Polynucleotide reagents containing modified deoxyribose moieties, and associated methods of synthesis and use

• US 5,597,909 A
• Filed: 08/25/1994
• Issued: 01/28/1997
• Est. Priority Date: 08/25/1994
• Status: Expired due to Fees

• Current Assignee: Chiron Diagnostics Corp (Bayer AG)
• Original Assignee: Chiron Corporation (Novartis Ag)
• Inventors: Urdea, Michael S., Horn, Thomas
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Atzel, Amy
• Application Number:
  US08/296,368
• Time in Patent Office: 887 Days
• Field of Search: 536/17.2, 536/18.2, 536/18.3, 536/18.7, 536/25.3, 536/55, 536/24.3, 536/24.31, 536/24.32, 536/25.31, 536/25.32, 435/6, 436/501, 935/77.78
• US Class Current: 536/24.3
• CPC Class Codes: C07F 9/65515 the oxygen atom being part of a five-membered ring
• CPC Class Codes: C07H 15/04 attached to an oxygen atom of the saccharide radical
• 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 1/6823 Release of bound markers

Abstract

Methods and reagents are provided for synthesizing polynucleotides containing modified deoxyribose residues. Monomeric reagents having the structural formula (I) ##STR1## wherein R¹, R², R³, R⁴ and R⁵ are as defined herein, are used to create polynucleotides having nonnucleotidic moieties -A-Z-(R⁹)_n at the 1 position of selected deoxyribose units. The polynucleotides so provided are useful in a variety of hybridization assay formats.

Claims

1. A reagent having the structural formula ##STR12## wherein:

R¹ is selected from the group consisting of hydrogen, acid-sensitive, base-stable protecting groups and acyl capping groups;

R² is a phosphorus derivative selected to enable addition of the reagent to a molecular species containing a free hydroxyl group, or is a linkage to a solid support;

R³ is selected from the group consisting of hydrogen, hydroxyl, sulfhydryl, halogeno, amino, alkyl, allyl, -OR⁶ wherein R⁶ is alkyl, allyl, silyl or phosphate;

R⁴ is either hydrogen or -(CH₂)_m OR⁷ wherein R⁷ is alkyl or -(CO)R⁸, R⁸ is alkyl and m is an integer in the range of 0 to 12 inclusive;

R⁵ is -A-Z-X(R⁹)n;

A is oxygen, sulfur or methylene;

Z is arylene, C₆ -C₁₈ aralkylene or C₁ -C₁₂ alkylene containing 0 to 6 heteroatoms selected from the group consisting of O, S, N, Si and Se and 0 to 6 linkages selected from the group consisting of -CO-, -COO-, -CONH-, -NHCO-, -S-S-, -SO₂ -, -CH(OH)-CH(OH)-, -CH(OR¹⁵)-CH(OR¹⁵)-,-O-PO(O³¹ )-O-, -O-P)(R¹⁵)-, -O-P)(OR¹⁵)-O-, -O-P)(OR¹⁵)-R¹⁶ - and -PO(OR¹⁵)-O-R¹⁶ - in which R¹⁵ is lower alkyl and R¹⁶ is lower alkylene, and, if Z is aralkylene or alkylene, containing 0 to 3 unsaturated bonds;

X is selected from the group consisting of -NH-, -CONH-, -NHCO-, -CO-, -S- and -Si≡

;

R⁹ is hydrogen, a protecting group, a detectable label, or, unless X is -Si≡

, a solid support; and

n is 1 when X is -NH-, -CONH-, -NHCO-, -CO-, or -S-, and is 3 when X is -Si≡

,

with the proviso that if R² represents a linkage to a solid support, R⁹ is hydrogen, a protecting group, or a detectable label.

2. The reagent of claim 1, wherein A is oxygen.

3. The reagent of claim 2, wherein Z is -(CH₂)_n - where n is an integer in the range of 1 to 12 inclusive.

4. The reagent of claim 3, wherein n is an integer in the range of 2 to 6 inclusive.

5. The reagent of claim 2, wherein Z is -(CH₂ CH₂ O)_n - where n is an integer in the range of 1 to 12 inclusive.

6. The reagent of claim 5, wherein n is an integer in the range of 2 to 6 inclusive.

7. The reagent of claim 2, wherein X is -NH-.

8. The reagent of claim 2, wherein X is -CONH-.

9. The reagent of claim 2, wherein X is -NHCO-.

10. The reagent of claim 2, wherein X is -S-.

11. The reagent of claim 2, wherein R⁹ is hydrogen.

12. The reagent of claim 2, wherein R⁹ is a protecting group.

13. The reagent of claim 12, wherein R⁹ is FMOC.

14. The reagent of claim 12, wherein R⁹ is NBOC.

15. The reagent of claim 12, wherein R⁹ is trityl.

16. The reagent of claim 2, wherein R⁹ is a label.

17. The reagent of claim 16, wherein R⁹ is an enzyme or enzyme fragment.

18. The reagent of claim 16, wherein R⁹ is a dye.

19. The reagent of claim 18, wherein R⁹ is fluorescein.

20. The reagent of claim 18, wherein R⁹ is 2,4-DNP.

21. The reagent of claim 16, wherein R⁹ is a hapten.

22. The reagent of claim 16, wherein R⁹ is a solid support.

23. The reagent of claim 2, wherein R¹ is selected from the group consisting of dimethoxytrityl, monomethoxytrityl, trityl and pixyl.

24. The reagent of claim 2, wherein R² is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, H-phosphonates and phosphorothioates.

26. A method for making branched DNA, comprising:

(a) sequentially coupling nucleotidic monomers to a growing oligonucleotide chain;

(b) introducing branch points into the chain during step (a) by replacing a fraction of the nucleotidic monomers with monomeric reagents having linker arms at the 1 position;

(c) sequentially adding nucleotidic monomers to the termini of the linker arms, wherein each of the monomeric reagents used in step (b) comprises a reagent of claim 1.

25. A reagent having the structural formula ##STR13## wherein:

R¹ is selected from the group consisting of hydrogen and acid-sensitive, base-stable protecting groups;

R² is is selected from the group consisting of phosphoramidites, phosphotriesters, phosphodiesters, phosphites, H-phosphonates and phosphorothioates;

A is oxygen, sulfur or methylene;

Z is a hydrocarbyl or oxyhydrocarbyl spacer moiety containing 1 to 18 carbon atoms and 0 to 6 oxygen atoms;

X is selected from the group consisting of -NH- and -S-; and

R⁹ is a protecting group.