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1. WO2006099050 - POLAR DYES

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]

CLAIMS

1. A polar fluorescent dye comprising a fluorescent compound substituted with one or more arsonate groups, the fluorescent compound having an excitation between 340 nm and 1100 nm and fluorescence emission maximum between 440 and 1150 nm.

2. A compound of Formula (I):



wherein,
R1 is hydrogen, alkyl, -CHO, -NO2, -CH2R11, -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2 or -L-Rx;
R2 is hydrogen, -CN, alkyl, aralkyl, haloalkyl Or -CH2R11;
R3 is hydrogen, alkyl, halogen, -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2;
R4 is -NR12R13, wherein R12 and R13 are the same or different and independently H, alkyl, aryl, alkylcarbonyl, arylcarbonyl or R12 and R13 together form a heterocycle;
R5 is hydrogen, halogen, -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2;
R6 and R7 are the same or different and independently H, alkyl, aryl, or heteroaryl; or
R6 and R7 are linked together to form a 5 or 6-member heterocycle comprising the arsenic and the two oxygen atoms to which R6 and R7 are attached, wherein -R6-R7- is -[C(R8)2]W- (w is 2 or 3), wherein each R8 is the same or different and independently hydrogen, hydroxy, cyano, -OC(O)-alkyl, -OC(O)-aryl, alkyl, aryl, alkylcarbonyl, arylcarbonyl, -CH2CN, -CH2SO2Me, -CH2SO2Ph1 or two R8 of adjacent carbons together with the carbons to which they are attached form an aryl or heteroaryl, or two R8 of the same carbon form an oxo;
R11 is -COOH, -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2;
L is a single covalent bond or a covalent linkage having 1-30 non-hydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds; and
Rx is a reactive group,
provided, at least one of R1, R3, R5 and R11 is -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2.

3. A compound of Formula (II):



wherein,
R15 is hydrogen, alkyl, aralkyl, haloalkyl or -CN;
R16 is -CH2As(=O)(OR6)(OR7) or -CH2As(OR6)2(OR7)2;
R6 and R7 are the same or different and independently H, alkyl, aryl, or heteroaryl; or
R6 and R7 are linked together to form a 5 or 6-member heterocycle comprising the arsenic and the two oxygen atoms to which R6 and R7 are attached, wherein -R6-R7~ is -[C(R8)2]W- (w is 2 or 3), wherein each R8 is the same or different and independently hydrogen, hydroxy, cyano, -OC(O)-alkyl, -OC(O)-aryl, alkyl, aryl, alkylcarbonyl, arylcarbonyl, -CH2CN, -CH2SO2Me, -CH2SO2Ph, or two R8 of adjacent carbons together with the carbons to which they are attached form an aryl or heteroaryl, or two R8 of the same carbon form an oxo;
R17 and R18 are the same or different and independently hydrogen, alkyl or haloalkyl;
R19 is hydrogen, alkyl optionally substituted with hydroxy, halogen, -COOH or -SO2OH, aryl optionally substituted with hydroxy, halogen, -COOH or -SO2OH1 Or -L-Rx;
L is a single covalent bond or a covalent linkage having 1-30 non-hydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds; and
Rx is a reactive group.

4. A compound of Formula (III):



wherein,
m is 1 , 2, 3, 4 or 5;
R24 and R25 are the same or different and independently hydrogen, alkyl, halogen, -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2;
R26, R27 R28 and R29 are the same or different and independently hydrogen, alkyl or aryl optionally substituted by -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2;
R31 are R32 are the same or different and independently hydrogen, alkyl, halogen, -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2;
each R30 is independently hydrogen, -C(O)R33, halogen, alkyl, aralkyl, aryl, alkylthio, alkylcarbonyl, arylcarbonyl, -SO2R33, -As(=O)(OR6)(OR7), -As(OR6)2(OR7)2 or -L-Rx;
R33 is hydroxy, -NH(CH2)jSO3R34, -NH(CH2)jAs(=O)(OR34)2, -NH(CH2)jCOOR34, -NH(CH2)jOR34 or -NH(CH2)jN(R34)2 0 is an integer between 2-6);
each R34 is the same or different and independently hydrogen, alkyl, aryl or heteroaryl;
R24 and R28 may optionally form a 5 or 6-member heterocycle;
R25 and R29 may optionally form a 5 or 6-member heterocycle;
R26 and R28 may optionally form a 5-9 member heterocycle optionally substituted by -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2;
R27 and R29 may optionally form a 5-9 member heterocycle optionally substituted by -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2;
R26 and R31 may optionally form a 5 or 6-member heterocycle optionally substituted with hydrogen, alkyl -CH2As(=O)(OR6)(OR7) or -CH2As(OR6)2(OR7)2;

R27 and R32 may optionally form a 5 or 6-member heterocycle optionally substituted with hydrogen, alkyl -CH2As(=O)(OR6)(OR7) or -CH2As(OR6)2(OR7)2;
R6 and R7 are the same or different and independently H, alkyl, aryl, or heteroaryl; or
R6 and R7 are linked together to form a 5 or 6-member heterocycle comprising the arsenic and the two oxygen atoms to which R6 and R7 are attached, wherein -R6-R7- is -[C(R8)2]W- (w is 2 or 3), wherein each R8 is the same or different and independently hydrogen, hydroxy, cyano, -OC(O)-alkyl, -OC(O)-aryl, alkyl, aryl, alkylcarbonyl, arylcarbonyl, -CH2CN, -CH2SO2Me, -CH2SO2Ph, or two R8 of adjacent carbons together with the carbons to which they are attached form an aryl or heteroaryl, or two R8 of the same carbon form an oxo; and
L is a single covalent bond or a covalent linkage having 1-30 non-hydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds; and
Rx is a reactive group,
provided, at least one of the R24, R25, R26 R27, R28, R29, R30, R31, R32 and R33 is or comprises -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2.

5. A compound of Formula (IV)



wherein,
each R35 is the same or different and independently hydrogen, halogen, -NO2, -CN, -SO3", alkoxy, amine, dialkylamine, monoalkylamine, -N=N-R39, -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2;
n is 1 , 2 or 3;
p is 1 or 2;
each R36 is the same or different and independently hydrogen, halogen, -NO2, -CN, -SO3", alkoxy, amine, dialkylamine, monoalkylamine, -N=N-R39, -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2;

R6 and R7 are the same or different and independently H, alkyl, aryl, or heteroaryl; or
R6 and R7 are linked together to form a 5 or 6-member heterocycle comprising the arsenic and the two oxygen atoms to which R6 and R7 are attached, wherein -R6-R7- is -[C(R8)2]W- (w is 2 or 3), wherein each R8 is the same or different and independently hydrogen, hydroxy, cyano, -OC(O)-alkyl, -OC(O)-aryl, alkyl, aryl, alkylcarbonyl, arylcarbonyl, -CH2CN, -CH2SO2Me, -CH2SO2Ph, or two R8 of adjacent carbons together with the carbons to which they are attached form an aryl or heteroaryl, or two R8 of the same carbon form an oxo;
R37 and R38 are the same or different and independently hydrogen, alkyl, aralkyl, aryl or -L-Rx;
R39 is aryl or heteroaryl optionally substituted with one or more -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2;
L is a single covalent bond or a covalent linkage having 1-30 non-hydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds; and
Rx is a reactive group,
provided, at least one of the R35 and R36 is -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2.

6. A compound of Formula (V):



wherein,
v is 0, 1, 2, 3, 4, 5 or 6;
Y represents atoms necessary to form one or two fused aromatic rings having 6 atoms in each ring, each ring atom is the same or different and independently =CR46-, =N+R47- or =N-;
W represents atoms necessary to form one or two fused aromatic rings having 6 atoms in each ring, each ring atom is the same or different and independently =CR46-, =N+R47- or =N-;

Z is -O-, -S-, -Se-, -C(R4V or -NR49-;
R20 and R23 are the same or different and independently alkyl, aralkyl or substituted alkyl substituted with hydroxy, cyano, nitro, amino, sulfonyl, phosphonyl, -As(=O)(OR6)(OR7), -As(OR6)2(OR7)2, -COOH, alkoxy, monoalkylamino, dialkylamino or trialkylamonium or L-Rx;
R52 and R53 are the same or different and independently hydrogen, alkyl, alkyl substituted hydroxy, -COOH, halogen, cyano, nitro, amine, alkylamino, dialkylamino, sulfonyl, phosphonyl, -As(=O)(OR6)(OR7), As(OR6)2(OR7)2 or L-Rx;
R21 and each R22 are the same or different and independently hydrogen, halogen, alkoxy, hydroxy, mercapto, aryloxy, heterocycle, iminium ion, L-Rx, or two adjacent R21 and R22 or any two adjacent R22 form a 4, 5 or 6 member carbocycle, optionally substituted with alkyl, halogen or oxo; or two R22 that are one carbon away from each other, together with the carbons to which they are attached, form a 4, 5 or 6 member carbocycle, optionally substituted with alkyl, halogen, alkylthio, arylthio, alkoxy, aryloxy, oxo (=O), =S or =C(R50)2;
each R46 and R47 is the same or different and independently hydrogen, alkyl, aryl, halogen, cyano, nitro, amine, alkylamino, dialkylamino, sulfonyl, phosphonyl, -As(=O)(OR6)(OR7), As(OR6)2(C-R7)2 or L-Rx;
each R48 and R49 is the same or different and independently hydrogen or alkyl optionally substituted with hydroxy, -COOH, halogen, cyano, nitro, amine, alkylamino, dialkylamino, sulfonyl, phosphonyl, -As(=O)(OR6)(OR7), -As(OR6)2(OR7)2 or L-Rx;
each R50 is the same or different and independently hydrogen, alkyl, cyano, -C(O)NHR48;
R6 and R7 are the same or different and independently H, alkyl, aryl, or heteroaryl; or
R6 and R7 are linked together to form a 5 or 6-member heterocycle comprising the arsenic and the two oxygen atoms to which R6 and R7 are attached, wherein -R6-R7- is -[C(R8)2]W- (w is 2 or 3), wherein each R8 is the same or different and independently hydrogen, hydroxy, cyano, -OC(O)-alkyl, -OC(O)-aryI, alkyl, aryl, alkylcarbonyl, arylcarbonyl, -CH2CN, -CH2SO2Me, -CH2SO2Ph, or two R8 of adjacent carbons together with the carbons to which they are attached form an aryl or heteroaryl, or two R8 of the same carbon form an oxo;
L is a single covalent bond or a covalent linkage having 1-30 non-hydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds; and
Rx is a reactive group,
provided, at least one of the R20, R23 R46, R47, R48, R49, R52 and R53 is -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2.

7. A compound of Formula (Vl):



wherein,
y is 1, 2 or 3;
x is O, 1 , 2, or 3;
each R56 is the same or different and independently hydrogen, halogen, hydroxy, -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2;
R6 and R7 are the same or different and independently H, alky), aryl, or heteroaryl; or
R6 and R7 are linked together to form a 5 or 6-member heterocycle comprising the arsenic and the two oxygen atoms to which R6 and R7 are attached, wherein -R6-R7- is -[C(R8)2]W- (w is 2 or 3), wherein each R8 is the same or different and independently hydrogen, hydroxy, cyano, -OC(O)-alkyl, -OC(O)-aryl, alkyl, aryl, alkylcarbonyl, arylcarbonyl, -CH2CN, -CH2SO2Me, -CH2SO2Ph, or two R8 of adjacent carbons together with the carbons to which they are attached form an aryl or heteroaryl, or two R8 of the same carbon form an oxo;
R57 is alkyl, aralkyl or alkyl substituted with hydroxy, cyano, nitro, amino, sulfonyl, phosphonyl, -As(=O)(OR6)(OR7), -As(OR6)2(OR7)2, -COOH, alkoxy, monoalkylamino, dialkylamino or trialkylamonium or L-Rx;
R5δa and R58b are the same or different and independently hydrogen, alkyl, alkyl substituted with hydroxy, -COOH, halogen, cyano, nitro, amine, alkylamino, dialkylamino, sulfonyl, phosphonyl, -As(=O)(OR6)(OR7), As(OR6)2(OR7)2 or L-Rx;
R59 is hydrogen, alkyl or aralkyl;

each R60 is the same or different and independently hydrogen, _NR6iaR6ibj a|ky|j ary|) _AS(=O)(OR6)(OR7), -As(OR6)2(OR7)2;
each R61a and Rδ1b is the same or different and independently hydrogen, alkyl, alky! substituted with hydroxy, -COOH, halogen, cyano, nitro, amine, alkylamino, dialkylamino, sulfonyl, phosphonyl, -As(=O)(OR6)(OR7), -As(OR6)2(OR7)2 or L-Rx;
L is a single covalent bond or a covalent linkage having 1-30 non-hydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds; and
Rx is a reactive group,
provided, at least one of R56, R58a, R58b, R60 and R61a and R61b is or comprises -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2.

8. A compound of Formula (VII):



wherein,
each R62 is the same or different and independently hydrogen, halogen, hydroxy, -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2, alkyl, alkyl substituted with hydroxy, -COOH, halogen, cyano, nitro, amine, alkylamino, dialkylamino, sulfonyl, phosphonyl, -As(=O)(OR6)(OR7), As(OR6)2(OR7)2 or L-Rx;
R6 and R7 are the same or different and independently H, alkyl, aryl, or heteroaryl; or
R6 and R7 are linked together to form a 5 or 6-member heterocycle comprising the arsenic and the two oxygen atoms to which R6 and R7 are attached, wherein -R6-R7- is -[C(R8)2]W- (w is 2 or 3), wherein each R8 is the same or different and independently hydrogen, hydroxy, cyano, -OC(O)-alkyl, -OC(O)-aryl, alkyl, aryl, alkylcarbonyl, arylcarbonyl, -CH2CN, -CH2SO2Me, -CH2SO2Ph, or two R8 of adjacent carbons together with the carbons to which they are attached form an aryl or heteroaryl, or two R8 of the same carbon form an oxo;
each R63 and R64 are the same or different and independently hydrogen, halogen, hydroxy, -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2, alkyl, alkyl substituted with hydroxy, -COOH, halogen, cyano, nitro, amine, alkylamino, dialkylamino, sulfonyl, phosphonyl, -As(=O)(OR6)(OR7), As(OR6)2(OR7)2 or L-Rx;
L is a single covalent bond or a covalent linkage having 1-30 non-hydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds; and
Rx is a reactive group,
provided, at least one of R62, R63 and R64 is or comprises -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2.

9. A compound of Formula (VIII):



wherein,
z is 1 , 2 or 3;
T is >C=O or >S(=O)2;
Q iS -OR71 Or -NR72R73;
U iS -OR74 Or -NR75R76;
each R68 and R65 are the same or different and independently hydrogen, halogen, hydroxy, alkyl, aryl or -CH2As(=O)(OR6)(OR7) or -CH2As(OR6)2(OR7)2;
each R66 and R67 are the same or different and independently hydrogen, halogen, hydroxy, alkyl or L-Rx;

no each R70 is the same or different and independently hydrogen, halogen, alkyl, -As(=O)(OR6)(OR7), -As(OR6)2(OR7)2 or L-Rx;
R6 and R7 are the same or different and independently H, alkyl, aryl, or heteroaryl; or
R6 and R7 are linked together to form a 5 or 6-member heterocycle comprising the arsenic and the two oxygen atoms to which R6 and R7 are attached, wherein -R6-R7- is -[C(R8)2]W- (w is 2 or 3), wherein each R8 is the same or different and independently hydrogen, hydroxy, cyano, -OC(O)-alkyl, -OC(O)-aryl, alkyl, aryl, alkylcarbonyl, arylcarbonyl, -CH2CN, -CH2SO2Me, -CH2SO2Ph, or two R8 of adjacent carbons together with the carbons to which they are attached form an aryl or heteroaryl, or two R8 of the same carbon form an oxo;
each R71 and R74 are the same or different and independently is hydrogen, alkylcarbonyl or arylcarbonyl;
each R72, R73, R75 and R76 are the same or different and independently hydrogen, alkyl or aryl; or
R71 and R68, together with the carbons to which they are attached, form a 5 or 6 member heterocycle; or
R74 and R65, together with the carbons to which they are attached, form a 5 or 6 member heterocycle; or
R72 and R68, together with the carbons to which they are attached, form a 5 or 6 member heterocycle; or
R73 and R66, together with the carbons to which they are attached, form a 5 or 6 member heterocycle; or
R75 and R65, together with the carbons to which they are attached, form a 5 or 6 member heterocycle; or
R76 and R67, together with the carbons to which they are attached, form a 5 or 6 member heterocycle;
L is a single covalent bond or a covalent linkage having 1-30 non-hydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds; and
Rx is a reactive group,
provided, at least one of R65, R66 R67, R68 and R71 is or comprises -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2.

Ill

10. A compound of Formula (IX):



wherein,
R40 and R43 are the same or different and independently hydrogen, hydroxy, amino, alkyl, aryl, alkoxy or -L-Rx, provided at least one of R40 and R43 is -L-Rx;
each R41 is independently hydrogen, halogen, hydroxy, sulfonyl, phosphonyl, -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2;
R6 and R7 are the same or different and independently H1 alkyl, aryl, or heteroaryl; or
R6 and R7 are linked together to form a 5 or 6-member heterocycle comprising the arsenic and the two oxygen atoms to which R6 and R7 are attached, wherein -R6-R7- is -[C(R8)2]W- (w is 2 or 3), wherein each R8 is the same or different and independently hydrogen, hydroxy, cyano, -OC(O)-alkyl, -OC(O)-aryl, alkyl, aryl, alkylcarbonyl, arylcarbonyl, -CH2CN, -CH2SO2Me, -CH2SO2Ph, or two R8 of adjacent carbons together with the carbons to which they are attached form an aryl or heteroaryl, or two R8 of the same carbon form an oxo;
q is O, 1 or 2;
R42 is hydrogen, halogen, hydroxy, -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2;
L is a single covalent bond or a covalent linkage having 1-30 non-hydrogen atoms selected from the group consisting of C, N, O and S and is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds; and
Rx is a reactive group,
provided, at least one of R41 and R42 is -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2.

12. An oligonucleotide conjugate comprising an oligonucleotide substituted with a dye having an arsonate ester group.

13. The oligonucleotide of claim 12 wherein the dye is a fluorescent dye having an excitation between 340 nm and 1100 nm and fluorescence emission maximum between 440 and 1150 nm.

14. The oligonucleotide of claim 12 wherein the dye is a non-fluorescent quencher being adapted to absorb fluorescence emitted from a reporting fluorophore prior to the binding of the oligonucleotide to a target.

15. The oligonucleotide of claim 12 wherein the dye is a compound of any of claims 2-10.

16. The oligonucleotide of any of claims 12-15 further comprising an MGB group.

17. The oligonucleotide of claim 16 wherein the MGB group is substituted with a polar arsonate group.

18. The oligonucleotide conjugate of any of claims 12-17 wherein the oligonucleotide is a probe selected from molecular beacons, Scorpion® probes, sunrise probes, conformationally assisted probes and TaqMan™ probes.

19. A method of directly detecting a target nucleic acid, comprising:
providing a dye-labeled oligonucleotide probe comprising an oligonucleotide conjugated to one or more dye compounds of Formulae (l-lll) and (V-VIII), the oligonucleotide further optionally comprising an MGB optionally substituted with one or more polar arsonate groups,
adding the dye-labeled oligonucleotide probe to a biological sample, wherein the probe is complementary or substantially complementary to the target nucleic acid;
allowing the probe to hybridize with the target nucleic acid under an appropriate condition and for sufficient amount of time; and
detecting fluorescence emitted from the dye.

20. A method of preparing an oligonucleotide in an automated synthesis, using a dye reagent of any of Formulae (I)-(VIII), wherein Rx is a phosphoramidite group, the method comprising:
performing automated synthesis of oligonucleotide using phosphoramidite method to provide a dye-labeled oligonucleotide;
deprotecting -As(=O)(OR6)(OR7) or -As(OR6)2(OR7)2 of the dye-labeled oligonucleotide to provide a dye-labeled oligonucleotide having negatively charged polar group selected from -AsO32' or -ASO3H"; and
isolating the dye-labeled oligonucleotide from the reaction medium.

21. The method of claim 20 further comprising a step of conjugating an MGB in the oligonucleotide.

22. The method of claim 21 wherein the MGB is substituted with an arsonate group.

23. A method of detecting amplification of a target sequence in real life using a TaqMan assay, the method comprising the steps of:
providing a dye-labeled oligonucleotide probe comprising an oligonucleotide conjugated to a quencher dye of Formula (IV) and a fluorescent dye of any of the Formulae (l-lll) and (V-VIII), each dye having one or more polar arsonate group, wherein the probe is in a conformation allowing the fluorescent dye to be quenched by the quencher dye;
adding the dye-labeled oligonucleotide probe to a PCR reaction medium;
running the PCR temperature cycles to allow the probe to hybridize with the target and get cleaved by Taq polymerase when the target is present; and
reading the fluorescence signal from the cleaved dye in the real time.

24. A method of detecting amplification of a target sequence in real life using a Molecular Beacon assay, the method comprising the steps of:
providing a dye-labeled oligonucleotide probe comprising an oligonucleotide conjugated to a quencher dye of Formula (IV) and a fluorescent dye of any of the Formulae (l-lll) and (V-VlH), each dye having one or more polar arsonate group, wherein the probe is in a conformation allowing the fluorescent dye to be quenched by the quencher dye;

adding the dye-labeled oligonucleotide probe to a PCR reaction medium;
running the PCR temperature cycles to allow the probe to hybridize with the target thereby separating the fluorescent dye and the quencher dye when the target is present; and
reading the fluorescence signal from the unquenched fluorescent dye in the real time.

25. A method of detecting amplification of a target sequence in real life using a Scorpion Probe assay, the method comprising the steps of:
providing a dye-labeled oligonucleotide probe comprising a primer, an oligonucleotide conjugated to a quencher dye of Formula (IV) and a fluorescent dye of any of the Formulae (l-lll) and (V-VIII), each dye having one or more polar arsonate group, wherein the probe is in a conformation allowing the fluorescent dye to be quenched by the quencher dye;
adding the dye-labeled oligonucleotide probe to a PCR reaction medium;
running the PCR temperature cycles to allow the primer to hybridize with a target DNA1 thereby extending the probe on target DNA;
heat denaturing the extended probe which causes the quencher dye to disassociate;
cooling the extended probe to allow for an internal rearrangement which causes the fluorescent dye to fluorescence in a target specific manner; and
reading the fluorescence signal from the fluorescent dye in the real time.