Substrate for detecting UL9 helicase activity

• US 6,096,502 A
• Filed: 03/30/1998
• Issued: 08/01/2000
• Est. Priority Date: 03/30/1998
• Status: Expired due to Term

• Current Assignee: Lee; Sam S.-K.
• Original Assignee: Lee; Sam S.-K.
• Inventors: Lee, Sam S.-K.
• Primary Examiner(s): Mosher, Mary E.
• Assistant Examiner(s): SALIMI, ALI REZA
• Application Number:
  US09/050,559
• Time in Patent Office: 855 Days
• Field of Search: 435/6, 435/5, 435/7.1, 436/501, 436/504, 536/22.1, 536/23.1, 935/77, 935/82
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/533 involving isomerase
• CPC Class Codes: G01N 2333/03 Herpetoviridae, e.g. pseudorabies virus

Abstract

A UL9 substrate for detecting helicase activity in a UL9 protein is provided. In one embodiment, the substrate includes a first strand including a herpes replication origin sequence and a first single stranded tail; and a second strand including a sequence complementary to the herpes replication origin sequence. In one variation, the first single stranded tail is 3'"'"' relative to the herpes replication origin sequence. The second strand may optionally further include a single stranded tail. The single stranded tail on the second strand may be 3'"'"' or 5'"'"' relative to the sequence complementary to the herpes replication origin sequence. In another variation, the first single stranded tail is 5'"'"' relative to the herpes replication origin sequence. The second strand may optionally further include a single stranded tail. The single stranded tail on the second strand may be 3'"'"' or 5'"'"' relative to the sequence complementary to the herpes replication origin sequence. The UL9 substrate may optionally further include at least one detectable marker attached to at least one of the first and second strands. The UL9 substrate may also optionally be attached to a solid support.

Claims

1. A UL9 substrate comprising:

a first strand including a UL9 herpes replication origin sequence and a first single stranded tail 3'"'"' relative to the herpes replication origin sequence; and

a second strand including a sequence complementary to the UL9 herpes replication origin sequence.

2. The UL9 substrate according to claim 1 wherein the first single stranded tail is at least 8 nucleotides in length.

3. The UL9 substrate according to claim 1 wherein the first single stranded tail is between 8 and 100 nucleotides in length.

4. The UL9 substrate according to claim 1 wherein the first strand is a plasmid.

5. The UL9 substrate according to claim 1 wherein the replication origin sequence is selected from the group consisting of SEQ. ID Nos. 13-29, and at least 10 nucleotide long subsequences of SEQ. ID Nos. 13-29.

6. The UL9 substrate according to claim 1 wherein the first strand further includes a second single stranded tail 5'"'"' relative to the herpes replication origin sequence.

7. The UL9 substrate according to claim 6 wherein the second single stranded tail is at least 8 nucleotides in length.

8. The UL9 substrate according to claim 6 wherein the second single stranded tail is between 8 and 100 nucleotides in length.

9. The UL9 substrate according to claim 1 wherein the second strand further includes a single stranded tail.

10. The UL9 substrate according to claim 9 wherein the single stranded tail on the second strand is 5'"'"' relative to the sequence complementary to the herpes replication origin sequence.

11. The UL9 substrate according to claim 9 wherein the single stranded tail on the second strand is 3'"'"' relative to the sequence complementary to the herpes replication origin sequence.

12. A UL9 substrate comprising:

a first strand including a UL9 herpes replication origin sequence and a first single stranded tail 3'"'"' relative to the herpes replication origin sequence;

a second strand including a sequence complementary to the UL9 herpes replication origin sequence; and

at least one detectable marker attached to at least one of the first and second strands.

13. The UL9 substrate according to claim 12 wherein the at least one detectable marker is attached to the first strand.

14. The UL9 substrate according to claim 12 wherein the at least one detectable marker is attached to the second strand.

15. The UL9 substrate according to claim 12 wherein at least one detectable marker is a fluorescent dye.

16. The UL9 substrate according to claim 12 wherein at least one detectable marker is a radiolabel.

17. The UL9 substrate according to claim 12 wherein the at least one detectable marker includes a reporter dye attached to one strand and a quencher dye attached to another strand of the substrate.

18. The UL9 substrate according to claim 12 wherein the first strand further includes a second single stranded tail 5'"'"' relative to the herpes replication origin sequence.

19. The UL9 substrate according to claim 12 wherein the second strand further includes a single stranded tail.

20. The UL9 substrate according to claim 19 wherein the single stranded tail on the second strand is 5'"'"' relative to the sequence complementary to the herpes replication origin sequence.

21. The UL9 substrate according to claim 19 wherein the first single stranded tail is 3'"'"' relative to the herpes replication origin sequence.

22. An immobilized UL9 substrate comprising:

a first strand including a UL9 herpes replication origin sequence and a first single stranded tail 3'"'"' relative to the herpes replication origin sequence;

a second strand including a sequence complementary to the UL9 herpes replication origin sequence; and

a solid support to which one of the first and second sequences is attached.

23. The immobilized UL9 substrate according to claim 22 wherein the first strand further includes a second single stranded tail 5'"'"' relative to the herpes replication origin sequence.

24. The immobilized UL9 substrate according to claim 22 wherein the second strand further includes a single stranded tail.

25. The immobilized UL9 substrate according to claim 24 wherein the single stranded tail on the second strand is 5'"'"' relative to the sequence complementary to the herpes replication origin sequence.

26. The immobilized UL9 substrate according to claim 24 wherein the single stranded tail on the second strand is 3'"'"' relative to the sequence complementary to the herpes replication origin sequence.

27. The immobilized UL9 substrate according to claim 22 wherein at least one detectable marker is attached to at least one of the first and second strands.

28. A UL9 substrate comprising:

a first strand including a UL9 herpes replication origin sequence and a first single stranded tail 5'"'"' relative to the herpes replication origin sequence; and

a second strand including a sequence complementary to the UL9 herpes replication origin sequence and a single stranded tail 3'"'"' relative to the sequence complementary to the herpes replication origin sequence.

29. The UL9 substrate according to claim 28 wherein the first single stranded tail is at least 8 nucleotides in length.

30. The UL9 substrate according to claim 28 wherein the first single stranded tail is between 8 and 1 00 nucleotides in length.

31. The UL9 substrate according to claim 28 wherein the first strand is a plasmid.

32. The UL9 substrate according to claim 28 wherein the replication origin sequence is selected from the group consisting of SEQ. ID Nos. 13-29, and at least 10 nucleotide long subsequences of SEQ. ID Nos. 13-29.

33. The UL9 substrate according to claim 28 wherein the substrate further includes at least one detectable marker attached to at least one of the first and second strands.

34. The UL9 substrate according to claim 33 wherein the at least one detectable marker is attached to the first strand.

35. The UL9 substrate according to claim 33 wherein the at least one detectable marker is attached to the second strand.

36. The UL9 substrate according to claim 33 wherein at least one detectable marker is a fluorescent dye.

37. The UL9 substrate according to claim 33 wherein at least one detectable marker is a radiolabel.

38. The UL9 substrate according to claim 33 wherein the at least one detectable marker includes a reporter dye attached to one strand and a quencher dye attached to another strand of the substrate.

39. An immobilized UL9 substrate comprising:

a first strand including a UL9 herpes replication origin sequence and a first single stranded tail 5'"'"' relative to the herpes replication origin sequence; and

a second strand including a sequence complementary to the UL9 herpes replication origin sequence and a single stranded tail 3'"'"' relative to the sequence complementary to the herpes replication origin sequence; and

a solid support to which one of the first and second sequences is attached.

40. The UL9 substrate according to claim 39 wherein the immobilized substrate further includes at least one detectable marker attached to at least one of the first and second strands.

41. The UL9 substrate according to claim 40 wherein the at least one detectable marker is attached to the first strand.

42. The UL9 substrate according to claim 40 wherein the at least one detectable marker is attached to the second strand.

43. The UL9 substrate according to claim 40 wherein at least one detectable marker is a fluorescent dye.

44. The UL9 substrate according to claim 40 wherein at least one detectable marker is a radiolabel.

45. The UL9 substrate according to claim 40 wherein the at least one detectable marker includes a reporter dye attached to one strand and a quencher dye attached to another strand of the substrate.