Cosmic quenchers
The present invention provides quenchers of excited state energy, probes and other conjugates comprising these quenchers, and methods of their use.
1. A compound having a structure according to Formula I or II:
wherein
R 1a , R 1b , R 7a , and R 7b are independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl;
and R 1a and R 1b , together with the carbon atom to which they are attached, are optionally joined to form a ring which is a member selected from substituted or unsubstituted C 3 -C 7 cycloalkyl and substituted or unsubstituted 3- to 7-membered heterocycloalkyl;
and R 7a , and R 7b , together with the carbon atom to which they are attached, are optionally joined to form a ring which is a member selected from substituted or unsubstituted C 3 -C 7 cycloalkyl and substituted or unsubstituted 3- to 7-membered heterocycloalkyl; and
R 8 is selected from H,
wherein L x is selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl;
L xs is selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl;
R s is selected from a protected or unprotected reactive functional group, a linkage site, and a solid support; and
R x is selected from a protected or unprotected reactive functional group and a linkage site;
wherein each linkage site is covalently bound to a member independently selected from a nucleoside, a linker to a nucleoside, a nucleotide, a linker to a nucleotide, an oligonucleotide, a linker to an oligonucleotide, a nucleic acid, a linker to a nucleic acid, a carrier molecule, a linker to a carrier molecule, a solid support, and a linker to a solid support.
2. The compound according to claim 1 , wherein R 1a and R 1b are independently selected from unsubstituted C 1 , C 2 , C 3 , C 4 , C 5 , and C 6 alkyl.
3. The compound according to claim 2 , wherein R 1a and R 1b are each methyl.
4. The compound according to claim 1 , wherein R 7a and R 7b are independently selected from unsubstituted C 1 , C 2 , C 3 , C 4 , C 5 , and C 6 alkyl.
5. The compound according to claim 4 , wherein R 7a and R 1b are each methyl.
6. The compound according to claim 1 , wherein R 1a , R 1b , R 7a , and R 7b are independently selected from unsubstituted C 1 , C 2 , C 3 , C 4 , C 5 , and C 6 alkyl.
7. The compound according to claim 6 , wherein R 1a , R 1b , R 7a , and R 7b are each methyl.
8. The compound according to claim 1 , wherein L xs is selected from unsubstituted C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , and C 10 alkyl.
9. The compound according to claim 1 , wherein L xs is substituted heteroalkyl.
10. The compound according to claim 1 , wherein R x is selected from a phosphoramidite, —OH,-ODMT, —COOH, an active ester, and —NH 2 .
11. The compound according to claim 1 , wherein R s is selected from —OH, —ODMT, and a linkage site covalently bound to a linker to a solid support.
12. The compound according to claim 11 , wherein R x is selected from a phosphoramidite, —OH,-ODMT, —COOH, an active ester, and —NH 2 .
13. The compound according to claim 1 , wherein R x is a linkage site covalently bound to a linker to a nucleoside, wherein said nucleoside has the structure:
wherein the ring labeled B is a nucleobase;
R n3 is OH or a phosphoramidite; and
R n5 is OH or -ODMT.
14. The compound according to claim 1 , wherein R 8 is selected from:
15. A method for detecting a nucleic acid target sequence, said method comprising:
(a) contacting said target sequence with a detector nucleic acid; comprising a single-stranded target binding sequence, said detector nucleic acid having linked thereto,
i) a fluorophore; and
ii) a compound according to claim 1 , wherein R 8 comprises a linkage site covalently bound (directly or through a linker) to said detector nucleic acid;
wherein said detector nucleic acid is in a conformation allowing donor-acceptor energy transfer between said fluorophore and said compound when said fluorophore is excited;
(b) hybridizing said target binding sequence to said target sequence, thereby altering said conformation of said detector nucleic acid, causing a change in a fluorescence parameter; and
(c) detecting said change in said fluorescence parameter, thereby detecting said nucleic acid target sequence.
16. A method of ascertaining whether a first nucleic acid and a second nucleic acid hybridize, said first nucleic acid comprising a compound according to claim 1 , wherein R 8 comprises a linkage site covalently bound (directly or through a linker) to said first nucleic acid, said method comprising:
(a) contacting said first nucleic acid with said second nucleic acid; and
(b) detecting an alteration in a fluorescent property of a member selected from said first nucleic acid, said second nucleic acid and a combination thereof, thereby ascertaining whether said hybridization occurs.
17. A method of monitoring a nucleic acid amplification reaction, said method comprising:
(a) preparing an amplification reaction mixture comprising a detector nucleic acid having linked thereto,
i) a fluorophore; and
ii) a compound according to claim 1 , wherein R 8 comprises a linkage site covalently bound (directly or through a linker) to said detector nucleic acid;
(b) subjecting the amplification reaction mixture to amplification conditions;
(c) monitoring the reaction mixture for a fluorescent signal from the detector nucleic acid to obtain an assay result; and
(d) employing the assay result to monitor the nucleic acid amplification reaction.
18. A method of detecting amplification of a target sequence, said method comprising:
(a) hybridizing a sample nucleic acid comprising the target sequence with PCR primers that flank the target sequence;
(b) extending the hybridized primers with a polymerase to produce the PCR product, and separating the two strands of the PCR product to make accessible the sense and antisense strands of the target sequence;
(c) hybridizing a detector nucleic acid to the sense or antisense strand of the target sequence in the PCR product, wherein the detector nucleic acid comprises:
i) a single stranded target binding sequence that is complementary to at least a portion of the sense or antisense strand of the target sequence in the PCR product, and hybridizes to a region between the PCR primers;
ii) a fluorophore; and
iii) a compound according to claim 1 , wherein R 8 comprises a linkage site covalently bound (directly or through a linker) to said detector nucleic acid;
wherein prior to its hybridization to the target sequence, the detector nucleic acid is in a conformation allowing donor-acceptor energy transfer between the fluorophore and the compound when the fluorophore is excited;
thereby altering the conformation of the detector nucleic acid, causing a change in a fluorescence parameter; and
(d) measuring the change in the fluorescence parameter to detect the target sequence and its amplification.
19. A compound having a structure according to Formula I:
wherein
R 1a , R 1b , R 7a , R 7b are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl;
and R 1a and R 1b , together with the carbon atom to which they are attached, are optionally joined to form a ring which is a member selected from substituted or unsubstituted C 3 -C 7 cycloalkyl and substituted or unsubstituted 3- to 7-membered heterocycloalkyl;
and R 7a , and R 7b , together with the carbon atom to which they are attached, are optionally joined to form a ring which is a member selected from substituted or unsubstituted C 3 -C 7 cycloalkyl and substituted or unsubstituted 3- to 7-membered heterocycloalkyl;
wherein at least one of R 1a , R 1b , R 7a , and R 7b is not H; and
R 8 is selected from H,
wherein L x is selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl;
L xs is selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl;
R s is selected from a protected or unprotected reactive functional group, a linkage site, and a solid support; and
R x is selected from a protected or unprotected reactive functional group and a linkage site;
wherein each linkage site is covalently bound to a member independently selected from a nucleoside, a linker to a nucleoside, a nucleotide, a linker to a nucleotide, an oligonucleotide, a linker to an oligonucleotide, a nucleic acid, a linker to a nucleic acid, a carrier molecule, a linker to a carrier molecule, a solid support, and a linker to a solid support.
20. The compound according to claim 19 , wherein R 1a and R 1b are independently selected from unsubstituted C 1 , C 2 , C 3 , C 4 , C 5 , and C 6 alkyl.
21. The compound according to claim 20 , wherein R 1a and R 1b are each methyl.
22. The compound according to claim 20 , wherein R 7a and R 7b are each H.
23. The compound according to claim 19 , wherein R 7a and R 7b are independently selected from unsubstituted C 1 , C 2 , C 3 , C 4 , C 5 , and C 6 alkyl.
24. The compound according to claim 23 , wherein R 7a and R 7b are each methyl.
25. The compound according to claim 23 , wherein R 1a and R 1b are each H.
26. The compound according to claim 19 , wherein R 1a , R 1b , R 7a , and R 7b are independently selected from unsubstituted C 1 , C 2 , C 3 , C 4 , C 5 , and C 6 alkyl.
27. The compound according to claim 26 , wherein R 1a , R 1b , R 7a , and R 7b are each methyl.
28. A compound according to Formula II;
wherein
R 1a , R 1b , R 7a , and R 7b are independently selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl;
and R 1a and R 1b , together with the carbon atom to which they are attached, are optionally joined to form a ring which is a member selected from substituted or unsubstituted C 3 -C 7 cycloalkyl and substituted or unsubstituted 3- to 7-membered heterocycloalkyl;
and R 7a , and R 7b , together with the carbon atom to which they are attached, are optionally joined to form a ring which is a member selected from substituted or unsubstituted C 3 -C 7 cycloalkyl and substituted or unsubstituted 3- to 7-membered heterocycloalkyl;
wherein a member selected from:
(i) R 1a and R 1b ;
(ii) R 7a and R 7b ; and
(iii) a combination thereof is independently selected from unsubstituted C 1 , C 2 , C 3 , C 4 , C 5 , and C 6 alkyl; and
R 8 is selected from H,
wherein L x is selected from a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl;
L xs is selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, and substituted or unsubstituted heterocycloalkyl;
R s is selected from a protected or unprotected reactive functional group, a linkage site, and a solid support; and
R x is selected from a protected or unprotected reactive functional group and a linkage site;
wherein each linkage site is covalently bound to a member independently selected from a nucleoside, a linker to a nucleoside, a nucleotide, a linker to a nucleotide, an oligonucleotide, a linker to an oligonucleotide, a nucleic acid, a linker to a nucleic acid, a carrier molecule, a linker to a carrier molecule, a solid support, and a linker to a solid support.
29. The compound according to claim 28 , wherein R 1a and R 1b are each methyl.
30. The compound according to claim 28 , wherein R 7a and R 7b are each methyl.
31. The compound according to claim 19 , wherein R 1a , R 1b , R 7a , and R 7b are independently selected from unsubstituted C 1 , C 2 , C 3 , C 4 , C 5 , and C 6 alkyl.
32. The compound according to claim 28 , wherein R 1a , R 1b , R 7a , and R 7b are each methyl.