Nonenzymatic catalytic signal amplification for nucleic acid hybridization assays

• US 7,005,265 B1
• Filed: 06/20/2002
• Issued: 02/28/2006
• Est. Priority Date: 06/20/2002
• Status: Active Grant

• Current Assignee: Alan M. Cassell, Jie Han, Wenhong Fan
• Original Assignee: Alan M. Cassell, Jie Han, Wenhong Fan
• Inventors: Cassell, Alan M., Han, Jie, Fan, Wenhong
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US10/178,683
• Time in Patent Office: 1,349 Days
• Field of Search: 435/6, 435/287.2, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00536 in the shape of disks
• CPC Class Codes: B01J 2219/00545 Colours
• CPC Class Codes: B01J 2219/00574 radioactive
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2531/119 : Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2565/513 : characterised by the pattern of the arrayed oligonucleotides

Abstract

Devices, methods, and kits for amplifying the signal from hybridization reactions between nucleic acid probes and their cognate targets are presented. The devices provide partially-duplexed, immobilized probe complexes, spatially separate from and separately addressable from immobilized docking strands. Cognate target acts catalytically to transfer probe from the site of probe complex immobilization to the site of immobilized docking strand, generating a detectable signal. The methods and kits of the present invention may be used to identify the presence of cognate target in a fluid sample.

Claims

1. A device for nonenzymatically amplifying the signal from a hybridization reaction between at least a first nucleic acid probe and its cognate nucleic acid target present in fluid solvent, the device comprising:

at least one substrate;

a first plurality of probe complexes, each having a probe nucleic acid strand and a displacement nucleic acid strand hybridized thereto, the plurality of probe strands being identical and the plurality of displacement strands being identical; and

a first plurality of identical docking nucleic acid strands,

wherein the displacement strands and the docking strands are immobilized to one of said at least one substrates, said immobilized displacement strands and said immobilized docking strands being spatially segregated from one another, separately addressable, and fluidly communicating upon contact of said device with fluid solvent,

wherein the probe strand of said probe complexes has at least three regions,

a first region that is substantially complementary to a region of each of the displacement strand, the target, and the docking strand;

a second region that is substantially complementary to a region of each of the target and the docking strand, but not substantially complementary to a region of the displacement strand; and

a third region that is substantially complementary to a region of the docking strand, but not substantially complementary to a region of either of the displacement strand or the target,

whereby contact of target to said device is capable of catalyzing transfer of probe strands from the displacement strands to the spatially segregated, separately addressable docking strands, the transfer of said probe generating a detectable signal.

2. The device of claim 1, wherein said at least one substrate is a unitary substrate.

3. The device of claim 2, wherein said unitary substrate has a substantially planar surface to which said displacement strands and docking strands are segregably attached.

4. The device of claim 2, wherein said unitary substrate is at least one internal wall of a capillary, channel, or chamber formed in said device.

5. The device of either of claim 3 or 4, wherein said substrate comprises glass, silicon, or plastic.

6. The device of claim 1, wherein said at least one substrate is an array of electrodes.

7. The device of claim 1, wherein said probe strands further comprise a signaling moiety.

8. The device of claim 7, wherein said signaling moiety is optically detectable.

9. The device of claim 8, wherein said signaling moiety is a fluorophore.

10. The device of claim 7, wherein said signaling moiety is electrochemically detectable.

11. The device of claim 10, wherein said signaling moiety is a polyguanidine polymer.

12. The device of claim 10, wherein said signaling moiety includes a ferrocenyl group.

13. The device of claim 7, wherein said signaling moiety is radioactive.

14. The device of claim 1, wherein either or both of the second and third regions of said probe strands is independently 2–

14 nucleotides in length.

15. The device of claim 14, wherein either or both of the second and third regions of said probe strands is independently 2–

10 nucleotides in length.

16. The device of claim 15, wherein either or both of the second and third regions of said probe strands is independently 2–

6 nucleotides in length.

17. The device of claim 1, wherein any or all of said probe strands, said displacement strands, and said docking strands are peptide nucleic acids.

18. The device of claim 1, wherein any or all of said probe strands, said displacement strands, and said docking strands include locked nucleic acid analogues.

19. The device of claim 1, further comprising a second plurality of probe complexes and a second plurality of docking strands,

wherein the displacement strands, probe strands, and docking strands of said second pluralities are respectively identical thereamong but differ from those of said first plurality, and said probe strand first and second regions are substantially complementary to a second nucleic acid target.

20. The device of claim 19, wherein said second plurality of probe strands are distinguishably labeled from said first plurality of probe strands.

21. The device of claim 20, wherein said first and second plurality of probe strands are respectively labeled with a first and second fluorophore, said first and second fluorophore having optically distinguishable excitation or emission spectra.

22. A method of detecting a hybridization reaction between a nucleic acid probe and its cognate nucleic acid target with a catalytically amplified signal, the method comprising:

contacting the target in a fluid solvent to a device according to claim 1,

wherein the first and second regions of the probe strands of said device are substantially complementary to said target and said target catalyzes transfer of the probe strand from the immobilized displacement strand to the spatially segregated, spatially addressable, immobilized docking strand, the transfer of said probe generating a detectable signal; and

then

detecting the detectable signal.

23. The method of claim 22, wherein said signal is detected as a diminution of signal at the displacement strand address.

24. The method of claim 22, wherein said signal is detected as an increase of signal at the docking strand address.

25. The method of claim 22, wherein said signal is detected both as a diminution of signal at the displacement strand address and increase of signal at the docking strand address.

26. The method of claim 22, wherein the signal is optically detectable.

27. The method of claim 26, wherein the signal is fluorescent.

28. The method of claim 22, wherein the signal is electrochemically detectable.

29. The method of claim 22, wherein the signal is mass spectrally detectable.

30. The method of claim 22, further comprising the step, after contacting and before detecting, of:

separating the immobilized docking strands from said fluid solvent.

31. A kit for nonenzymatically amplifying the signal from a hybridization reaction between at least a first nucleic acid probe and its cognate nucleic acid target present in fluid solvent, comprising:

a device, the device comprising;

at least one substrate;

a first plurality of identical displacement nucleic acid strands; and

a first plurality of identical docking nucleic acid strands,

wherein the displacement strands and the docking strands are immobilized to one of said at least one substrates, said immobilized displacement strands and said immobilized docking strands being spatially segregated from one another, separately addressable, and fluidly communicating upon contact of said device with fluid solvent; and

a composition comprising probe nucleic acid strands, the probe nucleic strands having at least three regions,

a first region that is substantially complementary to a region of each of the displacement strand, the target, and the docking strand;

a second region that is substantially complementary to a region of each of the target and the docking strand, but not substantially complementary to a region of the displacement strand; and

a third region that is substantially complementary to a region of the docking strand, but not substantially complementary to a region of either of the displacement strand or the target.

32. The kit of claim 31, wherein said probe strands are detectably labeled.

33. The kit of claim 31, further comprising reagents for detectably labeling the probe strands of said composition.

34. The kit of claim 31, further comprising a control composition, said composition comprising target nucleic acids capable of catalyzing transfer of said probes from said displacement strands to said docking strands.