Exonuclease-resistant oligonucleotides

• US 5,256,775 A
• Filed: 06/05/1990
• Issued: 10/26/1993
• Est. Priority Date: 06/05/1989
• Status: Expired due to Term

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: Gilead Sciences Inc.
• Inventors: Froehler, Brian C.
• Primary Examiner(s): Brown, Johnnie R.
• Assistant Examiner(s): LOW, CHRISTOPHER S F
• Application Number:
  US07/555,522
• Time in Patent Office: 1,239 Days
• Field of Search: 536/27-29
• US Class Current: 536/25.6
• 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

Abstract

A method is provided for making 3'"'"' and/or 5'"'"' end-capped oligonucleotides so as to render the oligonucleotide resistant to degradation by exonucleases. The exonuclease degradation resistance is provided by incorporating two or more phosphoramidate and phosphorocmonothioate and/or phosphorodithioate linkages at the 5'"'"' and/or 3'"'"' ends of the oligonucleotide, wherein the number of phosphoramidate linkages is less than a number which would interfere with hybridization to a complementary oligonucleotide strand and/or which would interfere with RNAseH activity when the oligonucleotide is hybridized to RNA.

Claims

1. An oligonucleotide resistant to degradation under physiological conditions, which oligonucleotide is a modified form of an oligonucleotide consisting essentially of nucleosides linked through phosphodiester linkages so that said phosphodiester linkages at the 3'"'"' terminus, or the 5'"'"' terminus, or both the 3'"'"' and 5'"'"' termini are replaced with N-substituted phosphoramidate linkages, the number of said replaced linkages being 1 to 10, wherein the phosphoramidate has the formula:

##STR14## wherein R¹ and R² are independently selected from the group consisting of hydrogen, hydrocarbyl substituents of 20 carbon atoms or less, and oxyhydrocarbyl substituents of 20 carbon atoms or less containing 1 to 3 oxy groups, and wherein said hydrocarbyl or oxyhydrocarbyl substituents are linear or branched alkyl of 1 to 20 carbon atoms, linear or branched alkenyl of 2 to 20 carbon atoms, cycloalkyl or cycloalkenyl of 3 to 20 carbon atoms, linear or branched alkoxy of 1 to 20 carbon atoms, or aryl of 6 to 18 carbon atoms, provided that R¹ and R² are not both hydrogens.

2. The oligonucleotide of claim 1 wherein one of R¹ and R² is hydrogen and the other is an oxyhydrocarbyl substituent having the structure CH₃ --(CH₂)_x -- wherein x is an integer in the range of 1 to 19, inclusive.

3. The oligonucleotide of claim 2 wherein x is 2 and the oxyhydrocarbyl substituent is 2-methoxyethyl.

4. The oligonucleotide of claim 1 wherein one of the R¹ and R² is hydrogen and the other is a straight-chain alkyl moiety having the formula CH₃ (Ch₂)_y -- wherein y is an integer in the range of 0 to 15, inclusive.

5. The oligonucleotide of claim 4 wherein y is 11 and said alkyl substituent is dodecyl.

6. The oligonucleotide of claim 4 wherein y is 2 and said alkyl substituent is n-propyl.

7. The oligonucleotide of claim 1 wherein the number of said phosphoramidate linkages is less than that which lowers the melting temperature of the duplex formed with complement by less than about 10 degrees.

8. The oligonucleotide of claim 7 wherein the number of said phosphoramidate linkages is less than that which lowers the melting temperature of the duplex formed with complement by less than about 5 degrees.

9. The oligonucleotide of claim 2 wherein x is an integer in the range of 1 to 10, inclusive.

10. The oligonucleotide of claim 1, wherein the number of said replaced linkages is in the range of about 2 to 8.

11. The oligonucleotide of claim 10, wherein the number of said replaced linkages is in the range of about 2 to 6.

12. The oligonucleotide having the formula selected from the group consisting of:

##STR15## wherein each n, m, i, j and s is independently an integer and each s is in the range of about 2 to 10;

each n and m is independently from 1 to about 50;

s+n in formulas I and II is less than 100; and

s+s+m in formula is less than 100;

each i varies from 1 to n;

each j varies from 1 to m;

T is hydrogen or a hydroxyl-protecting group;

R¹ and R₂ are independently selected from the group consisting of hydrogen, hydrocarbyl substituents of 20 carbon atoms or less, and oxyhydrocarbyl substituents of 20 carbon atoms or less containing 1 to 3 oxy groups, and wherein said hydrocarbyl or oxyhydrocarbyl substituents are linear or branched alkyl of 1 to 20 carbon atoms, linear or branched alkenyl of 2 to 20 carbon atoms, cycloalky or cycloalkenyl of 3 to 20 carbon atoms, linear or branched alkoxy of 1 to 20 carbon atoms, or aryl of 6 to 18 carbon atoms, with the proviso that R¹ to R² are not both hydrogen,

each B is independently a protected or unprotected heterocyclic base;

each X_i and X_j is independently O or S; and

each Y_i and Y_j is independently R, --SR or --OR, where R is as defined for R¹ and R².

13. The oligonucleotide of claim 12 wherein X_i and X_j are O and Y_i and Y_j are --OH.

14. The oligonucleotide of claim 12 wherein X_i and X_j are S and Y_i and Y_j are --OH.

15. The oligonucleotide of claim 12 wherein X_i and X_j are S and Y_i and Y_j are --SH.

16. The oligonucleotide of claim 12 wherein each n, m, i, j and s are integers in the range of about 2 to 10 and may be the same or different.

17. The oligonucleotide of claim 12 which is of formula (I) or (II).

18. The oligonucleotide of claim 17 wherein X_i and X_j are O and Y_i and Y_j are --OH.

19. The oligonucleotide of claim 17 wherein X_i and X_j are S and Y_i and Y_j are --OH.

20. The oligonucleotide of claim 17 wherein X_i and X_j are S and Y_i and Y_j are --SH.