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EP0210021A2
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EP
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Carbonic anhydrase inhibitor-tagged nucleic acid probes
Assignee: Siska Diagnostics Inc
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Filing: 1986-07-09
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Granted: —
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Expires: 2006-07-09
Abstract
Abstract Nucleic acid hybridization probes are provided which comprise nucleoside bases or terminal nucleotide phosphates chemically linked to aromatic sulfonamide inhibitors of carbonic anhydrase. 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. A probe of the invention is detected by binding to it a reporter group, such as a homopolymer or heteropolymer of enzymes, which includes a carbonic anhydrase which binds to the inhibitor linked to the probe, and then detecting the bound reporter group, as by production of a fluorescent or colored product in a reaction catalyzed by an enzyme component of the reporter group. Also provided are enzyme immunoassays wherein detection of antibody is by a process which comprises a chemical reaction catalyzed by a carbonic anhydrase.
Inventors
- 👤 Emil Thomas Kaiser
- 👤 Gary Fred Musso
- 👤 Soumitra Shankar Ghosh
- 👤 Leslie Eleazer Orgel
- 👤 Geoffrey Myles Wahl
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-30 13:17
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/53; G01N33/531; G01N33/532; G01N33/535; C; C07; C07F; C07F7/00; C07F7/02; C07F7/08; C07F7/18; C07F7/1804; C07H; C07H21/00
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
48 words
A nucleic acid probe which comprises a terminal nucleotide bound through a terminal phosphate to a modifying functional group, or a nucleoside base modified to include a modifying functional group, said modifying functional group comprising a tag moiety that is an aromatic sulfonamide inhibitor of a carbonic anhydrase.
Claim 2
Dependent Claim
48 words
A nucleic acid probe which comprises a terminal nucleotide bound through a terminal phosphate to a modifying functional group, or a nucleoside base modified to include a modifying functional group, said modifying functional group comprising a tag moiety that is an aromatic sulfonamide inhibitor of a carbonic anhydrase.
Claim 3
Dependent Claim
24 words
A nucleic acid probe according to Claim 1 wherein the tag moiety is an aromatic sulfonamide inhibitor of a mammalian erythrocyte carbonic anhydrase B.
Claim 4
Dependent Claim
30 words
A nucleic acid probe according to Claim 1 or claim 2 wherein the tag moiety is selected from and wherein X 3 is hydrogen, a halogen, or -NO 2 .
Claim 5
Dependent Claim
196 words
A nucleic acid probe according to Claim 3 which comprises (i) a cytosine or uracil modified by covalent attachment to position 5 of a group -R 1 , (ii) an adenine or guanine modified by covalent attachment to position 8 of a group -R 1 , or (iii) a cytosine modified by covalent attachment to the N 4 -nitrogen of a group -N=C(R 2 )-R 8 , -NH-CR 2 H-R 8 or -NH(C=R 3 )NH-R 8 , wherein -R l is -CH 2 CHR 5 R 6 , -CH 2 R 5 R 6 , -CH=CR 5 R 6 , -CH=CR5(CO)R6 or -CH=CR5(NHR6), wherein R 5 is hydrogen or alkyl of 1 to 6 carbon atoms and R 6 is -(CH 2 ) m R 7 , wherein m is 0 to 12 and R 7 is -(CO)(NH)R 8 or -(NH)(CO)R 8 , wherein R 8 is -(CH 2 ) p R 4 , wherein p is 2 to 20 and R 4 is wherein X 3 is hydrogen, a halogen or -NO 2 ; wherein R 2 is hydrogen or alkyl of 1 to 4 carbon atoms; and wherein R 3 is oxygen or sulfur.
Claim 6
Dependent Claim
86 words
A nucleic acid probe according to Claim 4 which comprises a cytosine modified by covalent attachment to the N 4- nitrogen of a group -N=C(R 2 )-R 8 , -NH-CR 2 H-R 8 or -NH(C=R 3 )NH-R 8 , wherein R 2 is hydrogen or alkyl of 1 to 4 carbon atoms, R 3 is oxygen or sulfur and R 8 is -(CH2) R 4 , wherein p is 2 to 20 and R 4 is or wherein X 3 is hydrogen, a halogen or -NO2.
Claim 7
Dependent Claim
19 words
A nucleic acid probe according to Claim 5 wherein p is 2 to 8 and R 4 is PABSAT.
Claim 8
Dependent Claim
28 words
7 . A nucleic acid probe according to any one of claims 1 to 6 wherein the probe is between about 12 and about 10,000 bases in length.
Claim 9
Dependent Claim
107 words
A compound of Formula R 10 (CO)R 11 R 12 , wherein R 10 is or wherein X 3 is hydrogen, halogen or -NO 2 ; wherein R 11 is alkyl of 2 to 20 carbon atoms; and wherein R 12 is -(C=O)R 2 , -C0 2 H, -(CHR 2 )OH, -NH 2 or -N=C=R 3 , wherein R 2 is hydrogen or alkyl of 1 to 4 carbon atoms, R 3 is oxygen or sulfur, R 58 and R 59 are the same or different and are each alkyl of 1 to 5 carbon atoms, and R 60 is alkyl of 2 or 3 carbon atoms.
Claim 10
Dependent Claim
107 words
A compound of Formula R 10 (CO)R 11 R 12 , wherein R 10 is or wherein X 3 is hydrogen, halogen or -NO 2 ; wherein R 11 is alkyl of 2 to 20 carbon atoms; and wherein R 12 is -(C=O)R 2 , -C0 2 H, -(CHR 2 )OH, -NH 2 or -N=C=R 3 , wherein R 2 is hydrogen or alkyl of 1 to 4 carbon atoms, R 3 is oxygen or sulfur, R 58 and R 59 are the same or different and are each alkyl of 1 to 5 carbon atoms, and R 60 is alkyl of 2 or 3 carbon atoms.
Claim 11
Dependent Claim
21 words
A compound according to Claim 8 wherein R 11 is n-alkyl of 2 to 8 carbon atoms, and R 12 is
Claim 12
Dependent Claim
23 words
A compound according to claim 8 or claim 9 wherein R 11 is - CHO or -NH 2 and R 10 is PABSAT.
Claim 13
Dependent Claim
89 words
A process for making a nucleic acid probe which comprises a modified cytosine moiety of Formula LXI wherein R 10 is or wherein X 3 is hydrogen, a halogen or -NO 2 ; and wherein R 11 is alkyl of 2 to 20 carbon atoms, 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 LXII wherein R 10 and R 11 are as in the compound of Formula LXI.
Claim 14
Dependent Claim
89 words
A process for making a nucleic acid probe which comprises a modified cytosine moiety of Formula LXI wherein R 10 is or wherein X 3 is hydrogen, a halogen or -NO 2 ; and wherein R 11 is alkyl of 2 to 20 carbon atoms, 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 LXII wherein R 10 and R 11 are as in the compound of Formula LXI.
Claim 15
Dependent Claim
34 words
A process according to Claim 11 wherein the nucleic acid has between about 12 and about 10,000 bases and between about 10% and about 60% of its cytosines are modified to N 4 -aminocytosines.
Claim 16
Dependent Claim
25 words
A process according to claim 11 or claim 12 wherein R 11 is n-alkyl of 2 to 8 carbon atoms and R 10 is PABSAT.
Claim 17
Dependent Claim
306 words
A process for making a nucleic acid probe which comprises (i) a cytosine or uracil modified by covalent attachement to position 5 of a group -R 1 or (ii) an adenine or guanine modified by covalent attachment to position 8 of a group -R 1 , wherein -R l is -CH 2 CHR 5 R 6 , -CH=CR 5 R 6 , -CHR 5 R 6 , -CH=CR 5 (CO)R 6 or -CH=CR 5 (NHR 6 ), wherein R 5 is hydrogen or alkyl of 1 to 6 carbon atoms and R 6 is -(CH2)mR7, wherein m is 0 to 12 and R 7 is -(CO)(NH)R 8 or -(NH)(CO)R 8 , wherein R 8 is -(CH 2 ) p R 4 , wherein p is 2 to 20 and R 4 is or wherein X 3 is hydrogen, a halogen or -NO 2 , which process comprises reacting a quantity of the nucleic acid with the same sequence as the probe and comprising (i) a cytosine or uracil modified by covalent attachment to position 5 of a group -R 14 or (ii) an adenine or guanine modified by covalent attachment to position 8 of a group -R 14 , wherein R 14 is -CH 2 CHR 5 (CH 2 ) m R 141 , -CH=CR 5 (CH 2 ) m R 141 , -CHR 5 (CH 2 ) m R 141 , -CH-CR 5 (CO)(CH 2 ) m R 141 or -CH=CR 5 (NH(CH 2 ) m R 141 ), with a compound of Formula R 143 (CH 2 ) p R 4 , wherein R 141 is -NH 2 , -CO 2 H, or and R 143 is NH 2 , if R 141 is -CO 2 H or , or - CHO or - C02H , if R 141 is - NH 2 .
Claim 18
Dependent Claim
306 words
A process for making a nucleic acid probe which comprises (i) a cytosine or uracil modified by covalent attachement to position 5 of a group -R 1 or (ii) an adenine or guanine modified by covalent attachment to position 8 of a group -R 1 , wherein -R l is -CH 2 CHR 5 R 6 , -CH=CR 5 R 6 , -CHR 5 R 6 , -CH=CR 5 (CO)R 6 or -CH=CR 5 (NHR 6 ), wherein R 5 is hydrogen or alkyl of 1 to 6 carbon atoms and R 6 is -(CH2)mR7, wherein m is 0 to 12 and R 7 is -(CO)(NH)R 8 or -(NH)(CO)R 8 , wherein R 8 is -(CH 2 ) p R 4 , wherein p is 2 to 20 and R 4 is or wherein X 3 is hydrogen, a halogen or -NO 2 , which process comprises reacting a quantity of the nucleic acid with the same sequence as the probe and comprising (i) a cytosine or uracil modified by covalent attachment to position 5 of a group -R 14 or (ii) an adenine or guanine modified by covalent attachment to position 8 of a group -R 14 , wherein R 14 is -CH 2 CHR 5 (CH 2 ) m R 141 , -CH=CR 5 (CH 2 ) m R 141 , -CHR 5 (CH 2 ) m R 141 , -CH-CR 5 (CO)(CH 2 ) m R 141 or -CH=CR 5 (NH(CH 2 ) m R 141 ), with a compound of Formula R 143 (CH 2 ) p R 4 , wherein R 141 is -NH 2 , -CO 2 H, or and R 143 is NH 2 , if R 141 is -CO 2 H or , or - CHO or - C02H , if R 141 is - NH 2 .
Claim 19
Dependent Claim
46 words
A process according to Claim 14 wherein the nucleic acid is between about 12 and about 100 bases; and/or wherein R 14 is -CH=CH-(CH 2 ) j NH 2 , wherein j is 1 to 4;and/or wherein p is 2 to 8;and/brwherein R 4 is PABSAT.
Claim 20
Independent Claim
294 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 nucleoside modified to include a group which comprises a tag moiety that is an aromatic sulfonamide inhibitor of a carbonic anhydrase or comprising a terminal nucleotide bound through a terminal phosphate to a group which comprises a tag moiety that is an aromatic sulfonamide inhibitor of a carbonic anhydrase, said probe being optionally as defined in any one of claims 1 to 7 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) combining the duplexed probe with a reporter group, which includes as conjugate protein to said tag moiety a carbonic anhydrase that is inhibited by 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 step (ii)(b), to produce a signal from any of said reporter group that is present; and (d) determining whether a detectable signal is generated by the treatment of step (ii)(c).
Claim 21
Dependent Claim
40 words
A method according to Claim 16 wherein the tag moiety is an aromatic sulfonamide inhibitor of a mammalian erythrocyte carbonic anhydrase B and the reporter group includes as conjugate protein to the tag moiety a mammalian erythrocyte carbonic anhydrase B.
Claim 22
Dependent Claim
40 words
A method according to claim 17 wherein, in the probe, the group covalently attached to the N 4- nitrogen has the formula -N=CH(CH 2 ) p R 4 , wherein p is 2 and 8 and R 4 is PABSAT.
Claim 23
Dependent Claim
28 words
An EIA, preferably an ELISA, wherein antibody is detected by a process which comprises a reaction catalyzed by a carbonic anhydrase, e.g. a mammalian erythrocyte carbonic anydrase B.
Claim 24
Dependent Claim
28 words
An EIA, preferably an ELISA, wherein antibody is detected by a process which comprises a reaction catalyzed by a carbonic anhydrase, e.g. a mammalian erythrocyte carbonic anydrase B.
Claim 25
Dependent Claim
24 words
The use of a probe as claimed in any one of claims 1 to 7 in a hybridisation assay for target DNA or RNA.
References Cited (Backward Citations)
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