Fluorescent universal nucleic acid end label
First Claim
1. A 5'"'"' universal end label for a nucleic acid sequence, wherein said label comprises a 5'"'"' homopolymer, said homopolymer comprising a fluorescent nucleoside analog not capable of forming hydrogen-bonded Watson-Crick base pairs, and a sequence complementary to a promoter region on a nucleic acid sequence, wherein said fluorescent nucleoside analog has the structure:
- ##STR3## wherein X1 ═
X6 ═
C or Si;
X2, X3, X4, X5 ═
C, S, N or Si;
provided that at least one of X1, X2, X3, X4, X5, or X6 ═
N;
X7 ═
--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 absent, H, or is part of an etheno linkage with R4 ;
R6 is H, NH2, SH, or ═
O;
R8 and R9 are independently absent, hydrogen, methyl, bromine, fluorine, or iodine;
an alkyl or aromatic substituent, 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 phosphorus derivative;
R11 ═
R13 ═
H;
R12 is hydrogen, OH, 3'"'"' amino, 3'"'"'-azido, 3'"'"'-thiol, 3'"'"'-unsaturated or a 3'"'"'-phosphorus derivative; and
R14 is H, OH, or OR3 where R3 is a reactive group, protecting group, or additional fluorophore;
provided that, excluded from such compound is any purine-like compound in which;
(i) X1═
X4═
C;
X2═
X3═
N;
R4═
NH2;
R5═
R8 which is absent;
R6═
H;
R9is H or absent;
R10═
H; and
R12═
R14═
OH;
or(ii) X1═
C;
X2═
X3═
X4═
N;
R4═
NH2or H;
R5═
R8 which is absent;
R6═
NH2;
R9 is H or is absent;
R10═
H; and
R12═
R14═
OH;
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)n R, 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 on-, 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)NH2, or --C(N-- NH2)NH2;
X1═
X2═
X3═
C; and
X4═
N.
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Accused Products
Abstract
Structural analogs of the six non-fluorescent N-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.
66 Citations
6 Claims
-
1. A 5'"'"' universal end label for a nucleic acid sequence, wherein said label comprises a 5'"'"' homopolymer, said homopolymer comprising a fluorescent nucleoside analog not capable of forming hydrogen-bonded Watson-Crick base pairs, and a sequence complementary to a promoter region on a nucleic acid sequence, wherein said fluorescent nucleoside analog has the structure:
- ##STR3## wherein X1 ═
X6 ═
C or Si;X2, X3, X4, X5 ═
C, S, N or Si;provided that at least one of X1, X2, X3, X4, X5, or X6 ═
N;X7 ═
--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 absent, H, or is part of an etheno linkage with R4 ; R6 is H, NH2, SH, or ═
O;R8 and R9 are independently absent, hydrogen, methyl, bromine, fluorine, or iodine;
an alkyl or aromatic substituent, 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 phosphorus derivative; R11 ═
R13 ═
H;R12 is hydrogen, OH, 3'"'"' amino, 3'"'"'-azido, 3'"'"'-thiol, 3'"'"'-unsaturated or a 3'"'"'-phosphorus derivative; and R14 is H, OH, or OR3 where R3 is a reactive group, protecting group, or additional fluorophore; provided that, excluded from such compound is any purine-like compound in which; (i) X1═
X4═
C;
X2═
X3═
N;
R4═
NH2;
R5═
R8 which is absent;
R6═
H;
R9is H or absent;
R10═
H; and
R12═
R14═
OH;
or(ii) X1═
C;
X2═
X3═
X4═
N;
R4═
NH2or H;
R5═
R8 which is absent;
R6═
NH2;
R9 is H or is absent;
R10═
H; and
R12═
R14═
OH;
or(iii) R4 and R5 in combination form an etheno linkage;
R6═
R8═
H;
R9 is absent;
X1═
X3═
C; andX2═
X4═
N;
or(iv) X1═
X2═
C;
X3═
X4═
N;
R4═
halogen or --S(CH2)n R, 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 on-, 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)NH2, or --C(N-- NH2)NH2;
X1═
X2═
X3═
C; and
X4═
N. - View Dependent Claims (2, 3, 4, 5, 6)
- ##STR3## wherein X1 ═
Specification