Chromophore probe for detection of nucleic acid

• US 5,595,726 A
• Filed: 06/07/1995
• Issued: 01/21/1997
• Est. Priority Date: 01/21/1992
• Status: Expired due to Fees

• Current Assignee: Pharmacyclics Incorporated (Abbvie Incorporated), Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Pharmacyclics Incorporated (Abbvie Incorporated), Board of Trustees University of TX Systems
• Inventors: Iverson, Brent, Sessler, Jonathan L., Magda, Darren
• Primary Examiner(s): Raymond, Richard L.
• Application Number:
  US08/486,311
• Time in Patent Office: 594 Days
• Field of Search: 540/145, 540/465, 540/472, 424/1.73, 424/9.6, 424/9.61, 436/98, 436/172, 436/800, 436/905, 435/6
• US Class Current: 424/9.61
• CPC Class Codes: A61K 41/0038 Radiosensitizing, i.e. administration of pharmaceutical agents that enhance the effect of radiotherapy radiotherapy per se A61N5/10
• CPC Class Codes: A61K 41/0076 PDT with expanded (metallo)porphyrins, i.e. having more than 20 ring atoms, e.g. texaphyrins, sapphyrins, hexaphyrins, pentaphyrins, porphocyanines
• CPC Class Codes: A61K 47/54 the modifying agent being an organic compound
• CPC Class Codes: A61K 47/546 Porphyrines; Porphyrine with an expanded ring system, e.g. texaphyrine
• CPC Class Codes: A61K 49/0021 the fluorescent group being a small organic molecule
• CPC Class Codes: A61K 49/0052 Small organic molecules oligomers, polymers, dendrimers A61K49/0054
• CPC Class Codes: A61K 49/0054 Macromolecular compounds, i.e. oligomers, polymers, dendrimers
• CPC Class Codes: A61K 49/06 Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
• CPC Class Codes: A61K 49/085 conjugated systems
• CPC Class Codes: A61K 49/10 Organic compounds
• CPC Class Codes: A61K 51/0485 Porphyrins, texaphyrins wherein the nitrogen atoms forming the central ring system complex the radioactive metal
• CPC Class Codes: A61L 2/0011 using physical methods
• CPC Class Codes: B01J 2531/025 Ligands with a porphyrin ring system or analogues thereof, e.g. phthalocyanines, corroles
• CPC Class Codes: B01J 2531/38 Lanthanides other than lanthanum
• CPC Class Codes: B01J 31/061 Chiral polymers
• CPC Class Codes: B01J 31/1658 immobilised by covalent linkages, i.e. pendant complexes with optional linking groups, e.g. on Wang or Merrifield resins
• CPC Class Codes: B01J 31/1815 with more than one complexing nitrogen atom, e.g. bipyridyl, 2-aminopyridine
• CPC Class Codes: C07D 487/22 in which the condensed system contains four or more hetero rings
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/15 : Nucleic acids forming more than 2 strands, e.g. TFOs
• CPC Class Codes: C12N 2310/321 : 2'-O-R Modification
• CPC Class Codes: C12N 2310/3511 : intercalating or cleaving agent
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: Y10S 436/80 : Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/905 : Photochemical activation of reactions
• CPC Class Codes: Y10T 436/147777 : Plural nitrogen in the same ring [e.g., barbituates, creatinine, etc.]

Abstract

The present invention is directed to chromophoric probes for use in detecting nucleic acid sequences. More particularly, the invention is directed to probes that comprise a texaphyrin chromophore molecule covalently coupled or conjugated to a site-directing molecule, the site-directing molecule having affinity for a biological receptor or a nucleic acid sequence. In one embodiment of the invention, the probe comprises a texaphyrin chromophore conjugated to an oligonucleotide having complementary binding affinity for a target nucleic acid sequence.

Claims

1. A probe for the detection of a target nucleic acid sequence, which probe comprises a texaphyrin chromophore conjugated to an oligonucleotide having complementary binding affinity for said targeted nucleic acid sequence.

4. The probe of claim 1 wherein the texaphyrin chromophore has the structure ##STR3## wherein M is H, a divalent metal cation, or a trivalent metal cation;

R₁ -R₄, R₇ and R₈ are independently hydrogen, halide, hydroxyl, alkyl, aryl, haloalkyl, nitro, formyl, acyl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, saccharide, carboxy, carboxyalkyl, carboxyamide, carboxyamidealkyl, aminoalkyl, sulfonatoalkyl, amidealkyl, aryl, or a couple that is coupled to the oligonucleotide;

R₆ and R₉ are independently selected from the groups of R₁ -R₄, R₇ and R₈, with the proviso that the halide is other than iodide and the haloalkyl is other than iodoalkyl;

R₅ and R₁₀ -R₁₂ are independently hydrogen, alkyl, aryl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, carboxyalkyl, carboxyamidealkyl or a couple that is coupled to a saccharide or to the oligonucleotide; and

Z is an integer value less than or equal to 5.

5. The probe of claim 4 wherein M is a trivalent metal cation, and the trivalent metal cation is Lu(III), Y(III), or La(III).

6. The probe of claim 4 wherein R₁ is CH₂ CH₃ or (CH₂)₂ CH₂ OH;

R₂ and R₃ are CH₂ CH₃ ;

R₄ is CH₃ ;

R₇ is a couple that is coupled to the oligonudeotide; and

R₈ is H, CH₃, or OCH₃.

7. The probe of claim 4 wherein R₁ is CH₂ CH₃ or (CH₂)₂ CH₂ OH;

R₂ and R₃ are CH₂ CH₃ ;

R₄ is CH₃ ;

R₇ is O(CH₂ CH₂ O)_t CH₂ CH₂ OR'"'"' where t is 2-100, and R'"'"' is H or CH₃ ; and

R₈ is a couple that is coupled to an oligonucleotide.

2. A probe for the detection of a target nucleic acid sequence, which probe comprises an oligonucleotide comprising a texaphyrin chromophore on either the 5'"'"'- or the 3'"'"'-end and an amine on the other end, said oligonucleotide having complementary binding affinity for said target nucleic acid and comprising a partial inverted repeat nucleic acid sequence.

8. The probe of claim 2 wherein the texaphyrin chromophore has the structure ##STR4## wherein M is H, a divalent metal cation, or a trivalent metal cation;

R₁ -R₄, R₇ and R₈ are independently hydrogen, halide, hydroxyl, alkyl, aryl, haloalkyl, nitro, formyl, acyl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, saccharide, carboxy, carboxyalkyl, carboxyamide, carboxyamidealkyl, aminoalkyl, sulfonatoalkyl, amidealkyl, aryl, or a couple that is coupled to the oligonucleotide;

R₆ and R₉ are independently selected from the groups of R₁ -R₄, R₇ and R₈, with the proviso that the halide is other than iodide and the haloalkyl is other than iodoalkyl;

R₅ and R₁₀ -R₁₂ are independently hydrogen, alkyl, aryl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, carboxyalkyl, carboxyamidealkyl or a couple that is coupled to a saccharide or to the oligonucleotide; and

Z is an integer value less than or equal to 5.

9. The probe of claim 8 wherein M is a trivalent metal cation, and the trivalent metal cation is Lu(III), Y(III), or La(III).

10. The probe of claim 2 wherein the partial inverted repeat nucleic acid sequence forms an intramolecular hairpin complex thereby positioning the texaphyrin and the amine into close proximity and causing quenching of fluorescence of the texaphyrin upon irradiation.

11. The probe of claim 10 further comprising the target nucleic acid wherein a duplex is formed between the probe and the target nucleic acid thereby separating the texaphyrin chromophore from the amine and allowing the texaphyrin chromophore to fluoresce upon irradiation.

12. The probe of claim 11 wherein the target nucleic acid is an invariant sequence from an HIV genome, the probe thereby serving as a means for screening for presence of HIV virus.

3. A probe for the detection of a target nucleic acid sequence, which probe comprises an oligonucleotide conjugated to a first texaphyrin, which exhibits fluorescence, on either the 5'"'"'- or the 3'"'"'-end and also conjugated to a second texaphyrin, which has lower excited state energetics as compared to the first texaphyrin, on the other end;

said oligonucleotide having complementary binding affinity for said target nucleic acid and comprising a partial inverted repeat nucleic acid sequence.

13. The probe of claim 3 wherein the first texaphyrin has the structure ##STR5## wherein M is H, a divalent metal cation, or a trivalent metal cation;

R₁ -R₄, R₇ and R₈ are independently hydrogen, halide, hydroxyl, alkyl, aryl, haloalkyl, nitro, formyl, acyl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, saccharide, carboxy, carboxyalkyl, carboxyamide, carboxyamidealkyl, aminoalkyl, sulfonatoalkyl, amidealkyl, aryl, or a couple that is coupled to the oligonucleotide;

R₆ and R₉ are independently selected from the groups of R₁ -R₄, R₇ and R₈, with the proviso that the halide is other than iodide and the haloalkyl is other than iodoalkyl;

R₅ and R₁₀ -R₁₂ are independently hydrogen, alkyl, aryl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, carboxyalkyl, carboxyamidealkyl or a couple that is coupled to a saccharide or to the oligonucleotide; and

Z is an integer value less than or equal to 5.

14. The probe of claim 13 wherein M is a trivalent metal cation, and the trivalent metal cation is Lu(III), Y(III), or La(III).

15. The probe of claim 3 wherein the partial inverted repeat nucleic acid sequence forms an intramolecular hairpin complex thereby positioning the first texaphyrin and the second texaphyrin into close proximity and causing quenching of fluorescence of the first texaphyrin upon irradiation.

16. The probe of claim 15 further comprising the target nucleic add wherein a duplex is formed between the probe and the target nucleic acid thereby separating the first texaphyrin from the second texaphyrin and allowing the first texaphyrin to fluoresce upon irradiation.

17. The probe of claim 16 wherein the target nucleic acid is an invariant sequence from an HIV genome, the probe thereby serving as a means for screening for presence of HIV virus.

18. The probe of claim 3 wherein the second texaphyrin has the structure ##STR6## wherein M is a paramagnetic metal cation or a diamagnetic metal cation having lower excited state energetics as compared to the first texaphyrin;

R₁ -R₄, R₇ and R₈ are independently hydrogen, halide, hydroxyl, alkyl, aryl, haloalkyl, nitro, formyl, acyl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, saccharide, carboxy, carboxyalkyl, carboxyamide, carboxyamidealkyl, aminoalkyl, sulfonatoalkyl, amidealkyl, aryl, or a couple that is coupled to the oligonucleotide;

R₆ and R₉ are independently selected from the groups of R₁ -R₄, R₇ and R₈, with the proviso that the halide is other than iodide and the haloalkyl is other than iodoalkyl;

R₅ and R₁₀ -R₁₂ are independently hydrogen, alkyl, aryl, hydroxyalkyl, oxyalkyl, oxyhydroxyalkyl, carboxyalkyl, carboxyamidealkyl or a couple that is coupled to a saccharide or to the oligonucleotide; and

Z is an integer value less than or equal to 5.

19. The probe of claim 18 wherein M is a paramagnetic metal cation, and the paramagnetic metal cation is Gd(III) or Eu(III).

20. The probe of claim 3 wherein the first texaphyrin is complexed with Lu(III) or Y(III), and the second texaphyrin is complexed with Gd(III).