Method for Identifying Drug-Sensitizing Antisense DNA Fragments and Use Thereof

Abstract

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 selecting bacterial strains containing specific drug-sensitizing DNA fragments, comprising:
 selecting those strains wherein the inserted DNA fragment is oriented antisense to some portion of the gene of interest;   selecting those strains from a. wherein the intact mRNA level expressed from the gene of interest is reduced at least 25% in the presence of inducer relative to the absence of inducer; and   selecting those strains from b wherein the magnitude of a discernible phenotype is dependent on the concentration of the inducer.   
     
     
         2 . The method of  claim 1 , wherein strains are subjected to a wide range of inducer concentrations to establish a growth response curve to the inducer, such that:
 at high concentrations of inducer, growth is maximally suppressed;   at low or no concentrations of inducer, there is no discernable growth inhibition; or   at moderate and empirically determined concentrations, inducer causes 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% growth reduction compared to untreated strains.   
     
     
         3 . The method of  claim 1 , wherein strains are grown in the presence of a wide range of concentrations of growth-inhibiting compound to establish a growth response curve and thereby a concentration that causes a 50% reduction in growth relative to untreated cells (EC50). 
     
     
         4 . The method of  claim 1 , in which the method is conducted both in the presence and in the absence of an empirically determined concentration of inducer that causes between 10% to 50% growth reduction. 
     
     
         5 . The method of  claim 3 , wherein a decrease in growth inhibitor EC50 values of at least four fold is evident in inducer-treated cells relative to cells without inducer treatment. 
     
     
         6 . The method of  claim 5 , wherein the result is an indication that the antibiotic works as a specific inhibitor of the gene of interest. 
     
     
         7 . The method of  claim 3 , wherein no significant change in antibiotic EC50 values is evident in inducer-treated cells relative to cells without inducer treatment. 
     
     
         8 . The method of  claim 7 , wherein the result is an indication that the antibiotic does not work as a specific inhibitor of the gene of interest.