Method for identifying drug-sensitizing antisense DNA fragments and use thereof
View Patent ↗The invention provides a method for generating and selecting drug-sensitizing antisense DNA fragments. In one embodiment, the method includes identifying a gene of interest using knowledge of bacterial physiology, biochemistry, genetics, genomics, and other means. The method includes PCR amplification of a gene of interest using genomic DNA as a template; fragmentation of the DNA by sonication or other means; selecting DNA fragments no longer than 400 base pairs; ligating the DNA fragments into a suitable expression plasmid with a selectable marker; transforming the plasmids containing the DNA fragments into the organism from which the gene of interest originated; and selecting clones from transformed cells that show a phenotypic difference of the clone grown in the presence of the inducer relative to the phenotype in the absence of inducer.
1. A method for generating and selecting drug-sensitizing antisense DNA fragments comprising:
selecting a gene of interest using means other than genomic surveys of antisense-defined proliferation genes,
amplification of the gene of interest using genomic DNA as a template,
fragmentation of the DNA by sonication or other means;
selecting DNA fragments no longer than 400 base pairs;
ligating the DNA fragments into a suitable expression plasmid with a selectable marker;
transforming the plasmids containing the DNA fragments into the organism from which the gene of interest originated; and
selecting clones from transformed cells that show a discernible phenotypic difference of the clone grown in the presence of the inducer relative to the phenotype in the absence of inducer.
2. The method of claim 1 , wherein the discernible phenotype is relative sensitivity to a growth inhibiting compound.
3. The method of claim 1 , wherein the discernible phenotype is requirement of a nutrient to the growth medium.
4. The method of claim 1 , wherein the discernible phenotype is visible morphology such as shape.
5. The method of claim 1 , wherein the discernible phenotype is relative sensitivity to osmotic stress.
6. The method of claim 1 wherein the discernible phenotype is colony size.
7. The method of claim 1 , wherein the drug is an antibiotic acting through inhibition of DNA biosynthesis.
8. The method of claim 1 , wherein the drug is an antibiotic acting through inhibition of RNA biosynthesis.
9. The method of claim 1 , wherein the drug is an antibiotic acting through inhibition of protein biosynthesis.
10. The method of claim 1 , wherein the drug is an antibiotic acting through inhibition of fatty acid biosynthesis.
11. The method of claim 1 , wherein the drug is an antibiotic acting through inhibition of cell wall biosynthesis.
12. The method of claim 1 , wherein the drug is an antibiotic acting through inhibition of amino acid biosynthesis.
13. The method of claim 1 , wherein the drug is an antibiotic acting through inhibition of nucleotide biosynthesis.
14. The method of claim 1 , wherein the drug is an antibiotic acting through inhibition of vitamin biosynthesis.
15. The method of claim 1 , wherein the drug is an antibiotic acting through inhibition of isoprenoid biosynthesis.
16. The method of claim 1 , wherein the drug is an antibiotic acting through inhibition of co-factor biosynthesis.
17. The method of claim 1 , wherein the drug is an antibiotic acting through inhibition of MurB.
18. The method of claim 1 , wherein the drug is an antibiotic acting through inhibition of UppS.
19. The method of claim 1 , wherein the drug is an antibiotic acting through inhibition of DHFR.
20. The method of claim 1 , wherein the drug is a beta-lactam antibiotic.
21. The method of claim 1 , wherein the drug is a diaminopyrimidine antibiotic.
22. The method of claim 1 , wherein the drug is macrolide antibiotic.
23. The method of claim 1 , wherein the drug is daptomycin.
24. The method of claim 1 , wherein the drug is a fluoroquinolone antibiotic including ciprofloxacin.
25. The method of claim 1 , wherein the drug is a tetracycline.
26. The method of claim 1 , wherein the drug is an oxazolidinone.
27. The method of claim 1 , wherein the drug is fosfomycin.
28. The method of claim 1 , wherein the drug is fosmidomycin.
29. The method of claim 1 , wherein the drug is mupirocin.
30. The method of claim 1 , wherein the drug is a lipopeptide.
31. The method of claim 1 , wherein the drug is a glycopeptide.
32. The method of claim 1 , wherein the drug is vancomycin.
33. The method of claim 1 , wherein the drug is trimethoprim.
34. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene murB-2 and is comprised of the sequence specified for SEQ ID:1 Ba-murB2-C1.
35. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene murB-2 and is comprised of the sequence specified for SEQ ID:2 Ba-murB2-H1.
36. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene murB-2 and is comprised of the sequence specified for SEQ ID:3 Ba-murB2-D1.
37. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene murB-2 and is comprised of the sequence specified for SEQ ID:4 Ba-murB2-D2.
38. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene metS and is comprised of the sequence specified for SEQ ID:5 Ba-metRS1-H1.
39. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene metS and is comprised of the sequence specified for SEQ ID:6 Ba-metRS1-H2.
40. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene metS and is comprised of the sequence specified for SEQ ID:7 Ba-metRS1-H6.
41. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene metS and is comprised of the sequence specified for SEQ ID:8 Ba-metRS1-E4.
42. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene uppS and is comprised of the sequence specified for SEQ ID:9 Ba-uppS-UG9.
43. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene uppS and is comprised of the sequence specified for SEQ ID:10 Ba-uppS-UA3.
44. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene dfrA and is comprised of the sequence specified for SEQ ID:11 Ba-dfrA-2G1.
45. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Bacillus anthracis gene dfrA and is comprised of the sequence specified for SEQ ID:12 Ba-dfrA-2G6.
46. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Staphylococcus aureus gene murB and is comprised of the sequence specified for SEQ ID:13 Sa-murB-E9.
47. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Staphylococcus aureus gene murB and is comprised of the sequence specified for SEQ ID:14 Sa-murB-F7.
48. The method of claim 1 , wherein said antisense-oriented fragment is a fragment of the Staphylococcus aureus gene murB and is comprised of the sequence specified for SEQ ID:15 Sa-murB-B9.