Base-modified nucleotides and cleavage of polynucleotides incorporating them

• US 6,610,492 B1
• Filed: 01/08/2002
• Issued: 08/26/2003
• Est. Priority Date: 10/01/1998
• Status: Expired due to Term

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Variagenics, Inc. (Arca Biopharma, Inc.)
• Inventors: Allerson, Charles, Verdine, Gregory L., Stanton, Vincent P. Jr., Wolfe, Jia Liu, Kawate, Tomohiko
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/043,511
• Time in Patent Office: 595 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 536/25.3
• US Class Current: 536/23.1
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

The present invention is directed to novel base-modified nucleotides and methods for their use in the preparation and cleavage of modified polynucleotides.

Claims

1. A method for cleaving a polynucleotide, comprising:

replacing a natural nucleotide at greater than 90% of its points of occurrence in a polynucleotide with a base-modified nucleotide to form a base-modified polynucleotide; and

,

contacting the base-modified polynucleotide with a reagent or combination of reagents that cleaves it at greater than 90% of the points of occurrence of the base-modified nucleotide, wherein;

the base-modified nucleotide has the chemical structure;

 

wherein

R is a ribose or 2′

-deoxyribose moiety of an oligonucleotide or of a polynucleotide;

R¹ and R² are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl and alkaryl, wherein,

if R¹ or R² contains two or more contiguous methylene (—

CH₂—

) groups, any two such methylene groups may have interjected between them another group selected from the group consisting of —

O—

, —

C(O)NH—

, —

C(O)NHC(O)—

, —

NH—

, —

C(S)NH—

, —

CO—

, —

CS—

, —

S— and

(—

CF₂—

)_m, wherein m is 1-10;

R³ is hydrogen or —

NH₂; and

,

n is 0, 1 or 2.

2. The method of claim 1, wherein contacting the base-modified polynucleotide with a reagent or reagents comprises contacting it with a chemical base.

3. The method of claim 2, wherein the chemical base is an amine.

4. The method of claim 3, wherein the amine has a boiling point greater than 100°

C. at atmospheric pressure.

5. The method of claim 3, wherein the amine has a boiling point greater than 200°

at atmospheric pressure.

6. The method of claim 5, wherein the amine is a secondary amine.

7. The method of claim 6, wherein the secondary amine is selected from the group consisting of 3-pyrrolidinol, 2-pyrrolidinemethanol, 3-pyrrolidinemethanol, 4-hydroxypiperidine and 4-piperidineethanol.

8. The method of claim 2, wherein the modified polynucleotide is contacted with a chemical oxidant prior to contact with the chemical base.

14. The method of claim 1, or claim 9, wherein the percentage replacement of a natural nucleotide with a modified nucleotide, the percentage cleavage of a modified polynucleotide or both the percentage replacement and the percentage cleavage is greater than 95%.

15. The method of claim 1 or claim 9, wherein the percentage replacement of a natural nucleotide with a modified nucleotide, the percentage cleavage of a modified polynucleotide or both the percentage replacement and the percentage cleavage is greater than 99%.

9. A method for cleaving a polynucleotide, comprising:

replacing a natural nucleotide at greater than 90% of its points of occurrence in a polynucleotide with a base-modified nucleotide to form a base-modified polynucleotide; and

,

contacting the base modified polynucleotide with a secondary amine having a boiling point greater than 100°

C. at atmospheric pressure whereby the polynucleotide is cleaved at greater than 90% of the sites of incorporation of the modified nucleotide.

10. The method of claim 9, wherein the secondary amine has a boiling point greater than 150°

at atmospheric pressure.

11. The method of claim 9, wherein the secondary amine has a boiling point greater than 200°

at atmospheric pressure.

12. The method of claim 9, wherein the secondary amine is selected from the group consisting of 3-pyrrolidinol, 2-pyrrolidinemethanol, 3-pyrrolidinemethanol, 4-hydroxypiperidine and 4-piperidineethanol.

13. The method of claim 9, wherein the modified polynucleotide is contacted with a chemical oxidant prior to contact with the secondary amine.