QUANTITATIVE MOLECULAR PROBES

• US 20090104614A1
• Filed: 03/15/2007
• Published: 04/23/2009
• Est. Priority Date: 03/15/2006
• Status: Abandoned Application

• Current Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Chen, Antony, Tsourkas, Andrew
• Application Number:
  US12/282,612
• Publication Number:
  US 20090104614A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

In accordance with this invention, a molecular probe for detection of a nucleic acid target containing a preselected target sequence is constructed and has at least two sources of a signal: a conventional reporter source and a reference source in a form of a luminescent material, e.g., a fluorophore, quantum dot, fluorescent nanoparticle, or other fluorescent reference dye/nanoparticle/microparticle conjugated to the molecular probe.

Claims

1. A quantitative molecular probe for detection of a nucleic acid target containing a preselected target sequence, said quantitative molecular probe being capable of assuming a closed conformation and an open conformation, said quantitative molecular probe comprising:

a) a target complement sequence of from 7 to about 140 nucleotides complementary to said preselected nucleic acid target sequence having a 5′

terminus and a 3′

terminus;

b) optionally comprising an affinity pair comprising a first affinity moiety covalently linked to said 5′

terminus and a second affinity moiety covalently linked to said 3′

terminus, said affinity pair interacting sufficiently to hold said quantitative molecular probe in the closed conformation in the absence of said nucleic acid target;

c) a label pair comprising a first label moiety conjugated to at least one of a first nucleotide of said preselected nucleic acid target sequence or said first affinity moiety and a second label moiety conjugated to at least one of a second nucleotide of said preselected nucleic acid target sequence or said second affinity moiety, provided that said first nucleotide and said second nucleotide are separated by at least 2 nucleotides and wherein said label moieties interact to affect a measurable characteristic of at least one of said label moieties such that a difference in a reporter signal is produced when said quantitative molecular probe is in the closed conformation, wherein hybridization of said target complement sequence to said target sequence causes said quantitative molecular probe to assume its open conformation, in which said label moieties do not so interact; and

d) a reference label moiety conjugated to at least one of said target complement sequence, said first affinity moiety, said second affinity moiety, said first label moiety, or said second label moiety, wherein said reference label moiety produces a reference signal which is a detectable signal qualitatively distinct from the reporter signal produced by any one of said first label moiety, said second label moiety alone or in combination with each other in the closed conformation.

2. The quantitative molecular probe according to claim 1, wherein said affinity pair is required and wherein said quantitative molecular probe is a quantitative molecular beacon and wherein said first label moiety is conjugated to said first affinity moiety and said second label moiety is conjugated to said second affinity moiety.

3. The quantitative molecular probe according to claim 1, wherein said affinity pair is not present and wherein said quantitative molecular probe is a quantitative linear oligonucleotide and wherein said first label moiety is conjugated to said first nucleotide of said preselected nucleic acid and said second label moiety is conjugated to said second nucleotide of said preselected nucleic acid target sequence.

4. The quantitative molecular probe according to claim 1, wherein said reference label moiety is a fluorophore selected from at least one of a nanoparticle, a microparticle, a quantum dot, a fluorescently labeled dendrimer, a fluorescent moiety or a fluorescently labeled moiety.

5. The quantitative molecular probe according to claim 1, wherein said first label moiety is a reporter fluorophore and said second label moiety is a quencher selected to substantially quench fluorescence of the reporter fluorophore in a closed conformation and said reference label moiety is a reference fluorophore, wherein said reference label moiety produces a signal at a wavelength sufficiently distinct from a first label moiety wavelength.

6. The quantitative molecular probe according to claim 5, wherein said reference label moiety is at least one of Cy3.5, Cy5, Cy5.5, ALEXA 660, ALEXA 680.

7. The quantitative molecular probe according to claim 1, wherein said target complement sequence is from 15 to 30 nucleotides.

8. The quantitative molecular probe according to claim 2, wherein said affinity pair comprises complementary oligonucleotide arm sequences 3 to 25 nucleotides in length.

9. The quantitative molecular probe according to claim 2, wherein said first label moiety is covalently linked to said first affinity moiety and said second label moiety is covalently linked to said second affinity moiety.

10. The quantitative molecular probe according to claim 1, wherein said reference label moiety is conjugated via covalent bonding or affinity bonding.

11. The quantitative molecular probe according to claim 1, wherein said label pair comprises either a FRET pair or a non-FRET pair.

12. The quantitative molecular probe according to claim 2, wherein said affinity pair comprises an antibody and an antigen.

13. The quantitative molecular probe according to claim 1 tethered to a solid surface.

14. The quantitative molecular probe according to claim 1, wherein said quantitative molecular probe is a unimolecular quantitative molecular probe.

15. The quantitative molecular probe according to claim 1, wherein said quantitative molecular probe is a bimolecular quantitative molecular probe consisting of a first molecule containing approximately half of said target complement sequence including said 5′

terminus, the first affinity moiety and the first label moiety; and

a second molecule containing approximately half of said target complement sequence including said 3′

terminus, the second affinity moiety and the second label moiety.

16. The quantitative molecular probe according to claim 1, wherein said reference label moiety further comprises a targeting ligand.

17. The quantitative molecular probe according to claim 1, wherein said quantitative molecular probe is encapsulated in a polymersome vesicle comprising a shell comprising an amphiphilic polymer or in a liposome comprising a phospholipid shell.

18. A method for a quantitative detection of a nucleic acid target, the method comprising:

providing the quantitative molecular probe of claim 1 and a nucleic acid target;

contacting said quantitative molecular probe with said nucleic acid target;

hybridizing said quantitative molecular probe to said preselected nucleic acid target sequence; and

detecting a reporter signal from at least one of said first label moiety and second label moiety and a reference signal from said reference label moiety to obtain a fluorescence ratio,

analyzing said fluorescence ratio and said reference fluorescence signal and thereby quantitatively detecting said nucleic acid target.

19. The method of claim 18, wherein said quantitative molecular probe is encapsulated in a polymersome vesicle comprising a shell comprising an amphiphilic polymer.

20. The method of claim 18, wherein said quantitative molecular probe is tethered to a solid surface.

21. The method of claim 18, wherein said nucleic acid target is provided in at least one of a live cell, an isolated tissue, a mammal, or an embrio.

22. The method of claim 18, wherein said fluorescence ratio is from 0 to 1,000,000.

24. A method for a quantitative determination of an effect of a substance on a nucleic acid target, the method comprising:

providing the quantitative molecular probe of claim 1 and a nucleic acid target;

contacting said quantitative molecular probe with said nucleic acid target;

hybridizing said quantitative molecular probe to said preselected nucleic acid target sequence to form a complex;

optionally contacting said nucleic acid target with said substance;

contacting a complex with said substance; and

detecting a reporter signal from at least one of said first label moiety and second label moiety and a reference signal from said reference label moiety to obtain a fluorescence ratio; and

thereby quantitatively determining the effect of said substance on said nucleic acid target.

25. The method of claim 24, wherein said quantitative molecular probe is encapsulated in a polymersome vesicle comprising a shell comprising an amphiphilic polymer or in a liposome comprising a phospholipid shell.

26. The method of claim 24, wherein said quantitative molecular probe is tethered to a solid surface.

27. The method of claim 24, wherein said nucleic acid target is provided in at least one of a live cell, an isolated tissue, a mammal, or an embrio.

28. The method of claim 24, wherein said fluorescence ratio is from 0 to 1,000,000.

29. The method of claim 24, wherein said substance is a chemical substance or a physical force.

30. The method of claim 24, wherein said substance is a drug and said method is applied to cells to identify cells which display drug resistance.

31. A calibration kit for detection of a signal from a quantitative molecular probe, said calibration kit comprising:

a plurality of encapsulated quantitative molecular probes comprising a quantitative molecular probes according to claim 1, wherein said quantitative molecular probe is encapsulated in at least one of a polymersome vesicle comprising a shell comprising an amphiphilic polymer or a liposome vesicle, wherein said encapsulated quantitative molecular probes have a predetermined amount of said quantitative molecular probes hybridized to a preselected nucleic acid target sequence.

23. In a molecular probe for detection of a nucleic acid target containing a preselected target sequence having:

a) a target complement sequence of from 10 to about 140 nucleotides complementary to said preselected nucleic acid target sequence, having a 5′

terminus and a 3′

terminus;

b) an affinity pair comprising a first affinity moiety covalently linked to said 5′

terminus and a second affinity moiety covalently linked to said 3′

terminus, said affinity pair interacting sufficiently to hold said molecular probe in the closed conformation in the absence of said nucleic acid target; and

c) a label pair comprising a first label moiety conjugated to said first affinity moiety and a second label moiety conjugated to said second affinity moiety, wherein said label moieties interact to affect a measurable characteristic of at least one of said label moieties such that a difference in a reporter signal is produced when said molecular probe is in the closed conformation, wherein hybridization of said target complement sequence to said target sequence causes said molecular probe to assume its open conformation, in which said label moieties do not so interact;

wherein the improvement comprises;

d) having a reference label moiety conjugated to at least one of said target complement sequence, said first affinity moiety, said second affinity moiety, said first label moiety, or said second label moiety, wherein said reference label moiety produces a reference signal which is a detectable signal qualitatively distinct from the reporter signal produced by any one of said first label moiety, said second label moiety alone or in combination with each other in the closed conformation so that said molecular probe is adapted to quantitative detect a nucleic acid target.