Nucleotides for prevention and treatment of bacterial and fungal pathologies

Abstract

A selectively inducible, single-stranded DNA (ssDNA) expression library, a method for constructing a ssDNA expression library, a method for screening ssDNA using the expression library, and a method for identifying ssDNA molecules that alter expression of bacterial and fungal gene(s) related to cell growth and toxin production and secretion. The screening library is used to, among other things, identify ODNs effective in stopping cell growth, killing bacteria or fungi, or preventing bacteria and/or fungi from synthesizing and secreting their toxins, and/or to discover ODNs effective in eukaryotic (e.g., mammalian) cells for targeted alteration of gene function. The library is also useful for identifying ssDNAs or ODNs that are used as therapeutic agents for, for instance, providing a method for treatment of bacterial infections such as sepsis.

1 . An oligodeoxynucleotide (ODN) library comprising a plurality of oligodeoxynucleotides of specific length, at least one of the oligodeoxynucleotides comprising said ODN library being capable of interacting with a target genomic DNA, mRNA or protein when inserted into a DNA expression vector with the specific calling sequence for said oligodeoxynucleotide being embedded in said expression vector, said expression vector being capable of being introduced into a target cell to produce at least one of said oligodeoxynucleotides when induced by exposure to a chemical agent for interacting with genomic DNA, mRNA or protein with observable result. 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . The plasmid pssXG. 
     
     
         5 . The plasmid of  claim 4  comprising a PBS having the sequence 5*-TGGTGCGTCCGAG-3′ [Seq. ED No. 3]. 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . The plasmid of  claim 4  comprising a sequence coding for in vivo expression of a single-stranded DNA enzyme targeted to the mRNA transcript of the bacterial FtsZ gene. 
     
     
         9 . The plasmid of  claim 8  wherein the single-stranded DNA enzyme is specific for a GU site at position 880 of the bacterial FtsZ gene. 
     
     
         10 . The plasmid of  claim 8  wherein the single-stranded DNA enzyme comprises 5′-Nj-GGCTAGCTACAACGA-N2-3′ [Seq. ED No. 7] where Ni and N2 represent any sequence of nucleotides ranging in size from about seven to about ten nucleotides that target a specific RNA. 
     
     
         11 . A cell having the plasmid of  claim 8  transformed therein. 
     
     
         12 . A single-stranded DNA enzyme comprising a 15 nucleotide catalytic domain flanked by random RNA target-binding domains of between about 7 and about 10 nucleotides each. 
     
     
         13 . The single-stranded DNA enzyme of  claim 12  wherein said catalytic domain comprises the sequence S′-Ni-GGCTAGCTACAACGA-NrS′ [Seq. ED No. 7], where Ni and N2 represent any sequence of nucleotides ranging in size from about seven to about ten nucleotides that target a specific RNA. 
     
     
         14 . A plasmid having a sequence coding for the DNA enzyme of  claim 12  contained therein. 
     
     
         15 . A cell having the plasmid of  claim 14  transformed therein. 
     
     
         16 - 65 . (canceled)