Fluorescent N-nucleosides and fluorescent structural analogs of N-nucleosides
First Claim
1. An improvement in methods of synthesis, amplification, enzymatic digestion, base-pairing, labeling, sequencing, replication, transcription, location, detection, or identification of DNA or RNA oligonucleotides, wherein said improvement comprises the incorporation of inherently fluorescent nucleosides, or structural analogs thereof, said inherently fluorescent nucleosides or structural analogs thereof being fluorescent under physiological conditions, having the following structures:
-
whereinX1, X2, X3, X4, X5, and X6═
N, O, C, S, or Si, wherein at least one of X1, X2, X3, X4, X5, or X6═
N, and wherein X7 is —
CH—
;
R4 is a reactive group derivatizable with a detectable label wherein said reactive group is selected from the group consisting of NH2, SH, ═
O, and optionally, a linking moiety selected from the group consisting of an amide, a thioether, a disulfide, a combination of an amide a thioether or a disulfide, R1—
(CH2)x—
R2 and R1—
R2—
(CH2)x—
R3 wherein x is an integer from 1 to 25 inclusive, and R1, R2, and R3 are H, OH, alkyl, acyl, amide, thioether, or disulfide, and wherein said detectable label is selected from the group consisting of radioisotopes, fluorescent or chemiluminescent reporter molecules, antibodies, haptens, biotin, photobiotin, digoxigenin, fluorescent aliphatic amino groups, avidin, enzymes, and acridinium;
R5 is H, absent, or part of an etheno linkage with R4;
R6 is H, NH2, SH, or ═
O;
R8 and R9 are hydrogen, methyl, bromine, fluorine, or iodine;
alkyl or aromatic substituents, or an optional linking moiety selected from the group consisting of an amide, a thioether, a disulfide linkage, and a combination thereof;
R10 is hydrogen, an acid-sensitive/base-stable blocking group, or a phosphorous derivative;
R11═
R13═
H;
R12 is hydrogen, OH, 3 amino, 3-azido, 3-thiol, 3-unsaturated or a 3-phosphorous derivative; and
R14 is H, OH, or OR3 where R3 is a reactive group, protecting group, or additional fluorophore;
provided that excluded from such structure is any purine-like compound in which;
(i) X1═
X4═
C;
X2═
X3═
N;
R4═
NH2;
R5═
R8 which is absent;
R6═
H;
R9 is H or is absent;
R10═
H; and
R12═
R14═
OH;
or (ii) X1═
C;
X2═
X3═
X4═
N;
R4═
NH2 or H;
R5═
R8 which is absent;
R6═
NH2;
R9 is H or is absent;
R10═
H; and
R12═
R14OH;
or (iii) R4 and R5 in combination form an etheno linkage;
R6═
R8═
H;
R9 is absent;
X1═
X3═
C; and
X2═
X4═
N;
or (iv) X1═
X2═
C;
X3═
X4═
N;
R4═
halogen or —
S(CH2)nR with n being an integer between 1-6 and R is lower alkoxy, alkylthio, phenoxy, phenylthio, unsubstituted or substituted phenyl, —
C═
C—
R′
wherein R′
is unsubstituted or mono-, di- or trisubstituted phenyl;
R9═
R10═
H; and
R12═
R14═
acyloxy;
or (v) R4═
NH2 or OH;
R5 is absent;
R9 is —
COOH, —
CONH2, —
C(S)NH2, —
C(NH)N2, or —
C(N—
NH2)NH2;
X1═
X2═
X3═
C; and
X4═
N;
and further provided that R8 is absent if X3═
N and R9 is absent if X2═
N;
and provided that the structure is not a nucleoside which is effectively non-fluorescent under physiological conditions and is selected from the group consisting of adenosine, cytidine, guanine, thymidine, uridine, and inosine.
0 Assignments
0 Petitions
Accused Products
Abstract
Structural analogs of the six non-fluorescentN-nucleosides commonly found in RNA and DNA, which are inherently fluorescent under physiological conditions, are identified and methods for their preparation provided. Such analogs may be incorporated into DNA and/or RNA oligonucleotides via either enzymatic or chemical synthesis to produce fluorescent oligonucleotides having prescribed sequences. Such analogous sequences may be identical to, or the analogous complement of, template or target DNA or RNA sequences to which the fluorescent oligonucleotides can be hybridized. Methods of preparing either RNA or DNA oligonucleotide probes of the invention, intermediates used in such methods, and methods of using the probes of the invention in oligonucleotide amplification, detection, identification, and/or hybridization assays are also provided.
-
Citations
2 Claims
-
1. An improvement in methods of synthesis, amplification, enzymatic digestion, base-pairing, labeling, sequencing, replication, transcription, location, detection, or identification of DNA or RNA oligonucleotides, wherein said improvement comprises the incorporation of inherently fluorescent nucleosides, or structural analogs thereof, said inherently fluorescent nucleosides or structural analogs thereof being fluorescent under physiological conditions, having the following structures:
-
wherein X1, X2, X3, X4, X5, and X6═
N, O, C, S, or Si, wherein at least one of X1, X2, X3, X4, X5, or X6═
N, and wherein X7 is —
CH—
;
R4 is a reactive group derivatizable with a detectable label wherein said reactive group is selected from the group consisting of NH2, SH, ═
O, and optionally, a linking moiety selected from the group consisting of an amide, a thioether, a disulfide, a combination of an amide a thioether or a disulfide, R1—
(CH2)x—
R2 and R1—
R2—
(CH2)x—
R3 wherein x is an integer from 1 to 25 inclusive, and R1, R2, and R3 are H, OH, alkyl, acyl, amide, thioether, or disulfide, and wherein said detectable label is selected from the group consisting of radioisotopes, fluorescent or chemiluminescent reporter molecules, antibodies, haptens, biotin, photobiotin, digoxigenin, fluorescent aliphatic amino groups, avidin, enzymes, and acridinium;
R5 is H, absent, or part of an etheno linkage with R4;
R6 is H, NH2, SH, or ═
O;
R8 and R9 are hydrogen, methyl, bromine, fluorine, or iodine;
alkyl or aromatic substituents, or an optional linking moiety selected from the group consisting of an amide, a thioether, a disulfide linkage, and a combination thereof;
R10 is hydrogen, an acid-sensitive/base-stable blocking group, or a phosphorous derivative;
R11═
R13═
H;
R12 is hydrogen, OH, 3 amino, 3-azido, 3-thiol, 3-unsaturated or a 3-phosphorous derivative; and
R14 is H, OH, or OR3 where R3 is a reactive group, protecting group, or additional fluorophore;
provided that excluded from such structure is any purine-like compound in which; (i) X1═
X4═
C;
X2═
X3═
N;
R4═
NH2;
R5═
R8 which is absent;
R6═
H;
R9 is H or is absent;
R10═
H; and
R12═
R14═
OH;
or(ii) X1═
C;
X2═
X3═
X4═
N;
R4═
NH2 or H;
R5═
R8 which is absent;
R6═
NH2;
R9 is H or is absent;
R10═
H; and
R12═
R14OH;
or(iii) R4 and R5 in combination form an etheno linkage;
R6═
R8═
H;
R9 is absent;
X1═
X3═
C; and
X2═
X4═
N;
or(iv) X1═
X2═
C;
X3═
X4═
N;
R4═
halogen or —
S(CH2)nR with n being an integer between 1-6 and R is lower alkoxy, alkylthio, phenoxy, phenylthio, unsubstituted or substituted phenyl, —
C═
C—
R′
wherein R′
is unsubstituted or mono-, di- or trisubstituted phenyl;
R9═
R10═
H; and
R12═
R14═
acyloxy;
or(v) R4═
NH2 or OH;
R5 is absent;
R9 is —
COOH, —
CONH2, —
C(S)NH2, —
C(NH)N2, or —
C(N—
NH2)NH2;
X1═
X2═
X3═
C; and
X4═
N;
and further provided that R8 is absent if X3═
N and R9 is absent if X2═
N;
and provided that the structure is not a nucleoside which is effectively non-fluorescent under physiological conditions and is selected from the group consisting of adenosine, cytidine, guanine, thymidine, uridine, and inosine. - View Dependent Claims (2)
-
Specification