Transcription factor decoys, compositions and methods

Abstract

Compositions and methods for identifying and using cis-regulatory and decoy sequences are disclosed.

What is claimed is: 
     
         1 . A method of treating a bacterial infection, the method comprising administering a decoy polynucleotide comprising a binding site for a transcription factor,
 wherein the transcription factor binding site in the decoy polynucleotide is not operably linked to a gene; and   wherein the decoy polynucleotide reduces binding of the transcription factor to a cis-regulatory sequence in a prokaryotic cell and causes an alteration in expression of gene or genes operably linked to the cis-regulatory sequence in the cell.   
     
     
         2 . The method according to  claim 1 , wherein the transcription factor is a regulator of expression of one or more genes that encode resistance to an antibiotic. 
     
     
         3 . The method according to  claim 2 , wherein the antibiotic is selected from one or more: the aminoglycosides; the carbapenems; the cephalosporins; the glycopeptides; the penicillins; the polypeptide antibiotics; the quinolines; the sulfonamides; or the tetracyclines. 
     
     
         4 . The method according to  claim 3 , wherein the antibiotic is selected from one or more of: kanamycin, meropenem, cefepime, vancomycin, daptomycin, ampicillin, carbenicillin, penicillin, polymixcin B, levaquin, Bactrim, tetracycline, chloramphenical, rifampicin and Zyvox. 
     
     
         5 . The method according to  claim 1 , wherein the gene or genes operably linked to the cis-regulatory sequence encode resistance to vancomycin. 
     
     
         6 . The method according to  claim 5 , wherein the transcription factor is the VanR transcription factor. 
     
     
         7 . The method according to  claim 1  wherein the prokaryotic cell is:
 (a) a gram positive bacterium of a genus selected from:  Actinomyces; Streptomyces ; Mycobacteria;  Clostridium; Bacillus; Listeria; Staphylococcus; Streptococcus; Enterococcus ; or 
 (b) a gram negative bacterium of a genus selected from: Enterobacteriaceae;  Pseudomonas; Moraxella; Helicobacter; Stenotrophomonas; Bdellovibio , Acetic acid bacteria; Legionella; Cyanobacteria; Spirochaetes; Green Sulphur bacteria; and Green Non-sulphur bacteria. 
 
     
     
         8 . The method according to  claim 1 , wherein the prokaryotic cell is a pathogen selected from:  Mycobacterium tuberculosis; Mycobacterium bovis; Mycobacterium africanum; Mycobacterium microti; Mycobacterium leprae; Clostridium difficile; Clostridium botulinum; Clostridium perfingens; Clostridium tetani; Salmonella  sp.;  Escherichia coli; Enterococcus faecium; Enterococcus faecalis; Neisseria gonorrhoeae; Nerisseria meningitides; Moraxella catarrhalis; Hemophilus influenza; Kebsiella pneumoniae; Legionella pneumophila; Pseudomonas aeruginosa; Proteus mirabilis; Enterobacter cloacae; Serratia marcescens; Helicobacter pylori; Salmonella enteritidis ; and  Salmonella typhi.    
     
     
         9 . The method according to  claim 1 , wherein the prokaryotic cell is a vancomycin resistant pathogen. 
     
     
         10 . The method according to  claim 1 , wherein the decoy polynucleotide comprises: circular double stranded DNA or a linear oligonucleotide; and/or at least one element of secondary structure; and/or more than one copy of the transcription factor binding site; and/or additional sequence to the binding site(s); and/or modified bases or sugars to increase nuclease resistance of the polynucleotide; and/or a plasmid or plasmid library. 
     
     
         11 . The method according to  claim 10 , wherein the decoy polynucleotide comprises a circular dumbbell. 
     
     
         12 . The method according to  claim 10 , wherein the decoy polynucleotide comprises multiple direct repeats of the binding site. 
     
     
         13 . The method according to  claim 10 , wherein the decoy polynucleotide comprises a linear oligonucleotide having at least one 5′ cholesterol modification. 
     
     
         14 . The method according to  claim 10 , wherein the decoy polynucleotide comprises a plasmid and wherein the plasmid has one or more copies of a monomer sequence comprising a snare sequence, the snare sequence comprising the transcription factor binding site wherein the binding site is not operably linked to a gene. 
     
     
         15 . The method according to  claim 14 , wherein the plasmid comprises:
 (a) two or more or at least 10, 20 or 30 copies of the monomer sequence; and/or   (b) tandem repeats of the monomer sequence.   
     
     
         16 . The method according to  claim 14 , wherein the monomer additionally comprises:
 (a) a binding site for a primer wherein the primer is suitable for use in rolling circle amplification; and/or   (b) a recognition and/or cutting site for one or more restriction enzymes.   
     
     
         17 . The method according to  claim 14 , wherein the monomer comprises from 10-1000 or from 10-500 nucleotides. 
     
     
         18 . The method according to  claim 14 , wherein the snare sequence comprises from 10 to 30 nucleotides or from 15 to 20 nucleotides. 
     
     
         19 . The method according to  claim 14 , wherein the plasmid is a broad host range or shuttle vector and/or is a high copy number plasmid. 
     
     
         20 . The method according to  claim 1 , wherein the transcription factor binding site in the decoy polynucleotide comprises the sequence of SEQ ID NO: 21 or SEQ ID NO: 26. 
     
     
         21 . The method according to  claim 1 , wherein treating the bacterial infection further comprises use of one or more antibiotics. 
     
     
         22 . A method for modulating antibiotic resistance of a prokaryotic cell, the method comprising administering a decoy polynucleotide comprising a binding site for a transcription factor;
 wherein the transcription factor binding site in the decoy polynucleotide is not operably linked to a gene; and   wherein the decoy polynucleotide reduces binding of the transcription factor to a cis-regulatory sequence in the prokaryotic cell and causes an alteration in expression of the operably linked gene or genes in the cell, thereby modulating antibiotic resistance of the cell.