Polynucleotide hybridization assays employing catalyzed luminescence

Claim 2

A polynucleotide probe as claimed in claim 1 wherein the transformation radical is OH⁻.

Claim 3

A polynucleotide probe as claimed in claim 2 wherein the apoluminescer is selected from activated diacetyldichlorofluorescein, homovanillic acid, p -hydroxyphenylacetic acid, tyrosine, tyramine, luminol and p -hydroxyproprionic acid.

Claim 4

A polynucleotide probe composition comprising two polynucleotide probes of the formulas (X₁---X n ) and (Y₁--Y n ) wherein X₁ through X n ,   which are the same or different, are nucleotide moieties which collectively form a polynucleotide sequence substantially complementary to a single-stranded region of a target polynucleotide analyte A; provided that at least one of nucleotide moieties X₁ through X n comprises a moiety Z where Z comprises a nucleotide having a chemical catalyst attached thereto capable of generation, from a substrate, of a transformation radical which, in turn, is capable of transforming an apoluminescer (as defined in claim 1) into a luminescer; and Y₁ through Y n ,   which are the same or different, are nucleotide moieties which collectively form a polynucleotide sequence substantially complementary to a single-stranded region of polynucleotide analyte A different from, but proximate to, the region to which X₁ through X n is complementary; provided that at least one of nucleotide moieties Y₁ through Y n comprises a moiety Z' where Z' comprises a nucleotide having an apoluminescer attached thereto capable to being transformed into a luminescer by the transformation radical generated by the activity of the chemical catalyst of (X₁---X n ); further provided that (X₁---X n ) and (Y₁---Y n ) are selected such that upon hybridization with A, the catalyst of (X₁---X n ) is sufficiently proximate the apoluminescer of (Y₁---Y n ) to effect the desired transformation of apoluminescer to luminescer upon introduction of the appropriate substrate.

Claim 5

A polynucleotide probe composition as claimed in claim 4 wherein n is an integer from 2 to about 500.

Claim 6

A polynucleotide probe composition as claimed in claim 4 or claim 5 wherein Z comprises a nucleotide having a chemical catalyst attached thereto capable of releasing an (OH⁻) radical from a substrate and Z' comprises a nucleotide having an apoluminescer attached thereto which when reacted with a (OH⁻) radical is converted to a luminescer.

Claim 7

A method for detecting the presence of a target polynucleotide analyte in a sample which comprises: (a) contacting the sample, under hybridization conditions, with a first and a second polynucleotide probe, the first probe having a chemical catalyst attached thereto and the second probe having an apoluminescer (as defined in claim 1) attached thereto, both probes being substantially complementary to substantially mutually exclusive single-stranded regions of the analyte, such that upon hybridization of both probes with the analyte, the chemical catalyst and the apoluminescer are located close enough to each other to permit the catalyst to release from a substrate a transformation radical that subsequently transforms the apoluminescer to a luminescer; (b) adding the substrate for the chemical catalyst, the substrate being capable of conversion to a transformation radical by the catalyst and the said radical being capable of converting the apoluminescer to a luminescer; (c) irradiating the sample with incident light within the absorption spectrum of the luminescer; and (d) measuring the light emitted by the luminescer.

Claim 8

A method as claimed in claim 7 wherein the said first and second polynucleotide probes are between 2 and about 500 nucleotides in length.

Claim 9

A method as claimed in claim 8 wherein the said first and second polynucleotide probes are between 5 and 50 nucleotides in length.

Claim 11

A method as claimed in claim 10 wherein the apoluminescer is as defined in claim 3.

Claim 12

A method as claimed in claim 10 or claim 11 wherein the chemical catalyst is selected from horseradish peroxidase, hematin, EDTA-Fe III complexes and microperoxidase, and the substrate is selected from molecular oxygen, hydrogen peroxide, Methylhydroperoxide, Ethylhydroperoxide, t -butylhydroperoxide, linoleic hydroperoxide, cholesterol 5-hydroperoxide and cumen hydroperoxide.

Claim 13

A kit for detecting the presence of a target polynucleotide analyte in a sample which comprises: (a) a first polynucleotide probe having a chemical catalyst attached thereto and which is substantially complementary to a first single-stranded region of the analyte; and (b) a second polynucleotide probe having an apoluminescer attached thereto which is substantially complementary to a second single-stranded region of the analyte; the second region being substantially mutually exclusive from the first region but close enough thereto such that upon hybridization of the first and second probes with the analyte, the chemical catalyst and the apoluminescer are close enough to each other to permit the catalyst to release from a substrate a transformation radical that subsequently converts the apoluminescer to a luminescer.

Claim 14

A kit as claimed in claim 13 which further comprises a substrate for the chemical catalyst, the substrate being capable of conversion to a transformation radical by the catalyst and the said radical being capable of converting the apoluminescer to a luminescer.

Claim 15

A kit as claimed in claim 13 or 14 wherein said apoluminescer and/or said chemical catalyst is as defined in any of claims 2, 3, 10 or 11.