Nucleic acid labelling compounds
View Patent ↗Nucleic acid labeling compounds are disclosed. The compounds are synthesized by condensing a heterocyclic derivative with a cyclic group (e.g. a ribofuranose derivative). The labeling compounds are suitable for enzymatic attachment to a nucleic acid, either terminally or internally, to provide a mechanism of nucleic acid detection.
1. A nucleic acid labeling compound represented by the structure
wherein:
A is hydrogen, monophosphate, diphosphate, triphosphate, α-thiotriphosphate, phosphoramidite, or H-phosphonate;
X 1 is O, S, NR 1 , or CHR 2 , wherein R 1 and R 2 are, independently, hydrogen, alkyl, or aryl;
R 15 is hydrogen, alkyl, or aryl;
Y 1 is hydrogen, N 3 , F, OR 9 , SR 9 , or NHR 9 , wherein R 9 is hydrogen, alkyl, or aryl;
Z 1 is hydrogen, N 3 , F, OR 9 , SR 9 , or NHR 10 , wherein R 10 is hydrogen, alkyl, or aryl;
one of Y 1 or Z 1 is a group having the formula —X 2 L-M-Q;
X 2 is O, S, or NR 16 , wherein R 16 is hydrogen, alkyl, or aryl;
L is a linker moiety that is sterically and electronically suitable for incorporation into a nucleic acid;
Q is a detectable moiety selected from the group consisting of biotin, carboxyfluorescein, colloidal gold, radiolabels, magnetic particles, fluorescent labels, chemiluminescent or bioluminescent labels, spin labels, dyes, and colored beads; and
M is a connecting group comprising a structure that does not interfere with the nucleic acid labeling compound's function.
2. The nucleic acid labeling compound of claim 1 , wherein:
L is selected from the group consisting of amido alkyl, functionalized alkyl, alkenyl alkyl, alkanoyl, and N-alkyl amido.
3. The nucleic acid labeling compound of claim 1 , wherein:
M is selected from the group consisting of amino alkyl, —CO(CH 2 ) 5 NH—, —CO—, —NH—, —CO(O)—, —CO(NH)—, —(CH 2 ) i O—, —(CH 2 ) j NH—, —C(O)(CH 2 ) h O—, —CO(CH 2 ) 5 NHCO(CH 2 ) 5 NH—, —NH(CH 2 CH 2 O) k NH—, and —CO(CH 2 ) 5 —, wherein h, i, j, and k are independently integers from 1 to 5.
4. The nucleic acid labeling compound of claim 1 , wherein:
Y 1 is selected from the group consisting of hydrogen, OR 9 , wherein R 9 is hydrogen or alkyl, —NR 16 -L-(M) m -Q, wherein R 16 is hydrogen, alkyl, or aryl, —O-L-(M) m -Q, and —S-L-(M) m -Q.
5. The nucleic acid labeling compound of claim 1 , wherein:
Z 1 is selected from the group consisting of hydrogen, OR 10 , wherein R 10 is hydrogen or alkyl, —NR 16 -L-(M) m -Q, wherein R 16 is hydrogen or alkyl, —O-L-(M) m -Q, and —S-L-(M) m -Q.
6. The nucleic acid labeling compound of claim 1 , wherein Q is:
a fluorescent group which absorbs light with a wavelength above 300 nm, and emits light with a wavelength above 310 nm, or
a fluorescent group which absorbs light with a wavelength above 400 nm, and emits light with a wavelength above 410 nm.
7. The nucleic acid labeling compound of claim 1 , wherein:
A is H or H 4 O 9 P 3 —;
X 1 is O;
Y 1 is hydrogen, OR 9 , wherein R 9 is hydrogen or alkyl, or —NR 16 -L-(M) m -Q, wherein R 16 is hydrogen, alkyl, or aryl;
Z 1 is hydrogen, OR 10 , wherein R 10 is hydrogen or alkyl, or —NR 16 -L-(M) m -Q, wherein R 16 is hydrogen or alkyl;
L is selected from the group consisting of amido alkyl, functionalized alkyl, alkenyl alkyl, alkanoyl, and N-alkyl amido;
R 15 is hydrogen or alkyl;
M is selected from the group consisting of amino alkyl, —CO(CH 2 ) 5 NH—, —CO—, —NH—, —CO(O)—, —CO(NH)—, —(CH 2 ) i O—, —(CH 2 ) j NH—, —C(O)(CH 2 ) h O—, —CO(CH 2 ) 5 NHCO(CH 2 ) 5 NH—, —NH(CH 2 CH 2 O) k NH—, and —CO(CH 2 ) 5 —, wherein h, i, j, and k are independently integers from 1 to 5; and
m is an integer from 1 to 15.
8. The nucleic acid labeling compound of claim 7 , wherein:
L is N-alkyl amido; and
M is —CH 2 —NR 3 —, wherein R 3 is hydrogen or alkyl.
9. The nucleic acid labeling compound of claim 7 , wherein:
Y 1 is hydrogen or OH;
Z 1 is —NH-L-(M) m -Q;
L is —(CH 2 ) n NHC(O)—, where n is an integer from 1 to 10;
M is —NH(CH 2 ) p —, where p is an integer from 1 to 10;
R 15 is hydrogen or methyl; and
Q is biotin or a carboxyfluorescein.
10. The nucleic acid labeling compound of claim 9 , wherein:
Y 1 is OH;
R 15 is hydrogen or methyl;
-L-(M) m - is —(CH 2 ) 4 NH—C(O)(CH 2 ) 5 NH—; and
Q is biotin or a carboxyfluorescein.
11. The nucleic acid labeling compound of claim 7 , wherein:
Y 1 is —NH-L-(M) m -Q;
Z 1 is hydrogen or OH;
L is selected from the group consisting of amido alkyl, functionalized alkyl, alkenyl alkyl, alkanoyl, and N-alkyl amido;
M is —NH(CH 2 ) p —, where p is an integer from 1 to 10;
R 15 is hydrogen or methyl; and
Q is biotin or a carboxyfluorescein.
12. The nucleic acid labeling compound of claim 7 , wherein:
A is H or H 4 O 9 P 3 —;
X 1 is O;
Y 1 is hydrogen, OR 9 , wherein R 9 is hydrogen or alkyl, or —O-L-(M) m -Q;
Z 1 is hydrogen, OR 10 , wherein R 10 is hydrogen or alkyl, or —O-L-(M) m -Q;
L is alkylene; and
m is an integer from 1 to 10.
13. The nucleic acid labeling compound of claim 12 , wherein:
Y 1 is hydrogen or OH;
Z 1 is —O-L-(M) m -Q;
L is —(CH 2 ) n —, where n is an integer from 1 to 12;
M is selected from the group consisting of amino alkyl, —CO(CH 2 ) 5 NH—, —CO—, —NH—, —CO(O)—, —CO(NH)—, —(CH 2 ) i O—, —(CH 2 ) j NH—, —C(O)(CH 2 ) h O—, —CO(CH 2 ) 5 NHCO(CH 2 ) 5 NH—, —NH(CH 2 CH 2 O) k NH—, and —CO(CH 2 ) 5 —, wherein h, i, j, and k are independently integers from 1 to 5;
R 15 is hydrogen or methyl; and
Q is biotin or a carboxyfluorescein.
14. The nucleic acid labeling compound of claim 13 , wherein:
M is —NH—.
15. The nucleic acid labeling compound of claim 14 , wherein:
Y 1 is OH;
R 15 is hydrogen or methyl;
-L-(M) m - is —(CH 2 ) 6 —NH—; and
Q is biotin or a carboxyfluorescein.
16. The nucleic acid labeling compound of claim 7 , wherein:
Y 1 is —O-L-(M) m -Q;
Z 1 is hydrogen or OH;
L is —(CH 2 ) n —, where n is an integer from 1 to 12;
M is selected from the group consisting of amino alkyl, —CO(CH 2 ) 5 NH—, —CO—, —NH—, —CO(O)—, —CO(NH)—, —(CH 2 ) i O—, —(CH 2 ) j NH—, —C(O)(CH 2 ) h O—, —CO(CH 2 ) 5 NHCO(CH 2 ) 5 NH—, —NH(CH 2 CH 2 O) k NH—, and —CO(CH 2 ) 5 —, wherein h, i, j, and k are independently integers from 1 to 5;
R 15 is hydrogen or methyl; and
Q is biotin or a carboxyfluorescein.
17. The nucleic acid labeling compound of claim 16 , wherein:
Z 1 is OH;
R 15 is hydrogen or methyl;
-L-(M) m - is —(CH 2 ) 6 —NH—; and
Q is biotin or a carboxyfluorescein.
18. The nucleic acid labeling compound of claim 7 , wherein:
A is H or H 4 O 9 P 3 —;
X 1 is O;
Y 1 is hydrogen, OR 9 , wherein R 9 is hydrogen or alkyl, or —S-L-(M) m -Q;
Z 1 is hydrogen, OR 10 , wherein R 10 is hydrogen or alkyl, or —S-L-(M) m -Q;
L is alkylene; and
m is an integer from 1 to 10.
19. The nucleic acid labeling compound of claim 18 , wherein:
Y 1 is hydrogen or OH;
Z 1 is —S-L-(M) m -Q;
L is —S—(CH 2 ) n —, where n is an integer from 1 to 10;
M is selected from the group consisting of amino alkyl, —CO(CH 2 ) 5 NH—, —CO—, —NH—, —CO(O)—, —CO(NH)—, —(CH 2 ) i O—, —(CH 2 ) j NH—, —C(O)(CH 2 ) h O—, —CO(CH 2 ) 5 NHCO(CH 2 ) 5 NH—, —NH(CH 2 CH 2 O) k NH—, and —CO(CH 2 ) 5 —, wherein h, i, j, and k are independently integers from 1 to 5;
R 15 is hydrogen or methyl; and
Q is biotin or a carboxyfluorescein.
20. The nucleic acid labeling compound of claim 19 , wherein:
Y 1 is OH;
R 15 is hydrogen or methyl;
-L-(M) m - is —S—(CH 2 ) 2 —NH—; and
Q is biotin or a carboxyfluorescein.
21. The nucleic acid labeling compound of claim 7 , wherein:
Y 1 is —S-L-(M) m -Q;
Z 1 is hydrogen or OH;
L is —S—(CH 2 ) n —, where n is an integer from 1 to 10;
M is selected from the group consisting of amino alkyl, —CO(CH 2 ) 5 NH—, —CO—, —NH—, —CO(O)—, —CO(NH)—, —(CH 2 ) i O—, —(CH 2 ) j NH—, —C(O)(CH 2 ) h O—, —CO(CH 2 ) 5 NHCO(CH 2 ) 5 NH—, —NH(CH 2 CH 2 O) k NH—, and —CO(CH 2 ) 5 —, wherein h, i, j, and k are independently integers from 1 to 5;
R 15 is hydrogen or methyl; and
Q is biotin or a carboxyfluorescein.
22. The nucleic acid labeling compound of claim 21 , wherein:
Z 1 is OH;
R 15 is hydrogen or methyl;
-L-(M) m - is —S—(CH 2 ) 2 —NH—; and
Q is biotin or a carboxyfluorescein.
23. A nucleic acid derivative produced by the process of:
attaching at least one nucleic acid to a support; and
coupling the at least one nucleic acid to the nucleic acid labeling compound of claim 1 .
24. The nucleic acid derivative produced by the process of claim 23 , wherein either a terminal transferase or a ligase performs the coupling step.
25. A hybridization product, comprising the nucleic acid derivative of claim 23 bound to a complementary probe.
26. The hybridization product of claim 25 , wherein the complementary probe is attached to a glass chip.