Carbonic anhydrase inhibitor-tagged nucleic acid probes

Claim 1

A single-stranded nucleic acid probe, of between about 12 to about 10,000 bases in length, which comprises: (I) a terminal nucleotide bound, through (A) the 3'-carbon, if said terminal nucleotide is at the 3'-end of said probe, or (B) the 5'-carbon, if said terminal nucleotide is at the 5'-end of said probe, to a group of formula --OPO 2 (NH) (CH 2 ) 2 R 6 ; or (II) (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 cystosine modified by covalent attachment to the N 4 -nitrogen of a group of formula --N═C(R 2 )--R 8 , --NH--CR 2 H--R 8 or --NH(C═R 3 )NH--R 8 , wherein --R 1 is --CH 2 CHR 5 R 6 , --CH 2 R 5 R 6 , --CH═CR 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 --(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 32 , wherein p is 2 to 20 and R 32 is: ##STR18## wherein X 32 is hydrogen, a halogen or --NO 2 , wherein R 2 is hydrogen or alkyl of 1 to 4 carbon atoms, wherein R 3 is oxygen or sulfur, and wherein R 32 is bound with a reporter group which is an heteropolymer or homopolymer of a carbonic anhydrase, in which said carbonic anhydrase is the conjugate protein and is capable of catalyzing a reaction which yields a detectable signal.

Claim 15

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 modified nucleic acid for the target DNA or RNA, said modified nucleic acid being single-stranded, being between about 12 to about 10,000 bases in length, comprising (a) a nucleotide modified to include a group which comprises a tag moiety that is an aromatic sulfonamide inhibitor of a carbonic anhydrase or (b) 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, provided that the sequence of said modified nucleic acid is such that, and the derivation of single-stranded nucleic acid from said sample and the combining of said single-stranded nucleic acid with said modified nucleic acid are carried out under conditions such that, stable duplexes form between said modified nucleic acid and at least a portion of the target DNA or RNA present in said sample but not significantly between modified nucleic acid and non-target DNA or RNA; and (ii) determining whether stable duplex was formed in step (i) by (a) separating unduplexed modified nucleic acid from duplexed probe formed in step (i); (b) binding the duplexed modified nucleic acid with a reporter group, which is a homopolymer or heteropolymer of a carbonic anhydrase, in which said carbonic anhydrase is the conjugate protein and is capable of catalyzing a reaction which yields a detectable signal, said binding being 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 duplexed modified nucleic acid so treated substantially all unbound 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) as an indicator of the presence of the biological entity in the sample.

Claim 16

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 modified nucleic acid for the target DNA or RNA, said modified nucleic acid being single-stranded, being between about 12 to about 10,000 bases in length, comprising (a) a nucleotide modified to include a group which comprises a tag moiety that is an aromatic sulfonamide inhibitor of a carbonic anhydrase or (b) 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, provided that the sequence of said modified nucleic acid is such that, and the derivation of single-stranded nucleic acid from said sample and the combining of said single-stranded nucleic acid with said modified nucleic acid are carried out under conditions such that, stable duplexes form between said modified nucleic acid and at least a portion of the target DNA or RNA present in said sample but not significantly between modified nucleic acid and non-target DNA or RNA; and (ii) determining whether stable duplex was formed in step (i) by (a) separating unduplexed modified nucleic acid from duplexed probe formed in step (i); (b) binding the duplexed modified nucleic acid with a reporter group, which is a homopolymer or heteropolymer of a carbonic anhydrase, in which said carbonic anhydrase is the conjugate protein and is capable of catalyzing a reaction which yields a detectable signal, said binding being 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 duplexed modified nucleic acid so treated substantially all unbound 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) as an indicator of the presence of the biological entity in the sample.