Improved probes using nucleosides containing 3-dezauracil analogs

• US 5,134,066 A
• Filed: 08/29/1989
• Issued: 07/28/1992
• Est. Priority Date: 08/29/1989
• Status: Expired due to Term

• Current Assignee: Monsanto Company (Bayer AG)
• Original Assignee: Monsanto Company (Bayer AG)
• Inventors: Devadas, Balekudru, Adams, Steven P., Rogers, Thomas E., Gray, Steven H.
• Primary Examiner(s): Moskowitz, Margaret
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/400,205
• Time in Patent Office: 1,064 Days
• Field of Search: 435/6, 435/91, 435/805, 436/501, 536/27-29, 536/23, 536/24, 536/122, 536/124, 536/126, 935/78, 935/88, 935/86, 546/290, 546/296, 546/302, 546/303, 546/345, 546/353
• US Class Current: 435/91.3
• 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: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: Y10S 435/805 Test papers

Abstract

The deazauracil containing probes of the invention are able to withstand higher temperatures, thereby allowing unmatched probes and mismatched probes to be washed off at higher hybridization stringency, thereby eliminating background readings and improving ease and accuracy of probe use.

Claims

1. A single-stranded nucleic acid probe which comprises a nucleoside residue of the formula:

##STR76## in which R₁ and R₂ independently are C₁ -C₅ alkyl, C₂ -C₅ alkenyl, halo or hydrogen, R₃ is hydrogen, hydroxy, C_6-14 aryloxy or C₁ -C₅ alkoxy and in which the nucleoside residue is at least five nucleosides from both ends of the probe and said probe is capable of hybridizing with a target nucleic acid and said hybridizing is mediated by the presence of nondeazauracil nucleotide which are contained within said probe residues.

2. The probe of claim 1 in which R₁, R₂ and R₃ are hydrogen.

3. The probe of claim 1 in which R₁ and R₂ are hydrogen and R₃ is hydroxy.

4. The probe of claim 1 in which R₁ is iodine and R₂ and R₃ are hydrogen.

5. The probe of claim 1 in which R₁ is iodine, R₂ is hydrogen and R₃ is hydroxy.

6. The probe of claim 1 in which R₁ is ethenyl and R₂ and R₃ are hydrogen.

7. The probe of claim 1 in which R₁ is ethenyl, R₂ is hydrogen and R₃ is hydroxy.

8. The probe of claim 1 in which R₁ and R₃ are hydrogen and R₂ is iodine.

9. The probe of claim 1 in which R₁ is hydrogen, R₂ is iodine and R₃ is hydroxy.

10. The probe of claim 1 in which the probe is 15 to 50 nucleotides in length.

11. The probe of claim 1 in which the probe is 15 to 25 nucleotides in length.

12. The probe of claim 1 in which the single-stranded nucleic acid is DNA.

13. The probe of claim 1 in which the single-stranded nucleic acid is RNA.

23. A nucleic acid duplex comprising a probe of claim 1.

24. The nucleic acid duplex of claim 23 in which R₁, R₂ and R₃ are hydrogen.

25. The nucleic acid duplex of claim 23 in which R₁ and R₂ are hydrogen and R₃ is hydroxy.

14. The probe which is selected from the group consisting of GGCAGCTTYCACATAGTCCA, AGCTTCCAYATGGTCC, AGCTTCCAYATGGTCCA, GCAGCTTYCACATGGTCC, GGCAGCTTYCACATGGTCCA, ATGAATCYGAACAATCG, ATGAATCCGAAYAATCG, TAGCGGCGACGYACGTACAC, CTCTAGGYTGTCGGCCAGCC, TAGGCAGCTTCCAYCTGGTCCAGTAGC, and GGCAGCTTCCACATGGTCYA in which Y=4-hydroxy-1(2'"'"'-deoxy-beta-D-erythro-pentofuranosyl)-2(1H)-pyridone.

15. The probe having the following sequence:

GGCAGCTTXCACATGGTCCA, in which X=3-iodo-4-hydroxy-1-(2'"'"'-deoxy-beta-D-erythro-pentofuranosyl)-2(1H)-pyridone.

16. The probe having the following sequence:

GGCAGCTTXCACATGGTCCA, in which X=5-iodo-4-hydroxy-1-(2'"'"'-deoxy-beta-D-erythro-pentofuranosyl)-2(1H)-pyridone.

17. The probe having the following sequence:

GGCAGCTTXCACATGGTCCA, in which X=4-0-diphenylcarbamoyl-3-ethenyl-1-(2'"'"'-1-deoxy-beta-D-erythropentofuranosyl)-2(1H)-pyridone.

18. A method for preparing hybrid DNA or RNA molecules which comprises hybridizing a target nucleic acid molecule to a single-stranded nucleic acid probe which comprises a nucleoside residue of the formula:

##STR77## in which R₁ and R₂ independently are C₁ -C₅ alkyl, C₂ -C₅ alkenyl, halo or hydrogen, R₃ is hydrogen, hydroxy, C₆ -C₁₄ aryloxy or C₁ -C₅ alkoxy, and in which the nucleoside residue is at least five nucleosides from both ends of the probe and said probe undergoes hybridization with a target nucleic acid and said hybridization is mediated by the presence of non-deazauracil nucleoside residues which are contained within said probe.

19. The method of claim 18 in which the hybrid molecule remains as a hybrid between said probe and target nucleic acid molecule when washed at temperatures of 50°

-100°

C.

20. The method of claim 18 in which the hybrid molecule remains as a hybrid between said probe and target nucleic acid molecule when washed at temperatures at 65°

-85°

C.

21. The method of claim 18 in which R₁ and R₂ are hydrogen and R₃ is hydroxy.

22. The method of claim 18 in which R₁, R₂ and R₃ are hydrogen.