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Single-stranded antimicrobial oligonucleotides and uses thereof

An oligonucleotide and insert technology, applied in biochemical equipment and methods, antibacterial drugs, drug combinations, etc., can solve the problems of RNA molecular difficulties and RNA molecular instability.

Inactive Publication Date: 2007-11-21
CYTOGENIX INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method can only be used to identify key genes, because antisense RNAs with a size of 200-800bp are useless for therapeutics because of 1) the instability of RNA molecules, and 2) the difficulty of synthesizing 200-800bp RNA molecules and 3) the problem of delivering RNA to appropriate cells

Method used

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  • Single-stranded antimicrobial oligonucleotides and uses thereof
  • Single-stranded antimicrobial oligonucleotides and uses thereof
  • Single-stranded antimicrobial oligonucleotides and uses thereof

Examples

Experimental program
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Embodiment 1

[0028] Embodiment 1 Construction of inducible prokaryotic ssDNA expression vector

[0029] Use pssXE plasmid as PCR template, carry out the PCR amplification of expression cassette, this expression cassette comprises inverted repeat sequence, the cloning site of meaningful sequence (sequence of interest, SOI), reverse transcriptase specific primer binding site and Murine Maloney reverse transcriptase gene. DNA primers used in PCR were synthesized by Integrated DNA Technologies (Coralville, IA):

[0030] 5'NheIPvuIATG

[0031] CTAGCTAGCT AGCGATCGAT GGGACCAATG GGGCAG [SEQ ID NO: 1]

[0032] and 3'KpnI

[0033] CGGGGTACCA GTATTCCCTG GTC [SEQ ID NO: 2].

[0034] The pssXE vector was constructed and described in detail in International Application No. PCT / US03 / 13593, and PCR amplification was previously described in International Application No. PCT / US04 / 17331, both of which are incorporated herein by reference .

[0035] The DNA fragment amplified by PCR was double-digested ...

Embodiment 2

[0039] The construction of the inducible expression library of embodiment 2 ssDNA or ODN

[0040] Library inserts were generated with annealed three ODNs in a 1:20:20 molar ratio: CY(SacII)-40,

[0041] CTCTCACTCC(N)40ACTGTTGAAAGGC [SEQ ID NO: 4]CY(SacII)-L,

[0042] CGGAGAGTGAGG [SEQ ID NO: 5] and CY(SacII)-R,

[0043] CTTTCAACAGT [SEQ ID NO: 6].

[0044] Here, "N" represents any base in A, T, C or G. The sequence with randomly synthesized 40-mers was therefore named CY(SacII)-40ODNS. All ODNs were mixed and denatured at 95°C for 3 minutes, followed by slow cooling to room temperature for about 1 hour. Since CY(SacII)-L is complementary to the left arm of CY(SacII)-40 and CY(SacII)-R is complementary to the right arm of the same ODN, partially double-stranded ODNs are formed during the annealing process. The annealed ODN formed a partially double-stranded DNA, and using DNA polymerase I, Large (Klenow) Fragment (New England Biolabs, Beverly, MA), those remaining single-s...

Embodiment 3

[0045] Example 3 Screening of ssDNA expression library

[0046] After overnight incubation at 37°C, transformants were recovered on LB plates containing 34 μg / ml Cm and 50 μg / ml Spec. LB plates were plated onto induced (100 ng / ml of aTc) and non-induced (no aTc) LB plates and incubated overnight at 37°C. Colonies that grew normally on LB plates but failed to grow and showed growth defects on induction plates were selected. Inducer-mediated growth inhibition was confirmed by retesting selected colonies on induced and non-induced plates for cell growth and inducible growth inhibition. Approximately 5000 transformants were screened. A total of 12 colonies were considered to have inducible growth inhibition in the presence of aTc.

[0047] Plasmids were isolated from these colonies and the inserts were sequenced:

[0048] 1. LIB0308:

[0049] GTAACGCCCA AACCTAAAAA ACCAGAATTA TTGCCCCCGT [SEQ ID NO: 7]

[0050] 2. LIB0309:

[0051] CGGGCATACA GGTCAAAATC GGGACAAGCG AAGGAATTAA AC...

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Abstract

The current invention is directed to oligonucleotide sequences isolated from a sequence designated rbl-1 [SEQ ID NO. 19] that either kill or inhibit growth, or prevent the production of endogenously expressed toxin, of microorganisms. These ssDNA sequences, identified through use of a screening method, appear to act as modulators of essential growth functions which may act at the level of triplex formation, antisense inhibition, or as aptamers that alter gene function. The sequences, referred to as minimum functional regions, or MFRs, are useful inter alia as therapeutic agents for treatment of sepsis and other pathologies caused by microorganisms such as sepsis and / or in which microorganisms are contributory agents.

Description

technical field [0001] The present invention relates to oligonucleotide sequences, in particular to oligonucleotide sequences that can kill microorganisms, inhibit the growth of microorganisms, or prevent microorganisms from producing endogenously expressed toxins. Background technique [0002] Currently, the need for new antimicrobial therapies is growing. Many microbial infections treated with antibiotics are acquiring multi-resistance to antibiotics, thus requiring new approaches to combat conditions caused by these microbial factors. Although many different approaches are under investigation, the present invention primarily involves the use of targeted single-stranded nucleotides. Oligonucleotide-mediated intervention (OMI) technology provides a powerful tool to alter the activity of any gene of known sequence. The ability to produce single-stranded DNA (ssDNA) of arbitrary sequence and length in cells of choice enables targeted alteration of gene expression at the gen...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/11C12N15/113
CPCC12N15/111C12N2320/30C12N2310/16C12N2310/11C12N2330/31C12N2330/50C12N2320/11C12N15/113C12N2310/111A61P31/00A61P31/04A61P31/10A61P31/12C12N15/09C12N15/10
Inventor 陈寅谭新星
Owner CYTOGENIX INC
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