Reagents for monitoring nucleic acid amplification and methods of using same

• US 20040053287A1
• Filed: 04/09/2003
• Published: 03/18/2004
• Est. Priority Date: 04/22/2002
• Status: Active Grant

• Current Assignee: Joseph F. Lawler Jr
• Original Assignee: Joseph F. Lawler Jr
• Inventors: Lawler, Joseph F. Jr.
• Granted Patent: US 7,118,871 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: C12Q 1/44 involving esterase
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

Described herein are novel indicator molecules of general formula (1):

wherein Q, F, N, Nuc, X₁ and X₂ are as defined herein, including their tautomeric forms and their additive salts. The present invention also concerns methods for the use of these molecules to monitor nucleic acid amplification in real time and their applications as diagnostics.

Claims

1. A molecule comprising the formula (1) shown below

2. The molecule according to claim 1, wherein said measurable signal is selected from fluorescence, magnetic field, and radiation intensity.

3. The molecule according to claim 1, wherein said F and Q are selected from the group consisting of fluorophores, quenchers, shift reagents, spin labels, radioisotooes, and magnetic reasonance contrast agents.

4. The molecule according to claim 1, wherein said F and Nuc cyclize to form an intrinsically fluorescent nucleobase analog.

5. The molecule according to claim 1;

wherein F is bound to Nuc by a cleavable linker.

6. The molecule according to claim 1, wherein the sugar shown in formula (1) is selected from the group consisting of optionally substituted riboses, deoxyriboses, acyclics, carbocyclics, dideoxyriboses, hexoses, and dideoxydidehydroriboses.

7. The molecule according to claim 1, wherein F is a fluorophore and Q is a quenching moiety and said change in measurable signal comprises a change in fluorescence intensity, lifetime or polarization.

8. The molecule according to claim 6, wherein said fluorophore is selected from the group consisting of optionally substituted pyrenes, anthracenes, naphthalenes, acridines, stilbenes, indoles, benzindoles, oxazoles, benzoxazoles, thiazoles, benzothiazoles, 4-amino-7-nitrobenz-2-oxa-1,3-diazoles, cyanines, carbocyanines, carbostyryls, porphyrins, salicylates, anthranilates, azulenes, perylenes, pyridines, quinolines, coumarins, polyazaindacenes, xanthenes, oxazines, benzoxazines, carbazines, phenalenones, benzphenalenones, carbazines, oxazines, 4-bora-3a,4a-diaza-s-indacenes, fluorophoresceins, rhodamines, rhodols, 5-carboxyfluorophoresceins (FAM), 5-(2′

-aminoethyl) aminonapthalene-1-sulfonic acids (EDANS), anthranilamides, terbium chelates, Reactive Red 4, Texas reds, and BODIPY dyes.

9. The molecule according to claim 7, wherein said quenching moiety is selected from the group consisting of optionally substituted phenyls, naphthyls, anthracenyls, benzothiazoles, benzoxazoles, or benzimidazoles, pyrenes, anthracenes, naphthalenes, acridines, stilbenes, indoles, benzindoles, oxazoles, benzoxazoles, thiazoles, benzothiazoles, 4-amino-7-nitrobenz-2-oxa-1,3-diazoles, cyanines, carbocyanines, carbostyryls, porphyrins, salicylates, anthranilates, azulenes, perylenes, pyridines, quinolines, coumarins, polyazaindacenes, xanthenes, oxazines, benzoxazines, carbazines, phenalenones, benzphenalenones, carbazines, oxazines, 4-bora-3a,4a-diaza-s-indacenes, fluorophoresceins, rhodamines, rhodols, 5-carboxyfluorophoresceins (FAM), 5-(2′

-aminoethyl) aminonapthalene-1-sulfonic acids (EDANS), anthranilamides, terbium chelates, Reactive Red 4, dabcyls, nitrotyrosines, malachite greens, Texas reds, dinitrobenzenes, and BODIPY dyes.

10. The molecule according to claim 1, wherein one or more of said phosphates of the molecule of formula (1) are replaced with phosphorothioate, phosphoramidate, phosphonate, or alkyl-phosphonate.

11. A method of detecting nucleic acid amplification comprising the steps of:

(a) contacting a polymerase with a primer, a template, nucleobase triphosphates and one or more molecules of the formula (1) shown below in a medium in which said polymerase is active;

 

wherein;

Nuc represents a nucleobase selected from the group consisting of adenine, cytosine, guanine, thymidine, uracil and analogs thereof;

N represents a number from 1 through 8 indicating the number of phosphate moieties present in said molecule;

X₁ and X₂ are independently selected from the group H, OH, NH₂, O-alkyl, N-alkyl, S-Alkyl, P-alkyl, a halogen, an alkyl, a cyclo-alkyl, and an aromatic containing group, wherein the alkyl and cycloalkyl possess varying degrees of unsaturation and are optionally substituted; and

F and Q comprise a pair of indicators, wherein cleavage of the molecule results in separation of the indicators which, in turn, yields a change in a measurable signal; and

(b) detecting a change a measurable signal.

12. The method according to claim 11, wherein said nucleic acid amplification process is selected from PCR, RCA, NASBA, 3SR.

13. A method of detecting a polymerase comprising the steps of:

(a) contacting said polymerase with a primer, a template, nucleobase triphosphates and one or more molecules of the formula (1) shown below in a medium in which said polymerase is active;

 

wherein;

Nuc represents a nucleobase selected from the group consisting of adenine, cytosine, guanine, thymidine, uracil and analogs thereof;

N represents a number from 1 through 8 indicating the number of phosphate moieties present in said molecule;

X₁ and X₂ are independently selected from the group H, OH, NH₂, O-alkyl, N-alkyl, S-Alkyl, P-alkyl, a halogen, an alkyl, a cyclo-alkyl, and an aromatic containing group, wherein the alkyl and cycloalkyl possess varying degrees of unsaturation and are optionally substituted; and

F and Q comprise a pair of indicators, wherein cleavage of the molecule results in separation of the indicators which, in turn, yields a change in a measurable signal; and

(b) detecting a change said measurable signal.

14. The method according to claim 13, wherein said polymerase is a DNA polymerase, an RNA polymerase or a reverse transcriptase.

15. The method according to claim 13, wherein polymerase is contained within virus particle.

16. The method according to claim 15, wherein said virus particle is an HIV particle and said method allows for the real time detection of the presence of HIV reverse transcriptase in a sample.

17. The method according to claim 13, wherein said medium is a body fluid selected from blood, plasma, serum, CSF, urine, semen, tears, saliva, bile, gastric secretions and breast milk.

18. A method of detecting a phosphodiesterase comprising the steps of:

(a) contacting one and one or more molecules of the formula (1) shown below with a phosphodiesterase in a medium in which said phosphodiesterase is enzymatically active;

 

wherein;

Nuc represents a nucleobase selected from the group consisting of adenine, cytosine, guanine, thymidine, uracil and analogs thereof;

N represents a number from 1 through 8 indicating the number of phosphate moieties present in said molecule;

X₁ and X₂ are independently selected from the group H, OH, NH₂, O-alkyl, N-alkyl, S-Alkyl, P-alkyl, a halogen, an alkyl, a cyclo-alkyl, and an aromatic containing group, wherein the alkyl and cycloalkyl possess varying degrees of unsaturation and are optionally substituted; and

F and Q comprise a pair of indicators, wherein cleavage of the molecule results in separation of the indicators which, in turn, yields a change in a measurable signal; and

(b) detecting a change in said measurable signal.

19. A method of detecting nucleobase cyclase activity comprising the steps of:

(a) contacting one or more molecules of the formula (1) shown below with said cyclase in a medium in which said cyclase is enzymatically active

 

wherein;

Nuc represents a nucleobase selected from the group consisting of adenine, cytosine, guanine, thymidine, uracil and analogs thereof;

N represents a number from 1 through 8 indicating the number of phosphate moieties present in said molecule;

X₁ and X₂ are independently selected from the group H, OH, NH₂, O-alkyl, N-alkyl, S-Alkyl, P-alkyl, a halogen, an alkyl, a cyclo-alkyl, and an aromatic containing group, wherein the alkyl and cycloalkyl possess varying degrees of unsaturation and are optionally substituted; and

F and Q comprise a pair of indicators, wherein cleavage of the molecule results in separation of the indicators which, in turn, yields a change in a measurable signal; and

(b) detecting a change in said measurable signal.

20. An assay for detecting the presence of an active viral polymerase in a sample comprising the following components:

a polymerase with a primer, a template, nucleobase triphosphates and one or more molecules of the formula (1) shown below;

21. The assay of claim 20, wherein said sample comprises a body fluid selected from blood, plasma, serum, CSF, urine, semen, tears, saliva, bile, gastric secretions and breast milk.

22. The assay of claim 20, wherein the viral polymerase comprises HIV reverse transcriptase.