe and the antiprobe; and

a low Tm level between the probe and an incorrect mismatched target;

wherein during hybridization the binding of the probe and a fully matching target is favored first, the binding of the probe and the antiprobe is favored second, and the binding of the probe and a mismatched target is effectively avoided or prevented even at low hybridization temperatures;

this composition selectively detecting target sequences complementary to the probe and selectively avoiding the detection of target sequences not complementary to the probe, said composition effective to discriminate small sequence differences within the target sequence such as SNPs or mutant bases.

16. The probe-antiprobe composition of claim 15, wherein the labeling components of the probe comprise a FRET donor-acceptor pair, wherein the probe is labeled at both ends;

wherein the composition inhibits mismatch detection and enables more precise detection of targets in situ and more precise detection of an internal target site with real-time pcr.

17. The probe-antiprobe composition of claim 15, wherein the probe comprises a TaqMan probe or a Molecular Beacon probe, said composition effective to inhibit mismatch detection and to enable more precise real-time PCR amplification and detection with said probes.

18. The probe-antiprobe composition of claim 15, wherein the antiprobe further comprises one or more labeling components, said probe and antiprobe labeling components comprise a FRET donor-acceptor pair;

wherein the labeling configuration is a 5′

donor-labeled probe and a 3′

acceptor-labeled antiprobe;

a 3′

donor-labeled probe and a 5′

acceptor-labeled antiprobe;

a 3′

acceptor-labeled probe and a 5′

donor-labeled antiprobe;

a 5′

acceptor-labeled probe and a 3′

donor-labeled antiprobe;

a 5′ and

3′

donor-labeled probe and a 5′ and

3′

acceptor-labeled antiprobe;

a 5′ and

3′

acceptor-labeled probe and a 5′ and

3′

donor-labeled antiprobe;

a probe with 5′

donor-label and 3′

acceptor-label and an antiprobe with 3′

acceptor-label and 5′

donor-label;

a probe with 5′

acceptor-label and 3′

donor-label and an antiprobe with 3′

donor-label and 5′

acceptor-label;

a probe with a central donor-label and an antiprobe with a central acceptor-label;

or a probe with a central acceptor-label and an antiprobe with central donor-label.

19. The probe-antiprobe composition of claim 18, wherein the probe is an internal probe comprising a sequence that is complementary to a target that is positioned between two primer binding sites, and wherein any unlabeled 3′

end of the probe or antiprobe is chemically modified to block polymerase extension;

wherein said 3′

modification is preferably selected from the group consisting of an amino modifier, a spacer or a phosphate, said composition effective for real-time PCR amplification and detection.

20. The probe-antiprobe composition of claim 15, wherein the paired probe and the antiprobe components substantially comprise sequences shown in SEQ ID NOS:

17 to 24 or the complements thereof, said composition effective to precisely detect, to precisely discriminate, or a combination thereof, M. tuberculosis and M. avium (paratuberculosis) species or samples.

21. The probe-antiprobe composition of claim 15, wherein the paired probe and antiprobe components substantially comprise sequences shown in SEQ ID NOS:

25 to 28 or the complements thereof, said composition effective to precisely detect, to precisely discriminate, or a combination thereof, HIV-1 at RT site K103N, wherein 103N comprises a common drug resistant mutant.

22. A universal probe-antiprobe composition, comprising:

(i) a universal linker/primer consisting of a 5′

linker sequence which does not match or complement a sequence in the target sample and a 3′

primer sequence;

(ii) a universal probe comprising a universal primer sequence that substantially corresponds to the linker sequence of the universal linker/primer; and

(iii) a universal antiprobe comprising a sequence complementary to a partial segment of the universal probe;

wherein the universal probe and antiprobe are labeled with the FRET donor-acceptor pair of claim 2;

wherein the labeling configuration preferably comprises a 5′

donor-labeled probe and a 3′

acceptor-labeled antiprobe or a 3′

acceptor-labeled probe and a 5′

donor-labeled antiprobe;

the composition suitable for real-time amplification and detection of a specific target sequence.

23. The universal probe-antiprobe composition of claim 22, comprising a universal primer sequence that does not match a known sequence in natural organisms as determined by a BLASTn analysis of the international GenBank database.

24. The universal probe-antiprobe composition of claim 22, wherein the composition does not comprise an antiprobe and the probe comprises a self-quenching universal probe having sequentially:

(i) a 5′

fluorescent labeling compound;

(ii) an inserted artificial cytidine-rich sequence comprising about 2 to about 8 cytidines; and

(iii) a 3′

universal primer sequence;

wherein the signaling potential of the universal probe switches from a high signaling state to a low signaling state when it is incorporated into amplified targets.

25. The universal probe-antiprobe composition of claim 22, wherein the composition comprises a common universal probe and antiprobe and two or more universal linker/primers;

wherein the universal linker/primers comprise different primer sequences specific to different target sites and a common universal linker;

said common universal probe effective to produce a combined detection signal such that false negative tests can be avoided in a multiplex amplification of different targets with the common universal probe, said composition effective to detect multiple target sites in a sample by real-time PCR and to prevent false negative tests due to small sequence variations that may occur in a target site.

26. The universal probe-antiprobe composition of claim 22, wherein the composition comprises two or more universal probes and antiprobes and two or more universal linker/primers;

wherein the universal linker/primers comprise different primer sequences specific to different target sites and different universal linkers specific to the different universal probes and antiprobes;

wherein each universal probe and antiprobe pair comprise different labeling with different colors effective to produce a pattern of two or more signals that confirm detection such that false positives can be avoided in a multiplex amplification of different targets with different universal probes.

27. The universal probe-antiprobe composition of claim 22, wherein false negative or false positive tests are avoided by (i) employing together a common self-quenching universal probe with two or more universal linker/primers that prime, amplify and label different targets with the same combined signal;

or by (ii) employing together two or more self-quenching universal probes and universal linker/primers that prime, amplify and label different targets with different signals.

28. The universal probe-antiprobe composition of claim 22, wherein the universal primer/probe substantially comprises a sequence that is one of SEQ ID NOS:

1 to 16, wherein said sequence is not significantly similar to any known genomic DNA sequence or expressed mRNA sequence in the GenBank international database as previously determined by BLASTn analysis, and the linker of the universal linker/primer comprises a substantial segment of the universal primer sequence, and comprises a segment of about 20 bases.

29. A method for detecting a polynucleotide target sequence in a sample, comprising:

(a) providing the polynucleotide probe and antiprobe composition of claim 1 at a ratio of about 1;

1 to about 1;

2;

(b) hybridizing the target and the probe-antiprobe composition together; and

(c) detecting changes in signaling to determine the presence and frequency of the target.

30. The method of claim 29, wherein different target sequences are detected in an array format, the method comprising:

(a) providing an array of target-specific probes that are labeled with a donor or acceptor moiety and that are attached to the substrate at known target-specific locations; and

providing known quantities of matching antiprobes that are labeled with a paired acceptor or donor moiety;

(b) hybridizing the antiprobes and the unlabeled targets of interest to the fixed probes in the array;

(c) detecting signaling changes per probe position in the array to indicate relative frequencies of the targets in the sample.

31. The method of claim 30, the method comprising:

(a) providing an array of two segment probes fixed to a substrate and a quantity of generic antiprobes;

wherein the probes and antiprobes are labeled with a donor-acceptor pair;

or

(b) providing an array of one segment probes fixed to a substrate and a quantity of target-specific antiprobes;

wherein the probes and antiprobes are labeled with a donor-acceptor pair.

32. A method for improving quantification with real-time PCR assays, wherein the self-quenching primer/probe of claim 12 is used at a concentration that exhausts the available primer/probes within the thermal cycling periods provided, the method comprising:

(a) providing the self-quenching probes at a highly restricted concentration;

preferably in the range of about 10% or less of the normal primer concentration;

(b) calculating the target copies that can be made with the restricted quantity of primer/probes;

(c) amplifying the targets with the starved primer/probe and a second primer; and

(d) monitoring and examining the signaling profile to determine the stages and quantity of target amplification that occurred, wherein the presence of a unique descending signaling curve, herein described as a Roller-Coaster curve, provides a quantitative indicator of the cycling time point when a defined quantity of target product was made, wherein the signature profile of this Roller-Coaster signaling curve comprises three or four phases;

(i) a level high, background signal, (ii) a rapidly descending signal, (iii) a low point or nadir in signaling, and (iv) optionally, a gradually re-ascending signal, wherein the nadir in the signaling curve marks the cycling time point when the number of primer/probes provided is exhausted, thereby providing a quantitative indicator of the amplified targets generated.

33. The method of claim 32, wherein the self-quenching primer/probe is provided in the concentration range of about 10 nM to about 50 nM, preferably at 20 nM, such that in a 25 μ

l reaction volume the resulting nadir in the signaling curve is indicative of the amplification time point when approximately 3×

10¹¹ copies of the product are present.

34. The method of claim 32, wherein the self-quenching primer/probe is a self-quenching universal primer/probe comprising a self-quenching universal probe having sequentially:

(i) a 5′

fluorescent labeling compound;

(ii) an inserted artificial cytidine-rich sequence comprising about 2 to about 8 cytidines; and

(iii) a 3′

universal primer sequence;

wherein the signaling potential of the universal probe switches from a high signaling state to a low signaling state when it is incorporated into amplified targets.

35. A method for providing double signaling or two-color signaling of the target product with real-time PCR or of other primer-based amplification methods, comprising:

(a) providing a pair of forward and reverse primer/probes according to the primer/probe composition of claim 9, wherein both the forward and the reverse primer/probes of each pair are labeled with the same color or a different color;

wherein universal probes of different color also comprise different universal sequences; and

(b) detecting the signaling;

wherein double signaling provides earlier and more sensitive detection; and

wherein two color signaling provides confirmation of correct target amplification and avoids false positive tests.