Process for detecting the presence or quantity of enzymatic activity in a sample
AssigneeEnzo Life Sciences, Inc.
InventorsJannis G. Stavrianopoulos, Elazar Rabbani
Life of this patent
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Abstract
This invention provides for labeling reagents, labeled targets and processes for preparing labeling reagents. The labeling reagents can take the form of cyanine dyes, xanthene dyes, porphyrin dyes, coumarin dyes or composite dyes. These labeling reagents are useful for labeling probes or targets, including nucleic acids and proteins. These reagents can be usefully applied to protein and nucleic acid probe based assays. They are also applicable to real-time detection processes.
Claims286
A labeling reagent for labeling a target, said labeling reagent comprising a marker moiety M and a reactive group R
- M—R
- wherein said marker moiety M and said reactive group R are covalently linked together, said M comprising at least one moiety that comprises a ligand, a dye, or both a ligand and a dye;
- and said reactive group R being capable of forming a carbon-carbon linkage with said target.
A process for labeling a target, said process comprising the steps of:
- (a) providing:
- (i) said target;
- (ii) a labeling reagent comprising a marker moiety M and a reactive group R
- M—R
- wherein said marker moiety M and said reactive group R are covalently linked together, said M comprising at least one moiety that comprises a ligand, a dye, or both a ligand and a dye;
- and said reactive group R being capable of forming a carbon-carbon linkage with said target;
- and
- (b) reacting said target (i) and said labeling reagent (ii) under conditions such that a carbon-carbon linkage forms between said target (i) and said labeling reagent (ii), thereby labeling said target (i) with said marker moiety M.
A labeled target, said target having been labeled by a process comprising the steps of:
- (a) providing:
- (i) said target;
- (ii) a labeling reagent comprising a marker moiety M and a reactive group R
- M—R
- wherein said marker moiety M and said reactive group R are covalently linked together, said M comprising at least one moiety that comprises a ligand, a dye, or both a ligand and a dye;
- and said reactive group R being capable of forming a carbon-carbon linkage with said target;
- (b) reacting said target (i) and said labeling reagent (ii) under conditions such that a carbon-carbon linkage forms between said target (i) and said labeling reagent (ii), thereby labeling said target (i) with said marker moiety M.
A process for preparing a cyanine dye labeling reagent, said process comprising the steps of:
- (a) providing:
- (i) a first intermediate compound comprising:
- 25
- wherein X1 comprises carbon, oxygen, nitrogen or sulfur;
- and
- (ii) a second intermediate compound comprising:
- 26
- wherein X1 comprises carbon, oxygen, nitrogen or sulfur;
- wherein at least one of R1 through R10 comprises a reactive group capable of forming a carbon-carbon linkage with a target, and
- (ii) linking reagents suitable for linking said first intermediate compound and said second intermediate compound;
- (b) forming a reaction mixture comprising said first intermediate compound (i), said second intermediate compound (ii), and said linking reagents under conditions to link (i) and (ii) to form
- 27
- wherein at least one of R1 through R10 comprises a reactive group capable of forming a carbon-carbon linkage with a target, and wherein n is an integer of 1, 2 or 3, and
- wherein X1 and X2 independently comprise carbon, oxygen, nitrogen or sulfur.
A labeling reagent comprising an aphenylic analog of a rhodamine dye, said analog comprising at least one reactive group for attaching said labeling reagent to a target, said at least one reactive group being attached directly to said analog or indirectly through a linker arm.
A labeled nucleotide comprising an aphenylic analog of a rhodamine dye, wherein said dye is attached directly to said nucleotide or indirectly through a linker.
A labeled target comprising
- T—L—M
- wherein T is a target, M is a marker moiety and L is a chemical group covalently linking said M to T, said chemical group L comprising a backbone that comprises at least one rigid group that comprises one or more of:
- 32
- (d) multimers of (a), (b) or (c), and
- (e) any combinations of (a), (b), (c) and (d).
A labeling reagent comprising
- R—L—M
- wherein R is a reactive group, M is a marker moiety and L is a chemical group covalently linking said M to R said chemical group L comprising a backbone that comprises at least one rigid group that comprises one or more of:
- 33
- (d) multimers of (a), (b) or (c), and
- (e) any combinations of (a), (b), (c) and (d).
A labeled target comprising
- T—L—M
- wherein T is a target, M is a marker moiety and L is a chemical group covalently linking said M to T, said chemical group L comprising a backbone that comprises at least two consecutive polar rigid units.
A labeling reagent comprising
- R—L—M
- wherein R is a reactive group, M is a marker moiety and L is a chemical group covalently linking said M to R, said chemical group L comprising a backbone that comprises at least two consecutive polar rigid units.
A labeled target comprising
- T—L—M
- wherein T is a target, M is a marker moiety and L is a chemical group covalently linking said M to T, said chemical group L comprising a backbone that comprises at least two consecutive peptide bonds.
The labeled target of claim 242, wherein said target T comprises a protein, a peptide, a nucleic acid, a nucleotide or a nucleotide analog, a receptor, a natural or synthetic drug, a synthetic oligomer, a synthetic polymer, a hormone, a lymphokine, a cytokine, a toxin, a ligand, an antigen, a hapten, an antibody, a carbohydrate, a sugar or an oligo- or polysaccharide.
A labeling reagent comprising
- R—L—M
- wherein R is a reactive group, M is a marker moiety and L is a chemical group covalently linking said M to R, said chemical group L comprising a backbone that comprises at least two consecutive peptide bonds.
A labeling reagent comprising a nonmetallic porphyrin, said reagent comprising:
- 34
- wherein R0 is a reactive group and is attached directly or indirectly to said nonmetallic porphyrin, and R1 through R8 independently comprise hydrogen, aliphatic, unsaturated aliphatic, cyclic, heterocyclic, aromatic, heteroaromatic, charged or polar groups, or any combinations of the foregoing.
A labeled target comprising a nonmetallic porphyrin, said reagent comprising:
- 35
- wherein T is a target molecule attached directly or indirectly to said nonmetallic porphyrin and R1 through R8 independently comprise hydrogen, aliphatic, unsaturated aliphatic, cyclic, heterocyclic, aromatic, heteroaromatic, charged or polar groups, or any combinations of the foregoing.
A process for determining the amount of a nucleic acid in a sample of interest, said process comprising the steps of:
- (a) providing:
- (i) said sample of interest;
- (ii) a dye comprising a first phenanthridinium moiety linked to a second phenanthridinium moiety through a phenyl group in each of said first and second phenanthridinium moieties;
- (iii) reagents for carrying out dye binding, hybridization, strand extension, or any combination thereof;
- (b) forming a mixture of (i), (ii) and (iii) above, to produce a complex comprising said dye (ii) and any nucleic acid that may be present in said sample of interest (i);
- (c) illuminating said mixture formed in step (b) at a wavelength below 400 nanometers (nm);
- and
- (d) measuring fluorescent emission from said illuminated mixture in step (c), said emission being proportional to the quantity of any nucleic acid present in said sample of interest (i).
The process of 183, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, well or depressions.
A composition comprising at least one of the following dye structures:
- 37
The process of 198, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, well or depressions.
A heterodimeric dye composition, said composition comprising a first dye that comprises a phenanthridinium moiety;
- and a second dye that is different from said first dye, said second dye being attached through the phenyl ring of said phenanthridium moiety.
The process of 212, wherein said solid support comprises beads, tubes, microtiter plates, glass slides, plastic slides, microchip arrays, well or depressions.
A chemiluminescent reagent having the structure:
- 39
- wherein Q comprises a cycloalkyl or polycycloalkyl group attached covalently to the 4-membered ring portion of said dioxetane above directly or indirectly through a linkage group;
- wherein Z comprises hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl or cycloheteroalkyl;
- and wherein R1 and R2 comprise chemical moieties attached to different sites of a cyclic ring attached to said dioxetane, and wherein R1 is enzymatically converted into R1* which comprises a chemical reactive group G1, and wherein R2 is attached to said cyclic ring through an oxygen atom and comprises a chemical reactive group G2 which reacts with said G1 to convert said dioxetane to an unstable light-emitting dioxetane form.
A chemiluminescent reagent having the structure:
- 40
- wherein Q comprises a cycloalkyl or polycycloalkyl group attached covalently to the 4-membered ring portion of said dioxetane above directly or indirectly through a linkage group;
- wherein Z comprises hydrogen, alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl or cycloheteroalkyl;
- and wherein R comprises a chemical linker having a reactive site attached to the aromatic ring in said structure;
- and wherein R′ comprises a substrate for an non-cleaving enzymatic process, wherein the product of said enzymatic process leads to further chemical rearrangements that generate an unstable light emitting dioxetane form.
A process for detecting the presence or quantity of enzymatic activity of interest in a sample, said process comprising the steps of:
- (a) providing:
- (i) said sample suspected of containing enzymatic activity;
- (ii) the chemiluminescent reagent of claim 235;
- (ii) reagents and buffers for carrying out chemiluminescent reactions;
- (b) forming a mixture of:
- (1) (i), (ii) and (iii);
- or
- (2) (ii) and (iii) and contacting said mixture of (ii) and (iii) with (i);
- (c) enzymatically converting the chemiluminescent reagent of claim 344 (ii) into an unstable light-emitting dioxetane form;
- and
- (d) measuring the quantity of light generated by said enzymatic conversion in step (c).
A dye composition of the formula
- R—Fluorescent Dye
- wherein R is covalently linked to said Fluorescent Dye comprises two or more members in combination from
- a) unsaturated aliphatic groups;
- b) unsaturated heterocyclic groups;
- c) aromatic groups;
- and wherein R is capable of providing a conjugated system or an electron delocalized system with said fluorescent dye.
A labeled target having the structure
- 41
- wherein said Dye is a fluorescent dye, wherein R is covalently linked to said Dye, and
- wherein R comprises two or more members in combination from
- a) unsaturated aliphatic groups;
- b) unsaturated heterocyclic groups;
- c) aromatic groups;
- and wherein R is capable of providing a conjugated system or an electron delocalized system with said Dye.
- comprises two or more unsaturated aliphatic groups, unsaturated heterocyclic groups, aromatic groups, or combinations of the foregoing groups and wherein R is covalently attached to said fluorescent dye and is capable of providing a conjugated system or an electron delocalized system with said fluorescent dye, and wherein said target is covalently attached to said Dye or said R.
Dependencies are inferred from the claim text.