Stabilized nucleic acids in gene and drug discovery and methods of use

• US 20030170694A1
• Filed: 12/20/2002
• Published: 09/11/2003
• Est. Priority Date: 12/21/2001
• Status: Abandoned Application

• Current Assignee: Merck & Co., Inc.
• Original Assignee: Merck & Co., Inc.
• Inventors: Froelich, Jamie, Wall, Daniel
• Application Number:
  US10/327,592
• Publication Number:
  US 20030170694A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2320/10 in screening processes

Abstract

Stabilized nucleic acids for use in gene and drug discovery are disclosed. Vectors and host cells useful in the production of stabilized nucleic acids are also disclosed. Cell-based assays which employ stabilized antisense nucleic acids to identify and develop antibiotics and to identify genes required for proliferation are described. The use of stabilized nucleic acids to identify homologous nucleic acids required for the proliferation of heterologous organisms is also described. Inhibition of the expression of genes required for proliferation in heterologous organisms through the use of stabilized antisense nucleic acids is disclosed.

Claims

1. A method for screening a candidate antibiotic compound which inhibits the proliferation of a cell said method comprising the steps of:

(a) sensitizing a cell by providing a sublethal level of an antisense nucleic acid complementary to at least a portion of a gene encoding a proliferation-required gene product in said cell, wherein said antisense nucleic acid is flanked on each end by at least one stem-loop structure;

(b) contacting said sensitized cell with a candidate antibiotic compound; and

(c) determining the degree to which said candidate antibiotic compound inhibits proliferation of said sensitized cell relative to a cell which has not been sensitized.

2. The method of claim 1, wherein said at least one stem-loop structure formed at the 5′

end of said antisense nucleic acid comprises a flush, double stranded 5′

end.

3. The method of claim 1, wherein the activity of at least one enzyme involved in RNA degradation has been reduced in said sensitized cell.

4. The method of claim 3, wherein said at least one enzyme involved in RNA degradation is selected from the group consisting of RNase E, RNase II, RNase III, polynucleotide phosphorylase, and poly(A) polymerase.

5. The method of claim 1, wherein said step of sensitizing said cell comprises transcribing said antisense nucleic acid from a promoter.

6. The method of claim 5, wherein said promoter is regulatable.

7. The method of claim 5, wherein the first transcribed nucleotide from said promoter is the first nucleotide of a 5′

stem-loop structure.

8. The method of claim 1, wherein said at least one stem-loop structure comprises SEQ ID NO.:

5.

9. The method of claim 1, wherein said antisense nucleic acid lacks RNase E recognition sites.

10. The method of claim 1, wherein said at least one stem-loop structure lacks RNase III recognition sites.

11. The method of claim 1, wherein said at least one stem-loop structure lacks a ribosome binding site.

12. The method of claim 1, wherein said at least one stem-loop structure formed at the 3′

end of said antisense nucleic acid comprises at least one rho independent terminator.

13. The method of claim 1, wherein said sensitized cell is a gram-negative bacterium.

14. The method of claim 1, wherein said sensitized cell is selected from a group consisting of Bacteroides fragilis, Bordetella pertussis, Burkholderia cepacia, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatus, Enterobacter cloacae, Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella haemolytica, Pasteurella multocida, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella bongori, Salmonella cholerasuis, Salmonella enterica, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Moxarella catarrhalis, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Treponema pallidum, Yersinia enterocolitica, Yersinia pestis or any species falling within the genera of any of the above species.

15. A candidate antibiotic compound identified using the method of claim 1.

16. A method for identifying a gene which is required for proliferation of a cell comprising:

(a) contacting a cell with an antisense nucleic acid flanked on each end by at least one stem-loop structure,

(b) determining whether said antisense nucleic acid inhibits proliferation of said cell; and

(c) identifying the gene in said cell which encodes the mRNA which is complementary to said antisense nucleic acid or a portion thereof.

17. The method of claim 16, wherein said step of determining whether said antisense nucleic acid inhibits the proliferation of said cell comprises comparing the proliferation of said cell transcribing a first level of said antisense nucleic acid to the proliferation of said cell which transcribes a lower level of said antisense nucleic acid or which does not transcribe said antisense nucleic acid.

18. The method of claim 16, wherein said at least one stem-loop structure formed at the 5′

end of said antisense nucleic acid comprises a flush, double stranded 5′

end.

19. The method of claim 16, wherein the activity of at least one enzyme involved in RNA degradation has been reduced in said cell.

20. The method of claim 19, wherein said at least one enzyme involved in RNA degradation is selected from the group consisting of RNase E, RNase II, RNase III, polynucleotide phosphorylase, and poly(A) polymerase.

21. The method of claim 16, wherein said antisense nucleic acid comprises a random genomic fragment from said organism.

22. The method of claim 16, wherein said step of contacting said cell with said antisense nucleic acid comprises transcribing said antisense nucleic acid from a promoter.

23. The method of claim 22, wherein said promoter is regulatable.

24. The method of claim 22, wherein the first transcribed nucleotide from said promoter is the first nucleotide of a 5′

stem-loop structure.

25. The method of claim 16, wherein said at least one stem-loop structure comprises SEQ ID NO.:

5.

26. The method of claim 16, wherein said antisense nucleic acid lacks RNase E recognition sites.

27. The method of claim 16, wherein said at least one stem-loop structure lacks RNase III recognition sites.

28. The method of claim 16, wherein said at least one stem-loop structure lacks a ribosome binding site.

29. The method of claim 16, wherein said at least one stem-loop structure formed at the 3′

end of said antisense nucleic acid comprises at least one rho independent terminator.

30. The method of claim 16, wherein said cell is a gram-negative bacterium.

31. The method of claim 16, wherein said sensitized cells are selected from a group consisting of Bacteroides fragilis, Bordetella pertussis, Burkholderia cepacia, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatus, Enterobacter cloacae, Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella haemolytica, Pasteurella multocida, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella bongori, Salmonella cholerasuis, Salmonella enterica, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Moxarella catarrhalis, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Treponema pallidum, Yersinia enterocolitica, Yersinia pestis or any species falling within the genera of any of the above species.

32. A method for manufacturing an antibiotic comprising the steps of:

(a) contacting sensitized cells which express a sublethal level of an antisense nucleic acid flanked on each end by at least one stem-loop structure with a compound;

(b) identifying a compound which substantially inhibits the proliferation of said sensitized cells relative to cells which have not been sensitized; and

(c) manufacturing the compound so identified.

33. The method of claim 32, wherein said at least one stem-loop structure formed at the 5′

end of said antisense nucleic acid comprises a flush, double stranded 5′

end.

34. The method of claim 32, wherein the activity of at least one enzyme involved in RNA degradation has been reduced in said sensitized cells.

35. The method of claim 32, wherein said antisense nucleic acid comprises a random genomic fragment from said sensitized cells.

36. The method of claim 32, wherein said sensitized cells comprise a gram-negative bacterium.

37. The method of claim 32, wherein said sensitized cells are selected from a group consisting of Bacteroides fragilis, Bordetella pertussis, Burkholderia cepacia, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatus, Enterobacter cloacae, Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella haemolytica, Pasteurella multocida, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella bongori, Salmonella cholerasuis, Salmonella enterica, Salmonella paratyphi, Salmonella typhi, Salmonella typhimurium, Moxarella catarrhalis, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Treponema pallidum, Yersinia enterocolitica, Yersinia pestis or any species falling within the genera of any of the above species.