Nucleic acid labeling compounds

Details Nucleic acid labeling compounds Nucleic acid labeling compounds

2001-02-09

2003-07-22

Wilson, James O. (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S022100, C536S024200, C536S025300, C435S006120

Reexamination Certificate

active

06596856

ABSTRACT:

BACKGROUND OF THE INVENTION
Gene expression in diseased and healthy individuals is oftentimes different and characterizable. The ability to monitor gene expression in such cases provides medical professionals with a powerful diagnostic tool. This form of diagnosis is especially important in the area of oncology, where it is thought that the overexpression of an oncogene, or the underexpression of a tumor suppressor gene, results in tumorogenesis. See Mikkelson et al.
J. Cell. Biochem.
1991, 46, 3-8.
One can indirectly monitor gene expression, for example, by measuring a nucleic acid (e.g., mRNA) that is the transcription product of a targeted gene. The nucleic acid is chemically or biochemically labeled with a detectable moiety and allowed to hybridize with a localized nucleic acid probe of known sequence. The detection of a labeled nucleic acid at the probe position indicates that the targeted gene has been expressed. See International Application Publication Nos. WO 97/27317, WO 92/10588 and WO 97/10365.
The labeling of a nucleic acid is typically performed by covalently attaching a detectable group (label) to either an internal or terminal position. Scientists have reported a number of detectable nucleotide analogues that have been enzymatically incorporated into an oligo- or polynucleotide. Langer et al., for example, disclosed analogues of dUTP and UTP that contain a covalently bound biotin moiety.
Proc. Natl. Acad. Sci. USA
1981, 78, 6633-6637. The analogues, shown below, possess an allylamine linker arm that is attached to the C-5 position of the pyrimidine ring. The dUTP and UTP analogues, wherein R is H or OH, were incorporated into a polynucleotide.
Petrie et al. disclosed a dATP analogue, 3-[5-[(N-biotinyl-6-aminocaproyl)-amino]pentyl]-1-(2-deoxy-&bgr;-D-erythro-pentofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine-5′-triphosphate.
Bioconjugate Chem.
1991, 2, 441-446. The analogue, shown below, is modified at the 3-position with a linker arm that is attached to a biotin moiety. Petrie et al. reported that the compound wherein R is biotin is incorporated into DNA by nick translation.
Prober et al. disclosed a set of four dideoxynucleotides, each containing a succinylfluorescein dye.
Science
1987, 238, 336-341. The dideoxynucleotides, one of which is shown below, were enzymatically incorporated into an oligonucleotide through a template directed extension of a primer. The compounds provided for a DNA sequencing method based on gel migration.
Herrlein et al. disclosed modified nucleoside trisphosphates of the four DNA bases.
Helv. Chim. Acta
1994, 77, 586-596. The compounds, one of which is shown below, contain a 3′-amino group containing radioactive or fluorescent moieties. Herrlein et al. further described the use of the nucleoside analogues as DNA chain terminators.
Cech et al. disclosed 3′-amino-functionalized nucleoside triphosphates.
Collect. Czech. Chem. Commun.
1996, 61, S297-S300. The compounds, one of which is shown below, contain a fluorescein attached to the 3′-position through an amino linker. Cech et al. proposed that the described functionalized nucleosides would be useful as terminators for DNA sequencing.
DISCLOSURE OF THE INVENTION
The present invention relates to nucleic acid labeling compounds. More specifically, the invention provides heterocyclic derivatives containing a detectable moiety. The invention also provides methods of making such heterocyclic derivatives. It further provides methods of attaching the heterocyclic derivatives to a nucleic acid.
The development of a novel nucleic acid labeling compound that is effectively incorporated into a nucleic acid to provide a readily detectable composition would benefit genetic analysis technologies. It would aid, for example, in the monitoring of gene expression and the detection and screening of mutations and polymorphisms. Such a compound should be suitable for enzymatic incorporation into a nucleic acid. Furthermore, the nucleic acid to which the labeling compound is attached should maintain its ability to bind to a probe, such as a complementary nucleic acid.
The present invention provides nucleic acid labeling compounds that are capable of being enzymatically incorporated into a nucleic acid. The nucleic acids to which the compounds are attached maintain their ability to bind to a complementary nucleic acid sequence.
The nucleic acid labeling compounds of the present invention are of the following structure:
A-O—CH
2
-T-H
c
-L-(M)
m
-Q
wherein A is hydrogen or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid; T is a template moiety; H
c
is a heterocyclic group; L is a linker moiety; Q is a detectable moiety; and M is a connecting group, wherein m is an integer ranging from 0 to about 5.
In one embodiment, the nucleic acid labeling compounds have the following structures:
wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid;
X is O, S, NR
1
or CHR
2
, wherein R
1
and R
2
are, independently, H, alkyl or aryl; Y is H, N
3
, F, OR
9
, SR
9
or NHR
9
, wherein R
9
is H, alkyl or aryl; Z is H, N
3
, F or OR
10
, wherein R
10
is H, alkyl or aryl; L is is amido alkyl; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.
In another embodiment, A is H or H
4
O
9
P
3
—; X is O; Y is H or OR
9
, wherein R
9
is H, alkyl or aryl; Z is H, N
3
, F or OR
10
, wherein R
10
is H, alkyl or aryl; L is —C(O)NH(CH
2
)
n
NH—, wherein n is an integer ranging from about 2 to about 10; Q is biotin or a carboxyfluorescein; and, M is —CO(CH
2
)
5
NH—, wherein m is 1 or 0.
In another embodiment, Y is H or OH; Z is H or OH; L is —C(O)NH(CH
2
)
4
NH—; Q is biotin; and, M is —CO(CH
2
)
5
NH, wherein m is 1.
In another embodiment, Y is H or OH; Z is H or OH; L is —C(O)NH(CH
2
)
4
NH—; Q is 5-carboxyfluorescein; and, m is 0.
In one embodiment, the nucleic acid labeling compounds have the following structures:
wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid; X is O, S, NR
1
or CHR
2
, wherein R
1
and R
2
are, independently, H, alkyl or aryl; Y is H, N
3
, F, OR
9
, SR
9
or NHR
9
, wherein R
9
is H, alkyl or aryl; Z is H, N
3
, F or OR
10
, wherein R
10
is H, alkyl or aryl; L is amino alkyl; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.
In another embodiment, A is H or H
4
O
9
P
3
—; X is O; Y is H or OR
9
, wherein R
9
is H, alkyl or aryl; Z is H, N
3
, F or OR
10
, wherein R
10
is H, alkyl or aryl; L is —NH(CH
2
)
n
NH—, wherein n is an integer ranging from about 2 to about 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH
2
)
5
NH— or —CO(CH
2
)
5
NHCO(CH
2
)
5
NH—, wherein m is 1 or 0.
In another embodiment, Y is H or OH; Z is H or OH; L is —NH(CH
2
)
4
NH—; Q is biotin; and, m is 0.
In another embodiment, Y is H or OH; Z is H or OH; L is —NH(CH
2
)
4
NH—; Q is 5-carboxyfluorescein; and, m is 0.
In one embodiment, the nucleic acid labeling compounds have the following structures:
wherein A is H or a functional group that permits the attachment of the nucleic acid labeling compound to a nucleic acid; X is O, S, NR
1
or CHR
2
, wherein R
1
and R
2
are, independently, H, alkyl or aryl; Y is H, N
3
, F, OR
9
, SR
9
or NHR
9
, wherein R
9
is H, alkyl or aryl; Z is H, N
3
, F or OR
10
, wherein R
10
is H, alkyl or aryl; L is alkynyl alkyl; Q is a detectable moiety; and, M is a connecting group, wherein m is an integer ranging from 0 to about 3.
In another embodiment, A is H or H
4
O
9
P
3
—; X is O; Y is H or OR
9
, wherein R
9
is H, alkyl or aryl; Z is H, N
3
, F or OR
10
, wherein R
10
is H, alkyl or aryl; L is —C≡C(CH
2
)
n
NH—, wherein n is an integer ranging from about 1 to about 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH
2
)
5
NH—, wherein m is 1 or 0.
In another embodiment, Y is H or OH; Z is H or OH

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