Methods and compositions for enhancing detection in determinations employing cleavable electrophoretic tag reagents

• US 20050053939A1
• Filed: 11/08/2002
• Published: 03/10/2005
• Est. Priority Date: 11/09/2001
• Status: Abandoned Application

• Current Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Inventors: Singh, Sharat, Chenna, Ahmed
• Application Number:
  US10/494,879
• Publication Number:
  US 20050053939A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals

Abstract

Probe sets for the multiplexed detection of the binding of, or interaction between, one or more ligands and target antiligands are provided. Detection involves the release of identifying tags as a consequence of target recognition. The probe sets include electrophoretic tag probes or e-tag probes, comprising a detection region and a mobility-defining region, both linked to a target-binding moiety. In a multiplexed assay, different released e-tag reporters may be separated and detected providing for target identification. The probes comprise interactive functionalities adjacent the cleaved portion positioned in the probes such that the interactive functionality does not form part of the e-tag reporters. Also described are biopolymers and nucleosides containing such interactive functionalities.

Claims

1. A method for isolating a signal produced by a detectable moiety of a released predetermined portion of a probe from signal obtained from intact probe and from other fragments thereof, the method comprising:

(a) combining a probe which comprises a first predetermined portion which is releasable and includes a detectable moiety, and a second portion which includes at least one interactive functionality, with a reagent that potentiates the release of the first portion from the probe;

(b) subjecting the resulting mixture to conditions under which the probe is cleaved, such that the first portion is released from the probe;

(c) contacting the mixture with a capture reagent which binds the interactive functionality, and

(d) separating the capture agent or the released predetermined first portion from the mixture.

2. The method of claim 1, wherein the interactive functionality is a ligand or a chelating moiety.

3. The method of claim 2, wherein the interactive functionality is a chelating moiety, and the capture agent comprises boron, arsenic, a transition metal, or a ferrocene.

4. The method of claim 1, wherein the interactive functionality comprises boron, arsenic, a transitions metal, or a ferrocene, and the capture agent comprises a chelating moiety.

5. The method of claim 2, wherein the interactive functionality is a ligand selected from the group consisting of a small molecule, a receptor for a small molecule, an antigen, an antibody, and an oligonucleotide.

6. The method of claim 1, wherein the probe comprises an oligonucleotide to which the releasable first portion is attached at a nucleotide thereof, and an organic moiety comprising the interactive functionality is attached at a nucleotide adjacent to the nucleotide to which the releasable first portion is attached.

7. The method of claim 6, wherein the nucleotide to which the releasable first portion is attached is a terminal nucleotide.

8. The method of claim 1, wherein the releasable first portion has a predetermined mass or mobility.

9. The method of claim 1, wherein the probe comprises a structure (D, M_j)-L, where

(i) D is a detection group comprising a detectable label;

(ii) L is a cleavable linkage, and

(iii) M_j is a mobility modifier having a mass or charge/mass ratio that imparts a unique and known electrophoretic mobility to a corresponding reporter of the form (D,M_j)-L′

, within a selected range of mobilities, with respect to other reporters of the same form in a set of such probes, where L′

is the residue of L following cleavage.

10. A method for detecting binding of or interaction between a target binding agent and any of a plurality of probes, comprising:

(a) subjecting a mixture comprising the target binding agent and the probes to conditions for interaction therebetween, wherein each probe comprises (i) a cleavable reporter group, comprising a detectable moiety and having a mobility, when cleaved, that is unique to the probe among the plurality of probes, and (ii) at least one interactive functionality adjacent the cleavable reporter group;

(b) subjecting the mixture to conditions under which the cleavable moiety is cleaved to release the reporter groups, wherein said cleavage is dependent on said interaction between the target binding agent and any of the plurality of probes;

and wherein the interactive functionality enables the isolation of signal produced by the detectable moieties of the released reporter groups from signal obtained from intact probes and from other fragments thereof,

(c) separating the released reporter groups by their differences in mobility; and

(d) detecting the detectable moieties of released reporter groups, to determine binding of or interaction between the target binding agent and each probe.

11. The method of claim 10, wherein the interactive functionality is effective to quench signal produced by the detectable moiety in intact probe but not in released reporter group.

12. The method of claim 10, wherein said isolation is effected by

contacting the mixture of (b) with a capture reagent which binds the interactive functionality, and

separating the capture agent or the released reporter groups from the mixture.

13. The method of claim 12, wherein the interactive functionality is a ligand or a chelating moiety.

14. The method of claim 12, wherein the interactive functionality is a chelating moiety, and the capture agent comprises boron, arsenic, a transition metal, or a ferrocene.

15. The method of claim 12, wherein the interactive functionality comprises boron, arsenic, a transition metal, or a ferrocene, and the capture agent comprises a chelating moiety.

16. The method of claim 13, wherein the interactive functionality is a ligand selected from the group consisting of a small molecule, a receptor for a small molecule, an antigen, an antibody, and an oligonucleotide.

17. The method of claim 10, wherein each probe comprises an oligonucleotide to which the cleavable reporter group is attached at a nucleotide thereof, and an organic moiety comprising the interactive functionality is attached at a nucleotide adjacent to the nucleotide to which the reporter group is attached.

18. The method of claim 17, wherein the nucleotide to which the reporter group is attached is a terminal nucleotide.

19. A kit for use in detecting the presence and/or amount of each of a plurality of target molecules, comprising in packaged combination:

a plurality of probes, each capable of target-specific binding to a binding site on a selected target molecule, and each having (i) a unique cleavable reporter group, having a detectable reporter moiety and a unique electrophoretic mobility which allows the reporter group to be uniquely identified among all other reporter groups associated with the plurality of probes, and (ii) at least one interactive functionality adjacent the reporter group, wherein the interactive functionality is effective in isolating the signal produced by released reporter groups from the signal produced by intact probes and other fragments thereof.

20. The kit of claim 19, further comprising a capture reagent effective to bind to the interactive functionality.

21. The kit of claim 20, wherein the interactive functionality is a chelating moiety, and the capture reagent comprises boron, arsenic, a transition metal, or a ferrocene.

22. The kit of claim 19, wherein each probe comprises an oligonucleotide to which the cleavable reporter group is attached at a nucleotide thereof, and an organic moiety comprising an interactive functionality attached at a nucleotide adjacent to the nucleotide to which the cleavable reporter group is attached.

23. The kit of claim 22, wherein the nucleotide to which the cleavable reporter group is attached is a terminal nucleotide.

24. A synthetic biopolymer or nucleoside having conjugated thereto a moiety having the formula Ya:

25. The synthetic biopolymer or nucleoside of claim 24, wherein R⁷ and R⁸ together form a benzene or anthracene ring.

26. The synthetic biopolymer or nucleoside of claim 24, wherein said moiety has the formula Yb:

27. The synthetic biopolymer or nucleoside of claim 26, wherein R¹⁰ and R¹¹ and R¹² together form a naphthalene ring.

28. The synthetic biopolymer or nucleoside of claim 24, wherein said moiety has the formula Y′

a;

29. The synthetic biopolymer or nucleoside of claim 24, wherein said moiety has the formula Y′

b;

30. The synthetic biopolymer or nucleoside of claim 24, wherein said moiety comprises a 1,2-diol, 1,3-diol, 1,2-aminoalcohol, 1,3-aminoalcohol, 1,2-hydroxy acid, 1,3-hydroxy acid, 1,2-hydroxy acid amide, 1,3-hydroxy acid amide or dioxime.

31. The synthetic biopolymer or nucleoside of claim 24, wherein said moiety is selected from the group consisting of ortho-hydroxybenzenes (catechols), ortho-hydroxybenzohydroxamic acids, ortho-hydroxybenzoic acids, ortho-hydroxybenzamides, imidazolyl phenols, dihydroxyfumaric acids, hydroxy pyridine aldehydes and hydroxyanthraquinone dioximes.

32. The synthetic biopolymer or nucleoside of claim 24, wherein said moiety is selected from the group consisting of catechol, salicylamide, N-phenylsalicylamidine, 2-(4′

imidazolyl)phenol, 1,8-dihydroxynaphthalene, dihydroxyfumaric acid, salicylaldehyde, 3-hydroxypyridine-4-aldehyde, pyridoxamine, 1-hydroxyanthraquinone dioxime and disalicylimide.

33. The synthetic biopolymer or nucleoside of claim 24, wherein the synthetic biopolymer is a polynucleotide or polypeptide.

34. A compound of the formula:

35. An oligonucleotide or polynucleotide having conjugated thereto a compound according to claim 34.

36. The compound of claim 34, wherein, in Ya, R⁷ and R⁸ together form a benzene or anthracene ring.

37. The compound of claim 34, wherein, in Yb, R¹⁰ and R¹¹ and R¹² together form a naphthalene ring.

38. The compound of claim 34, wherein Ya is of the form Ya′

;

39. The compound of claim 34, wherein Yb is of the form Yb′

;

40. The compound of claim 34, wherein, in Ya, R⁵ and R⁶ are hydroxy.

41. The compound of claim 34, wherein, in Yb, R⁵ and R⁶ are hydroxy.

42. The compound of claim 34, wherein Ya is selected from the group consisting of ortho-hydroxybenzenes (catechols), ortho-hydroxybenzohydroxamic acids, ortho-hydroxybenzoic acids, ortho-hydroxybenzamides, imidazolyl phenols, dihydroxyfumaric acids, and hydroxy pyridine aldehydes.

43. The compound of claim 34, wherein Yb is a hydroxyanthraquinone dioxime.

44. The compound of claim 34, wherein Ya is selected from the group consisting of catechol, salicylamide, N-phenylsalicylamidine, 2-(4′

-imidazolyl) phenol, 1,8-dihydroxynaphthalene, dihydroxyfumaric acid, salicylaldehyde, 3-hydroxypyridine-4-aldehyde, pyridoxamine and disalicylimide.

45. The compound of claim 34, wherein Yb is 1-hydroxyanthraquinone dioxime.

46. A method of synthesizing an oligonucleotide of predetermined length, said method comprising reacting activated nucleoside monomer reagents sequentially until said oligonucleotide of predetermined length is formed, wherein at least one of said nucleoside monomer reagents is a compound of claim 34.

47. The method of claim 46, further comprising purifying said oligonucleotide by contacting a reaction mixture comprising said oligonucleotide with a boronate, to form a boronate complex therewith, and separating said complex from said mixture.

48. The method of claim 47, wherein said boronate is attached to a solid support.