Nucleic acid melting analysis with saturation dyes
View Patent ↗Methods are provided for nucleic acid analysis wherein a target nucleic acid is mixed with a dsDNA binding dye to form a mixture. Optionally, an unlabeled probe is included in the mixture. A melting curve is generated for the target nucleic acid by measuring fluorescence from the dsDNA binding dye as the mixture is heated. Dyes for use in nucleic acid analysis and methods for making dyes are also provided.
1. A compound having the formula:
wherein
the moiety represents an optionally-substituted fused monocyclic or polycyclic aromatic ring or an optionally-substituted fused monocyclic or polycyclic nitrogen-containing heteroaromatic ring;
X is oxygen, sulfur, selenium, tellurium or a moiety selected from C(CH 3 ) 2 and NR 1 , where R 1 is hydrogen or C 1-6 alkyl;
R 2 is selected from the group consisting of C 1-6 alkyl, C 3-8 cycloalkyl, aryl, aryl(C 1-3 alkyl), hydroxyalkyl, alkoxyalkyl, aminoalkyl, mono and dialkylaminoalkyl, trialkylammoniumalkyl, alkylenecarboxylate, alkylenecarboxamide, alkylenesulfonate, optionally substituted cyclic heteroatom-containing moieties, and optionally substituted acyclic heteroatom-containing moieties;
t=0 or 1;
Z is a charge selected from 0 or 1;
R 3 is selected from the group consisting of hydrogen, C 1-6 alkyl, and arylcarbonyl, or R 2 and R 3 are taken together to form —CH 2 ) w —, wherein w is 1 to 5;
R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-6 alkyl, and arylcarbonyl;
n=0, 1, or 2; and
v=0 or 1; with the proviso that v=0 when R 2 and R 3 are not taken together to form —(CH 2 ) w —;
wherein when v=0, Q is an heterocycle selected from the group of structures consisting of:
and
wherein when v=1, Q is an heterocycle selected from the group of structures consisting of:
wherein R 4 , R 5 , R 6 , R 7 , R 8 , R 12 , and R 13 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, alkenyl, polyalkenyl, alkynyl, polyalkynyl, alkenylalkynyl, alkylnitrilethio, aryl, heteroaryl, alkoxy, arylcarbonylthio, cycloheteroalkylcarbonylthio, dialkylaminoalkylcarbonylthio, dialkylamino, cycloalkylthio, cycloheteroalkylthio, trialkylammoniumalkylthio, trialkylammoniumalkylcarbonylthio, and nucleosidylthio, each of which may be optionally substituted; an acyclic heteroatom-containing moiety, a cyclic heteroatom-containing moiety, a BRIDGE-DYE, and a reactive group, each of which optionally includes a quaternary ammonium moiety.
2. The compound of claim 1 , wherein at least one of R 4 , R 5 , R 6 , R 7 , and R 8 is selected from the group consisting of arylcarbonylthio, cycloheteroalkylcarbonylthio, dialkylaminoalkylcarbonylthio, trialkylammoniumalkylcarbonylthio, alkylnitrilethio, cycloalkylthio, cycloheteroalkylthio, trialkylammoniumalkylthio, and nucleosidylthio, each of which may be optionally substituted.
3. The compound of claim 2 , wherein R 4 is optionally substituted arylcarbonylthio and is selected from the group consisting of 4-(carbonylthio)-pyridinyl, 4-(carbonylthio)-nitrophenyl, 4-(carbonylthio)-phenyl, 4-(carbonylthio)-N,N-dimethylanilinyl, 2-(carbonylthio)-pyrazinyl, 6-(carbonylthio)-benzopyrazinyl, 5-(carbonylthio)-1-methyl-1,2,3-benzotriazolyl, and carbonylthio-pentafluorophenyl.
4. The compound of claim 2 , wherein R 4 is optionally substituted cycloheteroalkylcarbonylthio and is selected from the group consisting of 4-(carbonylthio)-N-methylpiperazinyl, 4-(carbonylthio)-N,N-dimethylpiperazinium, and 4-(carbonylthio)-morpholinyl.
5. The compound of claim 2 , wherein R 4 is the optionally substituted dialkylaminoalkylcarbonylthio group N,N-dimethylaminomethylcarbonylthio.
6. The compound of claim 2 , wherein R 4 is the optionally substituted trialkylammoniumalkylcarbonylthio group trimethylammoniummethylcarbonylthio.
7. The compound of claim 2 , wherein R 4 is the optionally substituted alkylnitrilethio group acetonitrilethio.
8. The compound of claim 2 , wherein R 4 is the optionally substituted cycloalkylthio group 1-(thio)-bicyclo[2.2.1]heptyl.
9. The compound of claim 2 , wherein R 4 is the optionally substituted cycloheteroalkylthio group 4-(thio)-N-methylpiperadinyl.
10. The compound of claim 2 , wherein R 4 is the optionally substituted trialkylammoniumalkylthio group trimethylammoniumpropylthio.
11. The compound of claim 2 , wherein R 4 is the optionally substituted nucleosidylthio group 5′-deoxy-adenosinyl-thio.
12. The compound of claim 2 , having the formula:
13. The compound of claim 1 , wherein at least one of R 4 , R 5 , R 6 , R 7 , and R 8 is selected from the group consisting of 4-(carbonylthio)-pyridinyl, 4-(carbonylthio)-nitrophenyl, 4-(carbonylthio)-phenyl, 4-(carbonylthio),N,N-dimethylanilinyl, 2-(carbonylthio)-pyrazinyl, 6-(carbonylthio)-benzopyrazinyl, 5-(carbonylthio)-1-methyl-1,2,3-benzotriazolyl, carbonylthio-pentafluorophenyl, 4-(carbonylthio)-N-methylpiperazinyl, 4-(carbonylthio),N,N-dimethylpiperazinium, 4-(carbonylthio)-morpholinyl, N,N-dimethylaminomethylcarbonylthio, trimethylammoniummethylcarbonylthio, 1-(thio)-bicyclo[2.2.1]heptyl, 4-(thio)-N-methylpiperadinyl,trimethylammoniumpropylthio, 5′-deoxy-adenosinyl-thio and acetonitrilethio.
14. The compound of claim 1 , with the proviso that at least one of R 4 , R 5 , R 6 , R 7 , and R 8 is not selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, alkenyl, polyalkenyl, alkynyl, polyalkynyl, alkenylalkynyl, aryl, heteroaryl, alkoxy, and dialkylamino, each of which may be optionally substituted; trimethylammoniumpropyl; amino; hydroxyl; N,N-dimethylpiperazinyl; pyrimidinylthio; a BRIDGE-DYE, and a reactive group, each of which optionally includes a quaternary ammonium moiety.
15. The compound of claim 1 , with the proviso that R 4 , R 5 , R 6 , and R 7 are not selected from the group consisting of halogen, cycloalkyl, alkenyl, polyalkenyl, alkynyl, polyalkynyl, alkenylalkynyl, alkoxy, and dialkylamino, each of which may be optionally substituted; a BRIDGE-DYE, and a reactive group, each of which optionally includes a quaternary ammonium moiety.
16. The compound of claim 1 , with the proviso that R 4 is not selected from the group consisting hydrogen, N,N-dimethylpiperazinyl, pyrimidinylthio, thiol, amino, and hydroxyl.
17. The compound of claim 1 , with the proviso that R 5 is not selected from the group consisting of trimethylammoniumpropyl and phenyl.
18. The compound of claim 1 , with the proviso that R 6 is not selected from the group consisting of hydrogen, methyl, and phenyl.
19. The compound of claim 1 , with the proviso that R 7 is not hydrogen.
20. The compound of claim 1 , having the formula:
wherein R 3 is hydrogen,and X, Y, R 2 , R 5 , and B are defined in the following table, with the proviso that the compound is not selected from the group consisting of dyes G5, H5, I5, K5, L5, D6, E6, P6, R6, Y6, Z6, F7, C8, E8, G8, L8, M8, N8, O8, and V8
Dye
Y
X
R 2
R 5
B
G5
S
Ph
H
H5
S
Me
H
I5
S
H
K5
S
Me
H
L5
S
Me
H
D6
S
Me
H
E6
O
Me
H
P6
S
H
R6
S
Me
H
Y6
O
Me
H
Z6
S
Me
H
F7
S
Me
Ph
2-[4-(N,N-dimethylpiperazine)]6-Me
C8
S
Me
Ph
2-NH 2 6-Me
E8
S
Me
Ph
2-OH6-Me
G8
S
Me
Ph
2-SCH 3 6-Me
L8
S
None
Ph
2-SCH 3 6-Me
M8
S
Me
Ph
2-[S-(2-pyrimidine)]6-Me
N8
S
Me
Ph
2-SMe6-Ph
O8
S
MeR 2 -C(O)Ph
H
V8
S
Me
Ph
2-SCH 3 6-Me
21. A compound having the formula:
wherein
the moiety represents an optionally-substituted fused monocyclic or polycyclic aromatic ring or an optionally-substituted fused monocyclic or polycyclic nitrogen-containing heteroaromatic ring;
X is oxygen, sulfur, selenium, tellurium or a moiety selected from C(CH 3 ) 2 and NR 1 , where R 1 is hydrogen or C 1-6 alkyl;
R 2 is selected from the group consisting of C 1-6 alkyl, C 3-8 cycloalkyl, aryl, aryl(C 1-3 alkyl), hydroxyalkyl, alkoxyalkyl, aminoalkyl, mono and dialkylaminoalkyl, trialkylammoniumalkyl, alkylenecarboxylate, alkylenecarboxamide, alkylenesulfonate, optionally substituted cyclic heteroatom-containing moietics, and optionally substituted acyclic heteroatom-containing moieties;
t=0 or 1;
Z is a charge selected from 0 or 1;
R 3 is selected from the group consisting of hydrogen, C 1-6 alkyl, and arylcarbonyl, or R 2 and R 3 are taken together to form —(CH 2 ) w —, wherein w is 1 to 5;
R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-5 alkyl, and arylcarbonyl;
n=0, 1, or 2; and
v=0 or 1; with the proviso that v=0 when R 2 and R 3 are not taken together to form —(CH 2 ) w —;
wherein when v=0, Q is an heterocycle selected from the group of structures consisting of:
and
wherein when v=1, Q is an heterocycle selected from the group of structures consisting of:
wherein R 4 , R 5 , R 6 , R 7 , R 8 , R 12 , and R 13 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, alkenyl, polyalkenyl, alkynyl, polyalkynyl, alkenylalkynyl, alkylnitrilethio, aryl, heteroaryl, alkoxy, alkylthio, arylcarbonylthio, cycloheteroalkylcarbonylthio, dialkylaminoalkylcarbonylthio, dialkylamino, cycloalkylthio, cycloheteroalkylthio, trialkylammoniumalkylthio, trialkylammoniumalkylcarbonylthio, and nucleosidylthio, each of which may be optionally substituted; an acyclic heteroatom-containing moiety, a cyclic heteroatom-containing moiety, a BRIDGE-DYE, and a reactive group, each of which optionally includes a quaternary ammonium moiety, and
wherein at least one of R 4 , R 5 ,R 6 , R 7 , and R 8 is selected from the group consisting of arylcarbonylthio, cycloheteroalkylcarbonylthio, dialkylaminoalkylcarbonylthio, trialkylammoniumalkylcarbonylthio, alkylnitrilethio, cycloalkylthio, cycloheteroalkylthio, trialkylammoniumalkylthio, and nucleosidylthio, each of which may be optionally substituted.
22. A compound having the formula:
wherein
the moiety represents an optionally-substituted fused monocyclic or polycyclic aromatic ring or an optionally-substituted fused monocyclic or polycyclic nitrogen-containing heteroaromatic ring;
X is oxygen, sulfur, selenium, tellurium or a moiety selected from C(CH 3 ) 2 and NR 1 , where R 2 is hydrogen or C 1-6 alkyl;
R 2 is selected from the group consisting of C 1-6 alkyl, C 3-8 cycloalkyl, aryl, aryl(C 1-3 alkyl), hydroxyalkyl, alkoxyalkyl, aminoalkyl, mono and dialkylaminoalkyl, trialkylammoniumalkyl, alkylenecarboxylate, alkylenecarboxamide, alkylenesulfonate, optionally substituted cyclic heteroatom-containing moieties, and optionally substituted acyclic heteroatom-containing moieties;
t=0 or 1;
Z is a charge selected from 0 or 1;
R 3 is selected from the group consisting of hydrogen, C 1-6 alkyl, and arylcarbonyl, or R 2 and R 3 are taken together to form —(CH 2 ) w —, wherein w is 1 to 5;
R 9 and R 10 are each independently selected from the group consisting of hydrogen, C 1-6 alkyl, and arylcarbonyl;
n=0, 1, or 2; and
v=0 or 1; with the proviso that v=0 when R 2 and R 3 are not taken together to form —(CH 2 ) w—;
wherein when v=0, Q is an heterocycle selected from the group of structures consisting of:
and
wherein when v=1, Q is an heterocycle selected from the group of structures consisting of:
wherein R 4 , R 5 , R 6 , R 7 , R 8 , R 12 , and R 13 are independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, alkenyl, polyalkenyl, alkynyl, polyalkynl, alkenylalkynyl, alkylnitrilethio, aryl, heteroaryl, alkoxy, alkylthio, arylcarbonylthio, cycloheteroalkylcarbonylthio, dialkylaminoalkylcarbonylthio, dialkylamino, cycloalkylthio, cycloheteroalkylthio, trialkylammoniumalkylthio, trialkylammoniumalkylcarbonylthio, and nucleosidylthio, each of which may be optionally substituted; an acyclic heteroatom-containing moiety, a cyclic heteroatom-containing moiety, a BRIDGE-DYE, and a reactive group, each of which optionally includes a quaternary ammonium moiety, with the proviso that R 4 is not selected from the group consisting hydrogen, N,N-dimethylpiperazinyl, pyrimidinylthio, methylthio, thiol, amino, and hydroxyl.