Nucleic acid probes and methods

• US 5,707,813 A
• Filed: 09/06/1996
• Issued: 01/13/1998
• Est. Priority Date: 05/15/1990
• Status: Expired due to Term

• Current Assignee: Diatron Corporation
• Original Assignee: Diatron Corporation
• Inventors: Devlin, Robert F., Dandliker, Walter B.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/709,285
• Time in Patent Office: 494 Days
• Field of Search: 435/5, 435/6, 435/91.2, 935/77, 935/78
• US Class Current: 435/6.13
• CPC Class Codes: A61K 41/0057 Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
• CPC Class Codes: A61K 41/0071 PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 49/0015 Phosphorescence
• CPC Class Codes: A61K 49/0021 the fluorescent group being a small organic molecule
• CPC Class Codes: A61K 49/0036 Porphyrins used in photodynamic therapy A61K41/0071 or A61K41/0076; used as targeting group or modifying agent for targeting a therapeutic compound A61K47/546
• CPC Class Codes: A61K 49/0054 Macromolecular compounds, i.e. oligomers, polymers, dendrimers
• CPC Class Codes: C07D 487/22 in which the condensed system contains four or more hetero rings
• CPC Class Codes: C07J 43/003 not condensed
• CPC Class Codes: C07J 51/00 Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
• CPC Class Codes: C09B 47/00 Porphines; Azaporphines non-dyeing compounds C07D487/22
• CPC Class Codes: C09B 47/045 Special non-pigmentary uses, e.g. catalyst, photosensitisers of phthalocyanine dyes or pigments
• CPC Class Codes: C09B 47/073 Preparation from isoindolenines , e.g. pyrrolenines
• CPC Class Codes: C09B 47/08 Preparation from other phthalocyanine compounds , e.g. cobaltphthalocyanineamine complex
• CPC Class Codes: C09B 47/24 Obtaining compounds having —COOH or —SO3H radicals, or derivatives thereof, directly bound to the phthalocyanine radical
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 2333/19 Rubella virus
• CPC Class Codes: G01N 33/533 : with fluorescent label
• CPC Class Codes: G01N 33/582 : with fluorescent label

Abstract

The present invention is directed to compositions comprising an oligonucleotide linked to a detectably labeled marker component comprising a fluorophore moiety which comprises a substantially planar, multidentate macrocyclic ligand coordinated to a central atom capable of coordinating with two axial ligands and two polyoxyhydrocarbyl moieties which are attached as axial ligands to the central atom. The present invention is also directed to nucleic acid hybridization and amplification methods employing such compositions.

Claims

1. A method for preparing a marker component-conjugated oligonucleotide comprising the steps of:

a) reacting an oligonucleotide having an attached linker terminating in an amino group with an N-hydroxysuccinimide ester or in an imidazolide of a marker component a composition comprising an oligonucleotide linked to a detectably labeled marker component which comprises a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of the planar molecular structure, to form a conjugate wherein said solubilizing polyoxyhydrocarbyl moieties are selected from a polyether, a polyol, or a water soluble polymer, and wherein said fluorophore moiety is a porphyrin derivative or an azaporphyrin derivative wherein one or more bridging carbon atoms has been replaced by nitrogen; and

b) separating the conjugate formed in step (a) from unreacted oligonucleotide and from unreacted marker component.

3. The method of claim 1 or 2 further comprising performing before step (a), the step of:

attaching to an oligonucleotide a linker terminating in an amino group.

7. A method for determining the presence or amount of a target nucleic acid sequence in a sample comprising the steps of:

a) contacting sample nucleic acid with a composition of claim 1 wherein said composition is hybridizing with said target nucleic acid sequence in homogenous solution; and

b) detecting the presence or amount of such hybridization by transient state polarized fluorescence.

9. A method for detection of a target nucleic acid sequence in a sample comprising the steps of:

a) contacting a sample suspected of containing a target nucleic acid sequence with a complementary oligonucleotide hybridizing with said target sequence;

b) contacting said sample with a composition of claim 1 wherein said composition is hybridizing to said complementary oligonucleotide or polynucleotide; and

c) detecting the presence or measuring the amount of hybridization of said conjugate with said complementary oligonucleotide or polynucleotide.

2. A method for preparing a dye-conjugated oligonucleotide comprising the steps of:

a) reacting an oligonucleotide having an attached linker terminating in an amino group with an N-hydroxysuccinimide ester or with an imidazolide of the dye shown in FIG. 1, to form a conjugate; and

b) separating the conjugate formed in step (a) from unreacted oligonucleotide and from unreacted dye.

4. A method for preparing marker component-conjugated oligonucleotide comprising the steps of:

a) reacting a marker component a composition comprising an oligonucleotide linked to a detectably labeled marker component which comprises a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of the planar molecular structure, with a carbodiimide in the presence of hydrobenzotriole and in the presence of an oligonucleotide, to form a conjugate wherein said solubilizing polyoxyhydrocarbyl moieties are selected from a polyether, a polyol, or a water soluble polymer, and wherein said fluorophore moiety is a porphyrin derivative or an azaporphyrin derivative wherein one or more bridging carbon atoms has been replaced by nitrogen; and

b) separating the resulting conjugate formed in step (a) from other components of the reaction mixture.

5. A method for preparing dye-conjugated oligonucleotide comprising the steps of:

a) reacting the dye having the structure depicted in FIG. 1 with a carbodiimide in the presence of hydroxybenzotriazole and in the presence of an oligonucleotide having an amino linker to form a conjugate wherein said solubilizing polyoxyhydrocarbyl moieties are selected from a polyether, a polyol, or a water soluble polymer, and wherein said fluorophore moiety is a porphyrin derivative or an azaporphyrin derivative wherein one or more bridging carbon atoms has been replaced by nitrogen; and

b) separating the resulting conjugate formed in step (a) from other components of the reaction mixture.

6. A method for determining the presence or amount of a target nucleic acid sequence in a sample comprising the steps of:

a) contacting sample nucleic acid with oligonucleotide-caged dicarboxy silicon phthalocyanine dye conjugate of FIG. 2 hybridizing with said target nucleic acid sequence in homogenous solution; and

b) detecting the presence or amount of such hybridization by transient state polarized fluorescence.

10. A method according to either one of claims 6-9 wherein said target nucleic acid sequence is a product of nucleic acid amplification.

11. A method according to either one of claims 6-9 wherein said target nucleic acid sequence is DNA.

12. A method according to either one of claims 6-9 wherein said target nucleic acid sequence is RNA.

13. A method according to either one of claims 6-9 wherein said nucleic acid amplification is performed by polymerase chain reaction (PCR).

14. A method according to either one of claims 6-9 wherein said nucleic acid amplification is performed by ligand chain reaction (LCR).

15. A method according to either one of claims 6-9 wherein said nucleic acid amplification is performed by self-sustained sequence replication (3SR).

16. A method according to either one of claims 6-9 wherein said nucleic acid amplification is performed by transcription-based amplification system (TAS).

8. A method for determination of the presence or amount of a target nucleic acid sequence in a sample comprising the steps of:

a) contacting a sample suspected of containing a target nucleic acid sequence with a complementary oligonucleotide hybridizing with said target sequence;

b) contacting said sample with an oligonucleotide-caged dicarboxy silicon phthalocyanine dye conjugate of FIG. 2 hybridizing to said complementary oligonucleotide; and

c) detecting the presence or measuring the amount of hybridization of said conjugate with said complementary oligonucleotide.

17. An improved nucleic acid amplification process, wherein the improvement comprising employing as a label a fluorescent probe which comprises a detectably labeled marker component which comprises a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of the planar molecular structure wherein said solubilizing polyoxyhydrocarbyl moieties are selected from a polyether, a polyol, or a water soluble polymer, and wherein said fluorophore moiety is a porphyrin derivative or an azaporphyrin derivative wherein one or more bridging carbon atoms has been replaced by nitrogen.

18. An improved nucleic acid amplification hybridization process, wherein the improvement comprising employing as a label a fluorescent probe which comprises a detectably labeled marker component which comprises a fluorophore moiety comprising a luminescent substantially planar molecular structure coupled to two solubilizing polyoxyhydrocarbyl moieties, one located on either side of the planar molecular structure wherein said solubilizing polyoxyhydrocarbyl moieties are selected from a polyether, a polyol, or a water soluble polymer, and wherein said fluorophore moiety is a porphyrin derivative or an azaporphyrin derivative wherein one or more bridging carbon atoms has been replaced by nitrogen.