Terminal-phosphate-labeled nucleotides and methods of use

• US 20030162213A1
• Filed: 02/05/2003
• Published: 08/28/2003
• Est. Priority Date: 08/29/2001
• Status: Active Grant

• Current Assignee: Global Life Sciences Solutions USA LLC (Danaher Corporation)
• Original Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Inventors: Nelson, John, Sood, Anup, Kumar, Shiv, Fuller, Carl
• Granted Patent: US 7,223,541 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C09B 15/00 Acridine dyes
• CPC Class Codes: C09B 57/02 Coumarine dyes
• CPC Class Codes: C09B 69/109 containing other specific dyes
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2521/525 Phosphatase

Abstract

The present invention relates to improved methods of detecting a target using a labeled substrate or substrate analog. The methods comprise reacting the substrate or substrate analog in an enzyme-catalyzed reaction which produces a labeled moiety with independently detectable signal only when such substrate or substrate analog reacts. The present invention, in particular, describes methods of detecting a nucleic acid in a sample, based on the use of terminal-phosphate-labeled nucleotides as substrates for nucleic acid polymerases. The methods provided by this invention utilize a nucleoside polyphosphate, dideoxynucleoside polyphosphate, or deoxynucleoside polyphosphate analogue which has a colorimetric dye, chemiluminescent, or fluorescent moiety, a mass tag or an electrochemical tag attached to the terminal-phosphate. When a nucleic acid polymerase uses this analogue as a substrate, an enzyme-activatable label would be present on the inorganic polyphosphate by-product of phosphoryl transfer. Cleavage of the polyphosphate product of phosphoryl transfer via phosphatase leads to a detectable change in the label attached thereon. When the polymerase assay is performed in the presence of a phosphatase, there is provided a convenient method for real-time monitoring of DNA or RNA synthesis and detection of a target nucleic acid.

Claims

1. A method of detecting a target using a labeled substrate or substrate analog comprising reacting said substrate or substrate analog in an enzyme-catalyzed reaction which produces a labeled polyphosphate that has independently detectable signal only when said substrate or substrate analog reacts, and wherein, when the target is a nucleic acid, the labeled polyphosphate has three or more phosphates.

2. The method of claim 1 wherein the labeled polyphosphate can further undergo a chemical change, to produce the detectable signal.

3. The method of claim 2 wherein the chemical change is catalyzed by one or more enzymes to produce the detectable signal.

4. The method of claim 1 wherein the target is a nucleic acid target.

5. The method of claim 1 wherein the target is a protein.

6. The method of claim 1 wherein labeled substrate analog is a terminal-phosphate labeled nucleoside polyphosphate.

7. The method of claim 1 wherein the said enzyme-catalyzed reaction is nucleic acid polymerization.

8. The method of claim 1 wherein the said enzyme-catalyzed reaction is catalyzed by a phosphodiesterase.

9. The method of claim 1 wherein the said enzyme-catalyzed reaction is catalyzed by a dinucleotide phosphorylase.

10. The method of claim 1 wherein the said enzyme-catalyzed reaction is catalyzed by a ligase.

11. The method of claim 1, wherein the polyphosphate contains 2 or more phosphate groups.

12. The method of claim 1 wherein the said labeled polyphosphate is represented by the formula

Label-x—

(p)_n—

p

wherein Label is a detectable moiety, x is a O, S, NH or a linker, n is 1-5 and p is a phosphate or phosphate derivative.

13. The method of claim 1 wherein the label in said labeled polyphosphate, is a calorimetric, a fluorescent, a chromogenic, a fluorogenic or a chemiluminescent compound or an electrochemical label.

14. The method of claim 2 wherein the said label is selected from the group consisting of chemiluminescent compounds, fluorogenic dyes, chromogenic dyes, electrochemical tags and combinations thereof.

15. The method of claim 1 wherein said detectable species is detectable by a property selected from the group consisting of color, fluorescence emission, chemiluminescence, mass change, reduction/oxidation potential and combinations thereof.

16. The method of claim 13 wherein the said colorimetric label is selected from the group consisting of cyanines, merrocyanines, phenoxazines, acridinones or nitrophenols.

17. The method according to claim 13 wherein the said fluorescent label is selected from fluoresceins, rhodamines, cyanines or merrocyanines.

18. The method of claim 14 wherein said label is a fluorogenic moiety is selected from the group consisting of 2-(5′

-chloro-2′

-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone, fluorescein diphosphate, fluorescein 3′

(6′

)-O-alkyl-6′

(3′

)-phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate, 4-methylumbelliferyl phosphate, resorufin phosphate, 4-trifluoromethylumbelliferyl phosphate, umbelliferyl phosphate, 3-cyanoumbelliferyl phosphate, 9,9-dimethylacirdin-2-one-7-yl phosphate, 6,8-difluoro-4-methylumbelliferyl phosphate, and derivatives thereof.

19. The method of claim 14 wherein said label is a chromogenic moiety selected from the group consisting of 5-bromo-4-chloro-3-indolyl phosphate, 3-indoxyl phosphate, p-nitrophenyl phosphate and derivatives thereof.

20. The method of claim 14 wherein said chemiluminescent compound is a phosphatase-activated 1,2-dioxetane compound.

21. The method of claim 20 wherein said 1,2-dioxetane compound is selected from the group consisting of 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2′

-(5-chloro-)tricyclo[3,3,1-1^3,7]-decan]-1-yl)-1-phenyl phosphate, chloroadamant-2′

-ylidenemethoxyphenoxy phosphorylated dioxetane, 3-(2′

-spiroadamantane)-4-methoxy-4-(3″

-phosphoryloxy)phenyl-1,2-dioxetane and derivatives thereof.

22. The method of claim 3 wherein the said enzyme is selected from a phosphatase, a glycosidase, a polyphosphate transferring enzyme, a peptidase, an oxidase, a peroxidase, a sulfatase, an esterase or a combination thereof.

23. The method of claim 2 wherein said chemical change is caused by chemical hydrolysis.

24. The method of claim 2 wherein the said chemical change is caused by a combination of chemical hydrolysis and enzymatic action.

25. The method according to claim 24 wherein the chemical hydrolysis is spontaneous.

26. A method of detecting a target nucleic acid sequence using a labeled nucleotide analog comprising incorporating said analog by a nucleic acid polymerase into a polynucleotide complimentary to the target sequence thereby, producing a species which is neither pyrophosphate nor a mono-substituted pyrophosphate that undergoes a chemical change to produce a detectable signal.

27. The method of claim 26 wherein said chemical change is catalyzed by one or more enzymes.

28. The method of claim 26 wherein the said species is a labeled polyphosphate with three or more phosphate groups.

29. The method of claim 26 wherein the said species is represented by the formula

Label-x—

p—

(p)_n—

p

wherein Label is a detectable moiety, x is a O, S, NH or a linker, n=1-4 and p is a phosphate or phosphate derivative.

30. The method of claim 29 wherein the linker is an enzyme-removable linker.

31. The method according to claim 29 wherein the linker is spontaneously removed after removal of phosphate.

32. A labeled phosphate containing compound comprising the formula

Label-(P)_n—

P

wherein the label is a detectable moiety selected from the group consisting of fluoresent, colored, fluorogenic, chromogenic, luminogenic moiety or an electrochemical label and n is 2-5.

33. A labeled phosphate containing compound comprising the formula

Label-(P)_n—

P

wherein the label is a detectable moiety selected from the group consisting of fluorogenic, luminogenic moiety or an electrochemical label and n is 1-5.