Nucleotides for prevention and treatment of bacterial and fungal pathologies
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. A vector for expressing a single-stranded oligonucleotide in a bacterial or fungal cell, comprising: a promoter; a set of inverted tandem repeats located 3′ to the promoter; a cloning site flanked by the set of inverted tandem repeats or located 3′ to the set of inverted tandem repeats; a primer binding site (PBS) for a reverse transcriptase located 3′ to the cloning site; and an expression termination sequence located 3′ to the PBS and wherein the primer binding site (PBS) has a sequence: TGGTGCGTCCGAG [SEQ ID NO: 3].
2. The cloning vector according to claim 1 , further comprising a gene coding for the reverse transcriptase.
3. The vector according to claim 2 , wherein the reverse transcriptase is a mouse Maloney virus reverse transcriptase.
4. The vector according to claim 1 , further comprising an origin of replication.
5. The vector according to claim 1 , wherein the promoter is a bacterial promoter.
6. The vector according to claim 1 , wherein the promoter is inducible.
7. The vector according to claim 6 , wherein the promoter is inducible by tetracycline or a tetracycline analog.
8. The vector according to claim 1 , wherein the vector is pssXG.
9. The vector according to claim 1 , further comprising an oligonucleotide insert inserted at the cloning site.
10. A library for expressing single-stranded oligodeoxynucleotides, comprising a plurality of vectors according to claim 9 , wherein the oligonucleotide inserts in the plurality of vectors have different nucleotide sequences.
11. The library according to claim 10 , wherein the oligonucleotide inserts have sequences of: 5′-N.sub.I-GGCTAGCTACAACGA-N.sub.2[SEQ ID NO: 7], wherein N.sub.1 and N.sub.2 each represent a nucleotide sequence having a random sequence and a length from 3 to 25 nucleotides long.
12. A cell having a vector according to claim 1 .
13. A method for screening an oligodeoxynucleotide that modulates a cell function using the library of claim 10 , wherein the promoter in the vector is inducible, the method comprising: transfecting the library into host cells; growing the transfected host cells on replica plates, one of the replica plates including an agent for inducing expression of single-stranded oligodeoxynucleotides from the oligonucleotide inserts in the vectors in the transfected host cells; comparing the induced and non-induced replica plates to identify a host cell having a different phenotype; and sequencing the oligonucleotide insert in the vector from the host cell having a different phenotype.
14. The vector of claim 9 , wherein the oligonucleotide insert is determined to have a sequence of: 5′-CTTTCAACAGTTTTGATGACCTTTGCTGACC [SEQ ID NO: 14] ATACAATTGCGATATCGTGGGGAGTGAGAG-3′, 5′-CTCATACTCT-3′, [SEQ ID NO: 33] 5′-GTTTCGAAGGCTAGCTACAACGATCATCCA [SEQ ID NO: 6], or 5′-CCTGCTTAGGCTAGCTACAACGATGGTCAC [SEQ ID NO: 8].
15. A cell having the vector according to claim 14 transfected.
16. A method for inhibiting bacterial, fungal or other microbial growth or reducing toxin activity, comprising contacting bacteria, fungi or other microorganism with the vector of claim 14 .
17. A method for inhibiting bacterial, fungal or other microbial growth or reducing toxin activity, comprising contacting bacteria, fungi or other microorganism with the vector of claim 9 .
18. The method of claim 16 , wherein the bacteria, fungi or other microorganism is a sepsis causative agent.
19. A method for reducing or blocking sepsis-related toxin activity or sepsis-induced immune responses, comprising contacting a bodily fluid with the vector of claims 9 .