EP0209996A2
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EP
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Nucleic acid probes comprising N4 -(substituted amino)cytidines
Assignee: Siska Diagnostics Inc
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Filing: 1986-06-25
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Granted: —
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Expires: 2006-06-25
Abstract
Abstract Nucleic acid hybridization probes are provided which comprise an N 4 -(substituted amino) cytosine moiety, wherein the substituted amino group comprises a tag moiety, whereby the probe is detected. Methods of preparing probes of the Invention, intermediates used in such methods, and methods of using the probes of the invention in hybridization assays are also provided. Typical tag moieties employed with the probes of the invention are biotinyl, aminothiadiazole and fluorescein derivatives, connected to N 4 -amino groups of modified cytosines of the probe through linker moieties.
Inventors
- 👤 Gary Fred Musso
- 👤 Soumitra Shankar Ghosh
- 👤 Leslie Eleazer Orgel
- 👤 Geoffrey Myles Wahl
- 👤 Emil Thomas Kaiser
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-29 16:39
CPC Classifications
C; C07; C07H; C07H21/00; C07F; C07F7/00; C07F7/02; C07F7/08; C07F7/18; C07F7/1804; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816
Claims Analysis
Total Claims: 34
Claim 1
Independent Claim
50 words
A nucleic acid probe which comprises a modified cytosine moiety of Formula I wherein R 1 is or wherein R 2 is hydrogen or alkyl of 1 to 4 carbon atoms; R 3 is sulfur or oxygen; R 5 is a linker moiety; and R 6 is a tag moiety.
Claim 2
Dependent Claim
50 words
A nucleic acid probe which comprises a modified cytosine moiety of Formula I wherein R 1 is or wherein R 2 is hydrogen or alkyl of 1 to 4 carbon atoms; R 3 is sulfur or oxygen; R 5 is a linker moiety; and R 6 is a tag moiety.
Claim 3
Dependent Claim
10 words
A probe according to Claim 1 which is a polydeoxyribonucleotide.
Claim 4
Dependent Claim
10 words
A probe according to Claim 1 which is a polyribonucleotide.
Claim 5
Dependent Claim
74 words
A probe according to any one of claims 1 to 3 wherein R 1 is -N=C(H)-R 15 -R 16 , wherein -R 16 is biotinyl, iminobiotinyl or sulfenylbiotinyl and -R 15 - is -(CH 2 ) n -R 151 , wherein -R 151 - is -NH- or -NH-N=C(H)- and is bonded to -R 16 through -NH-; wherein n is 2 to 20; and wherein the probe has between about 12 and about 10,000 bases.
Claim 6
Dependent Claim
21 words
A probe according to Claim 4 wherein -R 16 is biotinyl, -R 151 is -NH- and n is 5 to 8.
Claim 7
Dependent Claim
76 words
A probe according to any one of claims 1 to 3 wherein R 1 is -N=CH-R 25 -R 26 wherein -R 26 is PABSAT, aminothiadiasole or sulfanilamide bonded to R 25 through the amino group; wherein -R 25 - is -(CH 2 ) p CO-, wherein -(CH 2 ) p is bonded to -N=C(H)- and p is 2 to 20 preferably 5 to 8; and wherein the probe has between about 12 and about 10,000 bases.
Claim 8
Dependent Claim
75 words
A probe according to any of claims 1 to 3 wherein R 1 is -N=CH-R 35 -R 36 , wherein R 36 is fluorescein, tetramethylrhodamine or tetraethylrbodamine; wherein -R 35 - is -(CH 2 ) q (NH) (C-R 37 ) (NH)-, wherein -(CH 2 ) q - is bonded to -N=CH-, q is 2 to 20, and R 37 is sulfur or oxygen; and wherein the probe has between about 12 and about 10,000 bases.
Claim 9
Dependent Claim
17 words
A probe according to Claim 7. wherein q is 5 to 8 and R 37 is sulfur.
Claim 10
Independent Claim
285 words
A method for testing a sample for the presence of a biological entity, associated with a target DNA or RNA, which comprises: (i) combining single-stranded nucleic acid of the sample with a nucleic acid probe for the target DNA or RNA, said probe comprising a modified cytosine moiety of Formula I wherein R 1 is or wherein R 2 is hydrogen or alkyl of 1 to 4 carbon atoms; R 3 is sulfur or oxygen; R 5 is a linker moiety; and R 6 is a tag moiety, said probe being optionally as defined in any one of claims 1 to 8 provided that the derivation of single-stranded nucleic acid from said sample and the combining of said single-stranded nucleic acid with said probe are carried out under conditions whereby stable duplexes form between probe and at least a portion of the target DNA or RNA present in said sample but not significantly between probe and non-target DNA or RNA; and (ii) determining whether stable duplex was formed in step (i) by (a) separating unduplexed probe from duplexed probe formed in step (i); (b) if detectability of the tag moiety is provided by binding thereto a reporter group, combining the duplexed probe with a reporter group, conjugate to the tag moiety, under conditions whereby the reporter group binds to at least a portion of any of said tag moiety that is present, and separating from the product so treated substantially all uncomplexed reporter group; (c) treating the product of step (i), after treatment according to step (ii)(a) and, if used, step (ii)(b), to produce a signal from any of said tag moiety or, if step (ii) (b) is used, reporter group that is present; and
Claim 11
Dependent Claim
185 words
A method according to claim 9 wherein (a) when the probe is as defined in claim 4 or claim 5 the single-stranded nucleic acid of the sample is affixed to a solid support e.g. nitro-cellulose prior to combination with probe; and wherein detectability of the biotinyl, iminobiotinyl or sulfenylbiotinyl is provided by binding thereto a reporter group which includes as conjugate protein avidin or streptavidin. (b) when the probe is as defined in claim 6 the single-stranded nucleic acid of the sample is affixed to a solid support, e.g. nitro-cellulose, prior to combination with probe and wherein the detectability of the PABSAT, aminothiadiazole or sulfanilamide is provided by binding thereto a reporter group which includes as conjugate protein carbonic anhydrase, e.g. a mammalian erythrocyte carbonic anhydrase B. (c) when the probe is as defined in claim 7 or claim 8 the single-stranded nucleic acid of the sample is affixed to a solid support prior to combination with probe; and wherein any fluorescein, tetramethylrhodamine or tetraethylrhodamine tag moiety that is present is not bound by a reporter group and is detected by a fluorescence spectroscopic method.
Claim 12
Dependent Claim
144 words
A compound of Formula LII wherein -R 51 - is -NH- or -NH-N-C(R 511 )-, wherein the amino group is bonded to the carbonyl group and wherein R 511 ia hydrogen or alkyl of 1 to 4 carbon atoms; wherein R 52 is -CO 2 R 53 , -(CO)R 54 , wherein R 53 is alkyl of 1-5 carbon atoms; wherein R 54 is hydrogen, alkyl of 1-5 carbon atoms or chloro; wherein R 55 , R56 and R 57 are the same or different and are each alkyl of 1-6 carbon atoms; wherein R 58 and R 59 are the aame or different and are each alkyl of 1-5 carbon atoms; wherein R 60 is alkyl of 2 or 3 carbon atoms; wherein m is 2 to 20; wherein X 50 is S or S-0: and wherein X 52 is O or NH.
Claim 13
Dependent Claim
144 words
A compound of Formula LII wherein -R 51 - is -NH- or -NH-N-C(R 511 )-, wherein the amino group is bonded to the carbonyl group and wherein R 511 ia hydrogen or alkyl of 1 to 4 carbon atoms; wherein R 52 is -CO 2 R 53 , -(CO)R 54 , wherein R 53 is alkyl of 1-5 carbon atoms; wherein R 54 is hydrogen, alkyl of 1-5 carbon atoms or chloro; wherein R 55 , R56 and R 57 are the same or different and are each alkyl of 1-6 carbon atoms; wherein R 58 and R 59 are the aame or different and are each alkyl of 1-5 carbon atoms; wherein R 60 is alkyl of 2 or 3 carbon atoms; wherein m is 2 to 20; wherein X 50 is S or S-0: and wherein X 52 is O or NH.
Claim 14
Dependent Claim
32 words
A compound according to claim 11 wherein R 52 is -CHO, -COCl, R 51 is -NH- and/or X 50 is S and X 52 is 0 and/or m is 5 to 8.
Claim 15
Dependent Claim
44 words
A compound of Formula XXXV: wherein R 32 is or Wherein X 32 is hydrogen, halogen or -NO 2 ; wherein R 34 is -CHO, or wherein R 60 is alkyl of 2 or 3 carbon atoms; and wherein k is 2 to 20.
Claim 16
Dependent Claim
44 words
A compound of Formula XXXV: wherein R 32 is or Wherein X 32 is hydrogen, halogen or -NO 2 ; wherein R 34 is -CHO, or wherein R 60 is alkyl of 2 or 3 carbon atoms; and wherein k is 2 to 20.
Claim 17
Dependent Claim
15 words
14 . A compound according to Claim 13 wherein k is 5 to 8 and/or
Claim 18
Dependent Claim
42 words
A compound of Formula XLVII wherein R 45 is fluorescein, tetramethylrhodamine or tetraethylrhodamine; R 46 is oxygen or sulfur; R 48 is -CHO or wherein R 60 is alkyl of 2 or 3 carbon atoms; and wherein j is 2 to 20.
Claim 19
Dependent Claim
42 words
A compound of Formula XLVII wherein R 45 is fluorescein, tetramethylrhodamine or tetraethylrhodamine; R 46 is oxygen or sulfur; R 48 is -CHO or wherein R 60 is alkyl of 2 or 3 carbon atoms; and wherein j is 2 to 20.
Claim 20
Dependent Claim
17 words
A compound according to Claim 15 wherein j is 5 to 8 and/or R 46 is sulfur.
Claim 21
Dependent Claim
120 words
17 .. A process for making a nucleic acid probe, which comprises a modified cytosine moiety of Formula LIX wherein X 50 is S or S-0; wherein X 52 is O or NH, wherein -R 51 - is -NH- or -NH-N=C(R 511 )-, wherein the amino group is bonded to the carbonyl group and wherein R 511 is hydrogen or alkyl of 1 to 4 carbon atoms; and wherein m is 2 to 20, which process comprises reacting a nucleic acid, with the same sequence as the probe and comprising an N 4 -aminocytosine moiety, with a compound of Formula LX wherein X 50 , X 52 , R 51 and m are as in the compound of Formula LIX.
Claim 22
Dependent Claim
120 words
17 .. A process for making a nucleic acid probe, which comprises a modified cytosine moiety of Formula LIX wherein X 50 is S or S-0; wherein X 52 is O or NH, wherein -R 51 - is -NH- or -NH-N=C(R 511 )-, wherein the amino group is bonded to the carbonyl group and wherein R 511 is hydrogen or alkyl of 1 to 4 carbon atoms; and wherein m is 2 to 20, which process comprises reacting a nucleic acid, with the same sequence as the probe and comprising an N 4 -aminocytosine moiety, with a compound of Formula LX wherein X 50 , X 52 , R 51 and m are as in the compound of Formula LIX.
Claim 23
Dependent Claim
83 words
18 . A process for making a nucleic acid probe which comprises a modified cytosine moiety of Formula LVII wherein R 32 is or wherein X32 is hydrogen, halogen or -NO 2 ; and wherein k is 2 to 20, which process comprises reacting a quantity of the nucleic acid with the same sequence as the probe and comprising an N 4 -aminocytosine moiety with a compound of Formula XXXIV wherein R 32 and k are as in the compound of Formula LVII.
Claim 24
Dependent Claim
83 words
18 . A process for making a nucleic acid probe which comprises a modified cytosine moiety of Formula LVII wherein R 32 is or wherein X32 is hydrogen, halogen or -NO 2 ; and wherein k is 2 to 20, which process comprises reacting a quantity of the nucleic acid with the same sequence as the probe and comprising an N 4 -aminocytosine moiety with a compound of Formula XXXIV wherein R 32 and k are as in the compound of Formula LVII.
Claim 25
Dependent Claim
85 words
A process for making a nucleic acid probe which comprises a modified cytosine moiety of Formula LXII wherein R 45 is fluorescein, tetramethylrhodamine or tetraethylrhodamine; wherein R 46 is oxygen or sulfur; and wherein q is 2 to 20, which process comprises reacting a quantity of the nucleic acid with the same sequence as the probe and comprising an N 4- aminocytosine moiety with a compound of Formula LXIII wherein R 45 , R 46 and q are as in the compound of Formula LXII.
Claim 26
Dependent Claim
85 words
A process for making a nucleic acid probe which comprises a modified cytosine moiety of Formula LXII wherein R 45 is fluorescein, tetramethylrhodamine or tetraethylrhodamine; wherein R 46 is oxygen or sulfur; and wherein q is 2 to 20, which process comprises reacting a quantity of the nucleic acid with the same sequence as the probe and comprising an N 4- aminocytosine moiety with a compound of Formula LXIII wherein R 45 , R 46 and q are as in the compound of Formula LXII.
Claim 27
Dependent Claim
43 words
A process according to any one of claims 17 to 19 wherein the nucleic acid is an RNA or DNA with between about 12 and about 10,000 bases and with between about 10% and about 60% of its cytosines modified N 4 aminocytosines.
Claim 28
Dependent Claim
35 words
A process according to claims 17 or claim 20 when related to claim 17 wherein R 51 is -NH- and/or X 50 is S and X 52 is 0 and/or m is 5 to 8.
Claim 29
Dependent Claim
24 words
A process according to claim 18 or claim 20 when related to claim 18 wherein R 32 is and/or k is 5 to 8.
Claim 30
Dependent Claim
25 words
A process according to claim 19 or claim 20 when related to claim 18 wherein R 46 is sulfur and q is 5 to 8.
Claim 31
Dependent Claim
57 words
A process for preparing a probe according to claim 1 which comprises reacting a nucleic acid of the same sequence as the desired probe and comprising an N 4 -aminocytosine moiety with (a) a tag moiety of formula optionally followed by reduction of the hydrozone linkage to a hydrazine linkage or, (b) a tag moiety of formula
Claim 32
Dependent Claim
35 words
A process for preparing a compound of formula R 3 =C=N-R 5 -R 6 which comprises preparing an amino-derivatized compound of formula H 2 N-R 5 -R 6 followed by reaction with thiophosgene or phosgene.
Claim 33
Dependent Claim
35 words
A process for preparing a compound of formula R 3 =C=N-R 5 -R 6 which comprises preparing an amino-derivatized compound of formula H 2 N-R 5 -R 6 followed by reaction with thiophosgene or phosgene.
Claim 34
Dependent Claim
24 words
The use of a probe as claimed in any one of claims 1 to 8 in a hybridization assay for target RNA or DNA.
References Cited (Backward Citations)
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