γ-phosphoester nucleoside triphosphates

• US 6,399,335 B1
• Filed: 11/16/1999
• Issued: 06/04/2002
• Est. Priority Date: 11/16/1999
• Status: Expired due to Term

• Current Assignee: ADVANCED RESEARCH & TECHNOLOGY INSTITUTE
• Original Assignee: ADVANCED RESEARCH & TECHNOLOGY INSTITUTE
• Inventors: Epp, Jeffrey, Vassiliou, William, Kao, C. Cheng, Widlanski, Theodore
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/441,108
• Time in Patent Office: 931 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/25.3
• US Class Current: 435/91.1
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

The invention provides methods and compositions for polymerizing a particular nucleotide with a polymerase. In general, the method involves (a) forming a mixture of a polymerase and a nucleoside triphosphate (NTP) comprising α, β and γ phosphates and a γ-phosphate phosphoester-linked functional group; and incubating the mixture under conditions wherein the polymerase catalyzes cleavage of the NTP between the α and β phosphates, liberating a pyrophosphate comprising the functional group and polymerizing the resultant nucleoside monophosphate, i.e. incorporates the nucleoside monophosphate in a nascent polynucleotide. A variety of functional groups compatible with the polymerization reaction are provided.

Claims

1. A method for polymerizing a nucleotide comprising the steps of:

forming a mixture of a polymerase and a nucleoside triphosphate (NTP) comprising α

,β and

γ

phosphates and a γ

-phosphate phosphoester-linked functional group; and

incubating the mixture under conditions wherein the polymerase catalyzes cleavage of the NIP between the α and

β

phosphates, liberating a pyrophosphate comprising the functional group and polymerizing a resultant nucleoside monophosphate.

2. A method according to claim 1 wherein the functional group is a detectable label and the method further comprises, after the incubating step, the step of detecting the label.

3. A method according to claim 1 wherein the functional group is a detectable label and the method further comprises the step of detecting the label and the label is selected from the group consisting of the compounds of Table 1A.

4. A method according to claim 1, wherein the functional group is a cell delivery enhancing moiety, —

OR wherein R is selected from substituted or unsubstituted (C1-C18) alkyl, alkenyl, alkynyl and aryl.

5. A method according to claim 1, wherein the functional group is a cell delivery enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) alkyl.

6. A method according to claim 1, wherein the functional group is a cell delivery enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) alkyl selected from the group consisting of the compounds of Table 2A.

7. A method according to claim 1, wherein the functional group is a cell delivery enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) alkenyl.

8. A method according to claim 1, wherein the functional group is a cell delivery enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) alkenyl selected from the group consisting of the compounds of Table 3A.

9. A method according to claim 1, wherein the functional group is a cell delivery enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) alkynyl.

10. A method according to claim 1, wherein the functional group is a cell delivery enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) alkynyl selected from the group consisting of the compounds of Table 4A.

11. A method according to claim 1, wherein the functional group is a cell delivery enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) aryl.

12. A method according to claim 1, wherein the functional group is a cell delivery enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) aryl selected from the groups consisting of the compounds of Table 5A.

13. A method according to claim 1, wherein the functional group is a polymerase specificity enhancing moiety, —

OR wherein R is selected from substituted or unsubstituted (C1-C18) alkyl, alkenyl, alkynyl and aryl.

14. A method according to claim 1, wherein the functional group is a polymerase specificity enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) alkyl.

15. A method according to claim 1, wherein the functional group is a polymerase specificity enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) alkyl selected from the group consisting of the compounds of Table 6A.

16. A method according to claim 1, wherein the functional group is a polymerase specificity enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) alkenyl.

17. A method according to claim 1, wherein the functional group is a polymerase specificity enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) alkenyl selected from the group consisting of the compounds of Table 7A.

18. A method according to claim 1, wherein the functional group is a polymerase specificity enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) alkynyl.

19. A method according to claim 1, wherein the functional group is a polymerase specificity enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) alkynyl selected from the group consisting of the compounds of Table 8A.

20. A method according to claim 1, wherein the functional group is a polymerase specificity enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) aryl.

21. A method according to claim 1, wherein the functional group is a polymerase specificity enhancing moiety, —

OR wherein R is substituted or unsubstituted (C1-C18) aryl selected from the group consisting of the compounds of Table 9A.

22. A method according to claim 1, wherein the functional group is a pyrophosphate-prodrug liberating moiety, —

OR wherein R is selected from substituted or unsubstituted (C1-C18) alkyl, alkenyl, alkynyl and aryl.

23. A method according to claim 1, wherein the functional group is a a pyrophosphate-prodrug liberating moiety, —

OR wherein R is selected from substituted or unsubstituted (C1-C18) alkyl, alkenyl, alkynyl and aryl and selected from the group consisting of the compounds of Table 10.